Compositions and methods for the selective degradation of modified proteins

Abstract

The present disclosure relates to engineered polypeptides, compounds, compositions that contain degradation domains, and methods for their preparation and use to degrade engineered proteins within cells.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 230,225, filed August 6, 2021, which is incorporated by reference in its entirety for all purposes.

[0002] Field The present disclosure relates to engineered polypeptides, compounds, compositions that contain degradation domains, and methods for their preparation and use to degrade engineered proteins within cells. [Background technology]

[0003] Engineered cells containing modified heterologous polypeptides, such as chimeric antigen receptor T (CAR-T) cells, have been developed for therapeutic use. Modulation of the expression levels of such modified heterologous polypeptides may improve the therapeutic efficacy of the modified cells, for example, by reducing side effects and / or improving the efficacy of the modified cells.

[0004] Thus, in one embodiment, provided herein is a modified polypeptide and a degradation agent, wherein the modified polypeptide comprises a degradation domain that mediates ubiquitination within a cell when the degradation domain binds to the degradation agent. . Summary of the Invention

[0005] In certain embodiments, compounds and compositions thereof for regulating the level of heterologous polypeptides in cells are described herein. In various embodiments, the compounds and compositions thereof may be used to reduce the level of heterologous polypeptides in cells.

[0006] Embodiments of the present application can be more fully understood by reference to the detailed description and examples that are intended to illustrate non-limiting embodiments. [Brief description of the drawings]

[0007] [Figure 1A-1] Figure 1A shows a sequence alignment of human IKZF family proteins IKZF1 to IKZF5 (SEQ ID NOs: 48 to 52). Figure 1B shows a table of predicted G motif-containing zinc finger sequences of human IKZF1 to IKZF5. The G motif sequence is underlined. [Figure 1A-2] Figure 1A shows a sequence alignment of human IKZF family proteins IKZF1 to IKZF5 (SEQ ID NOs: 48 to 52). Figure 1B shows a table of predicted G motif-containing zinc finger sequences of human IKZF1 to IKZF5. The G motif sequence is underlined. [Figure 1B] Figure 1A shows a sequence alignment of human IKZF family proteins IKZF1 to IKZF5 (SEQ ID NOs: 48 to 52). Figure 1B shows a table of predicted G motif-containing zinc finger sequences of human IKZF1 to IKZF5. The G motif sequence is underlined. [Diagram 2] FIG. 2 is a diagram of a chimeric antigen receptor (CAR) containing a C-terminal IKZF1 ZNF2 degron. [Diagram 3] FIG. 3 is a schematic diagram of a Jurkat cell reporter-based model system for studying CAR activity and degradation. [Figure 4A] 4A-4C show that IKZF1 ZNF2-tagged CAR retains function (FIG. 4A) but is only partially degraded (FIG. 4C) by high concentrations of Compound A. The structure of Compound A is shown in FIG. 4B. [Figure 4B-4C] 4A-4C show that IKZF1 ZNF2-tagged CAR retains function (FIG. 4A) but is only partially degraded (FIG. 4C) by high concentrations of Compound A. The structure of Compound A is shown in FIG. 4B. [Figure 5A]Figure 5A shows a schematic of CARs containing C-terminal IKZF1 ZNF1, 2, and / or 3 degrons. Each of the CARs also contains an N-terminal CD19-binding scFv. Figure 5B shows that CARs with C-terminal IKZF1 degrons retain activity. Figure 5C shows the degradation of CARs with C-terminal IKZF1 degrons with increasing concentrations of Compound A. Figure 5D shows that the degradation is specific to wild-type IKZF1 degron. [Figure 5B] Figure 5A shows a schematic of CARs containing C-terminal IKZF1 ZNF1, 2, and / or 3 degrons. Each of the CARs also contains an N-terminal CD19-binding scFv. Figure 5B shows that CARs with C-terminal IKZF1 degrons retain activity. Figure 5C shows the degradation of CARs with C-terminal IKZF1 degrons with increasing concentrations of Compound A. Figure 5D shows that the degradation is specific to wild-type IKZF1 degron. [Figure 5C] Figure 5A shows a schematic of CARs containing C-terminal IKZF1 ZNF1, 2, and / or 3 degrons. Each of the CARs also contains an N-terminal CD19-binding scFv. Figure 5B shows that CARs with C-terminal IKZF1 degrons retain activity. Figure 5C shows the degradation of CARs with C-terminal IKZF1 degrons with increasing concentrations of Compound A. Figure 5D shows that the degradation is specific to wild-type IKZF1 degron. [Figure 5D] Figure 5A shows a schematic of CARs containing C-terminal IKZF1 ZNF1, 2, and / or 3 degrons. Each of the CARs also contains an N-terminal CD19-binding scFv. Figure 5B shows that CARs with C-terminal IKZF1 degrons retain activity. Figure 5C shows the degradation of CARs with C-terminal IKZF1 degrons with increasing concentrations of Compound A. Figure 5D shows that the degradation is specific to wild-type IKZF1 degron. [Figure 6A-6C] 6A-6C show the reduction of CAR levels and repression of CAR activity following degradation in the Jurkat reporter system. [Figure 7] FIG. 7 shows that endogenous Erk signaling is attenuated by CAR degradation. [Figure 8] FIG. 8 shows an alignment of the G motif-containing C2H2 zinc fingers of certain human IKZF family members (SEQ ID NOs: 21, 32, 27, 38, 40, 29, 47, 31, 23, 20, 26, and 37). [Figure 9-1] FIG. 9 shows predicted C2H2 zinc finger degrons derived from various human proteins (SEQ ID NOs: 72-109). [Figure 9-2] FIG. 9 shows predicted C2H2 zinc finger degrons derived from various human proteins (SEQ ID NOs: 72-109). [Figure 10A-10B] Figure 10A shows the structure of compound B. Figure 10B shows the ubiquitination of the modified G motif from Ikaros ZNF2 using an in vitro ubiquitination assay. [Figures 11A-11C] Figure 11A shows the structure of compound C. Figures 11B-11C show that degradation of IKZF1 ZNF2_3 Q1F degron-tagged CAR is CRBN- and ubiquitin-proteasome pathway (UPP)-dependent. [Figure 12A-12B] 12A-12B show that degradation of IKZF1 ZNF2_3 Q1F-tagged CAR reduces CAR levels and signaling in Jurkat reporter assays. [Figure 13A] Figure 13A shows the structure of IKZF1 ZNF2_3 Q1F-tagged CAR. Figure 13B shows that the expression of IKZF1 ZNF2_3 Q1F-tagged CAR can be regulated by Compound C in primary T cells. [Figure 13B] Figure 13A shows the structure of IKZF1 ZNF2_3 Q1F-tagged CAR. Figure 13B shows that the expression of IKZF1 ZNF2_3 Q1F-tagged CAR can be regulated by Compound C in primary T cells. [Figure 14A] Figures 14A-14D show that the function of IKZF1 ZNF2_3 Q1F-tagged CAR is tunable with Compound C in primary T cells. [Figure 14B]Figures 14A-14D show that the function of IKZF1 ZNF2_3 Q1F-tagged CAR is tunable with Compound C in primary T cells. [Figure 14C] Figures 14A-14D show that the function of IKZF1 ZNF2_3 Q1F-tagged CAR is tunable with Compound C in primary T cells. [Figure 14D] Figures 14A-14D show that the function of IKZF1 ZNF2_3 Q1F-tagged CAR is tunable with Compound C in primary T cells. [Figure 15] FIG. 15 is a schematic diagram of the chronic antigen stimulation assay used to test functional persistence. [Figure 16A-16B] Figure 16A shows the structure of compound D. Figures 16B-16C show that transiently rested CAR T cells are less activated by chronic antigen exposure and maintain a more naive phenotype. [Figure 16C] Figure 16A shows the structure of compound D. Figures 16B-16C show that transiently rested CAR T cells are less activated by chronic antigen exposure and maintain a more naive phenotype. [Figure 17A] Figures 17A-17B show that transiently rested CAR T cells produce more inflammatory cytokines and exhibit greater anti-tumor activity than non-rested cells after chronic antigen exposure. [Figure 17B] Figures 17A-17B show that transiently rested CAR T cells produce more inflammatory cytokines and exhibit better anti-tumor activity than non-rested cells after chronic antigen exposure. [Figure 18A-18B] Figures 18A-18C show that Q1F degron-tagged CAR can be reversibly downregulated in vivo. [Figure 18C] Figures 18A-18C show that Q1F degron-tagged CAR can be reversibly downregulated in vivo. [Figure 19A-19B] Figures 19A-19D show that downregulation of Q1F degron-tagged CAR reduces tumor reactive growth in vivo. [Fig. 19C-19D]Figures 19A-19D show that downregulation of Q1F degron-tagged CAR reduces tumor reactive growth in vivo. [Figure 20A] 20A-20D show that in-frame degron-tagged knock-in into the endogenous AURKA or TOX locus allows compound-mediated control of protein levels. [Figure 20B] 20A-20D show that in-frame degron-tagged knock-in into the endogenous AURKA or TOX locus allows compound-mediated control of protein levels. [Fig. 20C-20D] 20A-20D show that in-frame degron-tagged knock-in into the endogenous AURKA or TOX locus allows compound-mediated control of protein levels. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] Detailed Description As used herein, the terms "comprising" and "including" may be used interchangeably. The terms "comprising" and "including" should be interpreted as specifying the presence of the mentioned or described features or components, but do not exclude the presence or addition of one or more features, or components, or groups thereof. Furthermore, the terms "comprising" and "including" are intended to include examples encompassed by the term "consisting of". Consequently, the term "consisting of" may be used in place of the terms "comprising" and "including" to provide more specific embodiments of the present invention.

[0009] The term "consisting of" means that the subject matter of the patented invention has at least 90%, 95%, 97%, 98%, or 99% of the recited features or components that make it up. In another embodiment, the term "consisting of" excludes from the scope of any succeeding recitation any other features or components, excluding those that are not essential to the technical effect to be achieved.

[0010] The term "or" as used herein should be interpreted as an inclusive "or" meaning any one or any combination. Thus, "A, B, or C" means any of the following: "A; B; C; A and B; A and C; B and C; A, B, and C." Exceptions to this definition occur only when combinations of elements, features, steps, or acts are in some way mutually exclusive in nature.

[0011] Any concentration range, percentage range, ratio range, or integer range herein should be understood to include any integer value within the stated range, and fractions thereof (such as tenths and hundredths of integers) where appropriate, unless otherwise specified. Also, any numerical range described herein for any physical characteristic (such as macromolecular subunits, size, or thickness) should be understood to include any integer within the stated range, unless otherwise specified. As used herein, the terms "about" and "approximately" mean ±20%, ±10%, ±5%, or ±1% of the indicated range, value, or structure, unless otherwise specified.

[0012] As used herein, an "engineered polypeptide" is a polypeptide having an amino acid sequence that does not occur in nature. Although a portion of an engineered polypeptide may occur in nature, the engineered polypeptide as a whole does not occur in nature. In some embodiments, an engineered polypeptide comprises a naturally occurring amino acid sequence that has been modified, for example, by fusing to or inserting a degradation domain. In some such embodiments, the resulting engineered polypeptide substantially retains the activity of the original naturally occurring polypeptide. In some embodiments, an engineered polypeptide comprises two or more, three or more, or four or more domains derived from two or more, three or more, or four or more naturally occurring polypeptides. In some embodiments, an engineered polypeptide comprises a degradation domain.

[0013] As used herein, "degron" and "degradation domain" are used interchangeably and refer to an amino acid sequence that, when present in a polypeptide in a cell, causes ubiquitination of the polypeptide by ubiquitin ligase in the presence of a compound that binds to both the degradation domain and the ubiquitin ligase. In some embodiments, the compound binds to the degradation domain and to cereblon. In some embodiments, the modified polypeptide comprises a degradation domain. After ubiquitination by ubiquitin ligase, the polypeptide comprising the degradation domain may be degraded.

[0014] Representative Modified Polypeptides Provided herein is a modified polypeptide comprising a degradation domain. In some embodiments, the modified polypeptide is a CAR. In some embodiments, such a polypeptide comprises a transmembrane domain, an extracellular domain, and an intracellular domain. In some such embodiments, the degradation domain is located in the intracellular domain of the modified polypeptide. In some embodiments, the extracellular domain comprises a ligand, a ligand-binding domain, or an antigen-binding domain. In some embodiments, the antigen-binding domain binds to a cancer antigen. In some embodiments, the antigen-binding domain comprises an antibody light or heavy chain variable region, or an scFv. In some embodiments, the antigen-binding domain comprises a single-domain antibody antigen-binding domain. In some embodiments, the intracellular domain comprises at least one co-stimulatory domain. In some embodiments, the intracellular domain comprises at least one signaling domain, such as an ITAM signaling domain. In some embodiments, the modified polypeptide is a CAR comprising a degradation domain as further described below.

[0015] In some embodiments, the modified polypeptide is based on a naturally occurring protein into which a degradation domain has been genetically inserted or fused. The resulting modified polypeptide may contain additional naturally occurring or non-naturally occurring amino acid sequences. In some embodiments, the modified polypeptide is based on a naturally occurring nuclear or cytoplasmic protein. In some embodiments, the modified polypeptide substantially retains the activity of the naturally occurring protein. Degradation of the modified polypeptide may be achieved by contacting a cell expressing the modified polypeptide with a degradation agent, such as by administration. In some such embodiments, the degradation domain is derived from an Ikaros Family Zinc Finger (ZNF) amino acid sequence and the degradation agent is a small molecule that binds to a ubiquitin ligase, such as an E3 ligase. Administration of the degradation agent to a cell expressing a modified polypeptide containing a degradation domain causes ubiquitination of the modified polypeptide containing the degradation domain by the E3 ligase and degradation of the modified polypeptide. In some embodiments, the degradation agent is a compound that binds to cereblon and the degradation domain.

[0016] In some embodiments, the modified polypeptide comprises a naturally occurring protein and a degradation domain that is fused or inserted into the naturally occurring protein. When the degradation domain is "fused" to a protein, the modified polypeptide may include a linker that connects the degradation domain to the protein, such as an amino acid linker. Such an amino acid linker may be of any length, for example, 1-50, 1-40, 1-30, 1-20, 1-10, or 1-5 amino acids. In some embodiments, the amino acid linker is composed of glycine and serine.

[0017] Exemplary proteins to which degradation domains may be fused or inserted include, but are not limited to, PRDM1, TGFBR2, CASP8, CBLB, CD5, CISH, CGKA, DGKz, MAP4K1, ARID2, BACH2, CHX37, KLF2, KLF3, KLF6, MAF, SIGLEC9, TOX, ZBTB32, PTPN2, AKT1, PIK3CD, MT1E, MT2A, CSK, ITK, PAG1, PDCD4, ZC3H12A, DNMT1, DNMT3A, PRBM1, ST K4, TET2, BNIP3, FAS, CBL, BGAT5, RNF128, STK17B, TRIB1, TXNIP, UBASH3A, BATF, FLI1, IKZF1, IKZF2, IRF4, NFATC1, NR4A1, MAP2K1, MAP2K2, MAP4K4, PPARGC1A, RELB, TMEM173, USP10, MT1A, PP2A family members, RASA2, NR4A2, NR4A3, AHR, CD70, LHALS1, SOCS1, SOCS2, SOCS3, TAZ, USP21, or YAP1. In some embodiments, the protein is a mammalian protein, such as a human protein.

[0018] In some embodiments, the degradation domain is fused to or inserted into an endogenous protein in the cell. In some such embodiments, the sequence encoding the degradation domain may be inserted into the genome of a cell expressing an endogenous protein that includes the degradation domain fused to or inserted into the endogenous protein, such that the modified polypeptide is expressed. Various methods of inserting a nucleic acid sequence (such as a sequence encoding a degradation domain) into the genome of a cell are known in the art, including, for example, CRISPR / Cas, adeno-associated virus (AAV)-mediated or non-viral homologous recombination, lentiviral transduction, or transposon delivery. In some embodiments, the nucleic acid sequence encoding the degradation domain is fused to or inserted into an endogenous protein in an immune cell (such as a T lymphocyte). In some such embodiments, the T cell is isolated, modified to express the modified polypeptide, and administered to the patient. After administration to the patient, a degradation drug may be subsequently administered if degradation of the modified polypeptide is desired.

[0019] In some embodiments, the nucleic acid sequence encoding the modified polypeptide is introduced into the cell. Methods for introducing nucleic acids into cells are known in the art, and include, for example, synthetic vectors, lentiviral or retroviral vectors, self-replicating plasmids, viruses (e.g., retroviruses, lentiviruses, adenoviruses, or herpes viruses), or the like, that contain the nucleic acid (polynucleotide) encoding the modified polypeptide described herein.

[0020] Representative degradation domains The modified polypeptides provided herein comprise a degradation domain. In some embodiments, the degradation domain comprises an amino acid sequence that binds to a degradation agent. The degradation agent binds to the degradation domain and a ubiquitin ligase, causing ubiquitination of the modified polypeptide.

[0021] In some embodiments, the degradation domain comprises an amino acid sequence derived from a G motif of an Ikaros family protein (such as Ikaros, Helios, Aiolos, Eos, or Pegasus). Non-limiting representative G motifs are underlined in the sequence shown in FIG. 1B. In some embodiments, the degradation domain provided herein comprises an amino acid sequence that is modified from a native G motif sequence by substituting phenylalanine (F) for the amino acid at position 1. In some embodiments, the degradation domain is derived from a G motif that naturally contains a Q at position 1, such that the degradation domain comprises a Q1F substitution.

[0022] In some embodiments, the degradation domain comprises the amino acid sequence FCX1X2CGX3X4 (SEQ ID NO: 1). In some embodiments, X1 is selected from asparagine, aspartic acid, glycine, glutamine, methionine, histidine, tryptophan, isoleucine, arginine, leucine, valine, threonine, and phenylalanine; X2 is selected from glutamine, arginine, histidine, leucine, phenylalanine, tyrosine, tryptophan, isoleucine, valine, and methionine; X3 is selected from alanine, serine, cysteine, arginine, leucine, isoleucine, methionine, and glycine; and X4 is selected from serine, methionine, lysine, isoleucine, valine, histidine, glutamine, arginine, phenylalanine, and tryptophan. In some embodiments, X1 is selected from asparagine, glutamine, methionine, histidine, tryptophan, isoleucine, arginine, leucine, valine, threonine, and phenylalanine. In some embodiments, X2 is selected from glutamine, arginine, histidine, leucine, phenylalanine, tyrosine, tryptophan, isoleucine, and methionine. In some embodiments, X3 is selected from alanine, serine, cysteine, and glycine. In some embodiments, X4 is selected from serine, methionine, histidine, glutamine, arginine, phenylalanine, and tryptophan. In some embodiments, X1 is asparagine. In some embodiments, X2 is glutamine. In some embodiments, X3 is alanine or serine. In some embodiments, X3 is alanine. In some embodiments, X4 is serine. In some embodiments, the degradation domain of the modified polypeptide comprises the amino acid sequence FCNQCGAS (SEQ ID NO: 3).

[0023] In some embodiments, the degradation domain comprises the amino acid sequence FCX1X2CGX3X4X5 (SEQ ID NO: 2), where X1, X2, X3, and X4 are as defined above. In some embodiments, X5 is selected from phenylalanine, tryptophan, methionine, arginine, histidine, leucine, tyrosine, cysteine, and glutamine. In some embodiments, X5 is selected from phenylalanine, tryptophan, methionine, arginine, histidine, leucine, tyrosine, and glutamine. In some embodiments, X5 is selected from phenylalanine, tryptophan, methionine, leucine, tyrosine, and glutamine. In some embodiments, X5 is phenylalanine.

[0024] In various embodiments, the degradation domain comprises at least one zinc finger domain comprising a modified G motif as described above. In some embodiments, the at least one zinc finger domain is derived from an Ikaros family protein (such as Ikaros, Helios, Aiolos, Eos, or Pegasus). Non-limiting representative zinc fingers comprising a G motif are shown in FIG. 1B. In various embodiments, the degradation domain comprises one, two, three, or four zinc finger domains. In some embodiments, the degradation domain comprises one zinc finger domain comprising a G motif and at least one zinc finger domain not comprising a G motif. The zinc finger domains may or may not be derived from the same protein. In some embodiments, the degradation domain comprises two zinc finger domains.

[0025] In some embodiments, the degradation domain comprises an amino acid sequence having at least 85%, at least 90%, or at least 95% identity to zinc finger 2 (ZNF2) of human Ikaros. In some embodiments, the degradation domain comprises an amino acid sequence having at least 85%, at least 90%, or at least 95% identity to zinc finger 2 (ZNF2) of human Ikaros and at least one additional zinc finger domain (such as at least one additional zinc finger domain of an Ikaros family protein). In some embodiments, the degradation domain comprises an amino acid sequence having at least 85%, at least 90%, or at least 95% identity to zinc finger 2 (ZNF2) of human Ikaros and ZNF1 or ZNF3 of human Ikaros. In some embodiments, the degradation domain comprises an amino acid sequence having at least 85%, at least 90%, or at least 95% identity to zinc finger 2 (ZNF2) and zinc finger 3 (ZNF3) of human Ikaros.

[0026] In some embodiments, the degradation domain comprises an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to amino acids 145-167 of human Ikaros (FQCNQCGASFTQKGNLLRHIKLH; SEQ ID NO:21). In some embodiments, the degradation domain comprises an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to amino acids 140-162 of human Helios (FHCNQCGASFTQKGNLLRHIKLH; SEQ ID NO:27). In some embodiments, the degradation domain comprises an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to amino acids 146-168 of human Aiolos (FQCNQCGASFTQKGNLLRHIKLH; SEQ ID NO:32). In some embodiments, the degradation domain comprises an amino acid sequence having at least 85%, at least 90%, or at least 95% identity to amino acids 187 to 209 of human Eos (FHCNQCGASFTQKGNLLRHIKLH; SEQ ID NO: 38).

[0027] In some embodiments, the degradation domain comprises an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to amino acids 141-168 of human Ikaros (GERPFQCNQCGASFTQKGNLLRHIKLHS; SEQ ID NO: 15). In some embodiments, the degradation domain comprises an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to amino acids 136-163 of human Helios (GERPFHCNQCGASFTQKGNLLRHIKLHS; SEQ ID NO: 60). In some embodiments, the degradation domain comprises an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to amino acids 142-169 of human Aiolos (GERPFQCNQCGASFTQKGNLLRHIKLHT; SEQ ID NO: 61). In some embodiments, the degradation domain comprises an amino acid sequence having at least 85%, at least 90%, or at least 95% identity to amino acids 183 to 210 of human Eos (GERPFHCNQCGASFTQKGNLLRHIKLHS; SEQ ID NO: 62).

[0028] In some embodiments, the degradation domain comprises an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to amino acids 141-196 of human Ikaros (GERPFQCNQC GASFTQKGNL LRHIKLHSGE KPFKCHLCNY ACRRRDALTG HLRTHS; SEQ ID NO: 6). In some embodiments, the degradation domain comprises an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to amino acids 136-191 of human Helios (GERPFHCNQC GASFTQKGNL LRHIKLHSGE KPFKCPFCSY ACRRRDALTG HLRTHS; SEQ ID NO: 63). In some embodiments, the degradation domain comprises an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to amino acids 142-197 of human Aiolos (GERPFQCNQC GASFTQKGNL LRHIKLHTGE KPFKCHLCNY ACQRRDALTG HLRTHS; SEQ ID NO: 64). In some embodiments, the degradation domain comprises an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to amino acids 183-238 of human Eos (GERPFHCNQC GASFTQKGNL LRHIKLHSGE KPFKCPFCNY ACRRRDALTG HLRTHS; SEQ ID NO: 65).

[0029] In some embodiments, the degradation domain comprises the amino acid sequence: GERPFFCX1X2CGX3X4X5TQKGNLLRHIKLHSGEKPFKCHLCNYACRRRDALTGHLRTHS (SEQ ID NO:5), where X1, X2, X3, and X4, and X5 are as defined above. In some embodiments, the degradation domain comprises the amino acid sequence: GERPFFCX1X2CGX3X4X5TQKGNLLRHIKLHSGEKPFKCPFCSYACRRRDALTGHLRTHS (SEQ ID NO:66), where X1, X2, X3, and X4, and X5 are as defined above. In some embodiments, the degradation domain comprises the amino acid sequence: GERPFFCX1X2CGX3X4X5TQKGNLLRHIKLHTGEKPFKCHLCNYACQRRDALTGHLRTHS (SEQ ID NO:67), where X1, X2, X3, and X4, and X5 are as defined above. In some embodiments, the degradation domain comprises the amino acid sequence: GERPFFCX1X2CGX3X4X5TQKGNLLRHIKLHSGEKPFKCPFCNYACRRRDALTGHLRTHS (SEQ ID NO: 68), where X1, X2, X3, and X4, and X5 are as defined above.

[0030] In some embodiments, the degradation domain comprises the amino acid sequence: GERPFFCNQCGASFTQKGNLLRHIKLHSGEKPFKCHLCNYACRRRDALTGHLRTHS (SEQ ID NO: 7). In some embodiments, the degradation domain comprises the amino acid sequence: GERPFFCNQCGASFTQKGNLLRHIKLHSGEKPFKCPFCSYACRRRDALTGHLRTHS (SEQ ID NO: 69). In some embodiments, the degradation domain comprises the amino acid sequence: GERPFFCNQCGASFTQKGNLLRHIKLHTGEKPFKCHLCNYACQRRDALTGHLRTHS (SEQ ID NO: 70). In some embodiments, the degradation domain comprises the amino acid sequence: GERPFFCNQCGASFTQKGNLLRHIKLHSGEKPFKCPFCNYACRRRDALTGHLRTHS (SEQ ID NO: 71).

[0031] Representative chimeric antigen receptor constructs The ability to regulate the expression of a CAR by degrading it in the presence of a degradation drug has many advantages over the absence of the ability to regulate CAR expression. For example, on-target off-tumor effects brought about by therapeutic immune cells expressing a CAR, which can result in toxicity, can be reduced or eliminated by degrading the CAR. A too strong CAR-mediated immune response can be reduced or eliminated by degrading the CAR. T cell dysfunction caused by chronic activation and overexpression of checkpoints can be avoided by cycling the expression of the CAR and / or titrating the expression of the CAR. Such CAR degradation is achieved by expressing a CAR comprising a degradation domain as provided herein and administering a degradation drug as needed. In some such embodiments, the degradation drug is a small molecule that binds to a ubiquitin ligase, such as an E3 ligase. Administration of the degradation drug to a cell expressing a CAR polypeptide comprising a degradation domain causes ubiquitination of the CAR polypeptide comprising the degradation domain by the E3 ligase and degradation of the CAR polypeptide. In some embodiments, the degradation drug is a compound that binds to cereblon and the degradation domain.

[0032] Provided herein are engineered polypeptides that include or consist of chimeric antigen receptors (CARs) that include (a) components of a CAR, such as an antigen-binding domain, a transmembrane domain, a cell signaling domain, and / or a costimulatory domain, and (b) a degradation domain. When a CAR fused to a degradation domain is expressed in an immune cell (e.g., a T lymphocyte or a natural killer cell) in the presence of a degradation agent, such as a cereblon-binding compound, an E3 ligase, such as cereblon, and the degradation domain of the CAR bind to the degradation agent, forming an E3 ligase complex that ubiquitinates the degradation domain. Thus, the activity (e.g., in vivo activity) of the CAR described herein can be controlled by contacting a cell expressing a CAR that includes a degradation domain (e.g., a T lymphocyte modified to express the CAR polypeptide) with a degradation agent, such as a cereblon-binding compound.

[0033] In some embodiments, provided herein is a modified polypeptide that is a CAR comprising an antigen-binding domain, a transmembrane domain, an intracellular primary signaling domain, and a degradation domain. In some embodiments, the degradation domain comprises an amino acid sequence provided herein.

[0034] In some embodiments, the modified polypeptide is a CAR that comprises, in order from amino terminus to carboxy terminus, an antigen-binding domain, a transmembrane domain, a primary T cell signaling domain, and / or a costimulatory domain, and a degradation domain. In some embodiments, the degradation domain is located at the C-terminus of the CAR. In some embodiments, the degradation domain comprises the amino acid sequence provided herein. In some embodiments, the CAR comprises a costimulatory domain.

[0035] In some embodiments, the modified polypeptide is a CAR that comprises, from amino to carboxy terminus, (i) extracellular domain [ECD]-transmembrane domain [TM]-costimulatory domain [CoD]-signaling domain [SigD]-degradation domain [DD]. In some embodiments, the modified polypeptide is a CAR that comprises, from amino to carboxy terminus, ECD-TM-CoD-DD-SigD. In some embodiments, the modified polypeptide is a CAR that comprises, from amino to carboxy terminus, ECD-TM-DD-CoD-SigD. The degradation domain may also be inserted into another domain (such as a costimulatory domain or signaling domain), preferably such that the desired activity of the domain is retained.

[0036] Representative antigen-binding domains The antigen binding domain of the CAR provided herein can be any polypeptide domain, motif, or sequence that binds to an antigen.

[0037] In certain embodiments, the antigen-binding domain of the CAR described herein is the antigen-binding portion of a receptor. In some embodiments, the antigen-binding domain of the CAR described herein is a receptor for a ligand produced by a tumor cell.

[0038] In certain embodiments, the antigen-binding domain of the CAR described herein is an antigen-binding portion of an antibody. In some embodiments, the antigen-binding domain of the CAR described herein is an antibody, an antibody chain, a single-chain antibody, or an antigen-binding portion thereof, an Fc domain, a glycophosphatidylinositol anchor domain, or an scFv antibody fragment.

[0039] In certain embodiments, the antigen-binding domain of a CAR described herein is a peptide-based polymeric antigen-binding agent, e.g., a phage-displayed protein.

[0040] In certain embodiments, antigen binding by the antigen binding domain of a CAR described herein is restricted to antigen presentation in the context of the major histocompatibility complex (MHC). In certain embodiments, antigen binding by the antigen binding domain of a CAR described herein is unrestricted by MHC.

[0041] The antigen bound / recognized by the antigen-binding domain of the CAR described herein can be any antigen of interest. In some embodiments, the antigen is an antigen expressed on the surface of a cell (e.g., a tumor cell, such as a solid tumor cell or a blood cancer tumor cell).

[0042] In some embodiments, the antigen bound / recognized by the antigen binding domain of the CAR described herein is an antigen on a tumor cell, e.g., the antigen is a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). Exemplary tumor cell antigens that can be recognized by the CAR described herein (i.e., can be bound by the antigen binding domain of the CAR) include, but are not limited to, 4-1BB, 5T4, 8H9, B7-H6, adenocarcinoma antigen, a-fetoprotein, B cell maturation antigen (BCMA), BAFFR, B lymphoma cells, C242 antigen, CA9, carcinoembryonic antigen, CA-125, carbonic anhydrase 9 (CA-IX), CCR4, CD3, CD4, CD19, CD20, CD22, CD23 (IgE receptor), CD28, CD30 (TFRSF8), CD33, CD38, CD40, CD44v6, CD44v7 / 8, CD51, CD52, CD56, CD70 CD74, CD80, CD123, CD152, CD171, CD200, CD221, CE7, CEA, C-MET, CLAUDIN6, CLAUDIN18.3, CNT0888, CTLA-4, DRS, EpCAM, ErbB2, ErbB3 / 4, EGFR, EGFRγIII, EphA2, EGP2, EGP40, FAP, Fetal AchR, Fibronectin extra domain B, Folate receptor a, Folate receptor 1, G250 / CAIX, GD2, GD3, Glycoprotein 75, GP MB, HER2 / neu, HGF, HLA-AI MAGE Al, HLA-A2 NY-ESO-1, HMW-MAA, Human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgGl, IL-6, IL-13, IL-13 receptor a2, IL-11 receptor a, Insulin-like growth factor I receptor, Integrin a5I31, Integrin avI33, Kappa light chain, Ll-CAM, Lambda light chain, Lewis Y, mesothelin, MORAb-009, MS4A1, MUCl, MUCl 6, mucin CanAg, NCAM, N-glycolylneuraminic acid, NKG2D ligand, NPC-IC, PDGF-R a, PDL192, phosphatidylserine, prostate specific cancer antigen (PSCA), prostate cancer cells, PSMA, PSC1, RANKL, RON, ROR1, SCH 900105, SDC1, SLAMF7, spl7, TAG72, tenascin C, TGF beta2, TGF-I3, TL1A, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, UPK1B, VEGF-A, VEGF receptor, VEGFR-1, VEGFR2, TEM1, TEM8, and / or vimentin.

[0043] In some embodiments, the antigen bound / recognized by the antigen binding domain of a CAR described herein is an antigen expressed on or associated with tumor cells of lymphoma / leukemia, lung cancer, breast cancer, prostate cancer, adrenal cortical carcinoma, thyroid cancer, nasopharyngeal carcinoma, melanoma, e.g., malignant melanoma, skin cancer, colorectal carcinoma, desmoid tumor, desmoplastic small round cell tumor, endocrine tumor, Ewing's sarcoma, peripheral primitive neuroectodermal tumor, solid germ cell tumor, hepatoblastoma, neuroblastoma, non-rhabdomyosarcoma soft tissue sarcoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, Wilms' tumor, glioblastoma, myxoma, fibroma, lipoma, or the like.

[0044] In some embodiments, the antigen bound / recognized by the antigen binding domain of a CAR described herein is selected from the group consisting of chronic lymphocytic leukemia (small lymphocytic lymphoma), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, Waldenstrom's macroglobulinemia, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, extranodal marginal zone B-cell lymphoma, MALT lymphoma, nodal marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary coagulable lymphoma, primary pulmonary ... and / or an antigen expressed on or associated with the tumor cells of lymphoma, Burkitt's lymphoma, T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, aggressive NK-cell leukemia, adult T-cell leukemia / lymphoma, extranodal NK / T-cell lymphoma, nasal-type, enteropathy-type T-cell lymphoma, hepatosplenic T-cell lymphoma, blastic NK-cell lymphoma, mycosis fungoides, Sezary syndrome, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T-cell lymphoma, peripheral T-cell lymphoma (unspecified), anaplastic large cell lymphoma, Hodgkin's lymphoma, or non-Hodgkin's lymphoma.

[0045] In some embodiments, the antigen bound / recognized by the antigen-binding domain of the CAR described herein is a non-tumor-associated or non-tumor-specific antigen. In certain embodiments, the antigen is associated with the tumor context, e.g., the tumor environment. For example, tumors can cause an inflammatory state in the tissue surrounding the tumor and release angiogenic growth factors, interleukins, and / or cytokines that promote angiogenesis in and around the tumor. Thus, in certain embodiments, the antigen is a growth factor, cytokine, or interleukin (e.g., a growth factor, cytokine, or interleukin associated with angiogenesis or vasculogenesis). Such growth factors, cytokines, and interleukins can include, but are not limited to, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), and interleukin-8 (IL-8).

[0046] In some embodiments, the antigens bound / recognized by the antigen-binding domain of the CAR described herein are damage-associated molecular pattern molecules (DAMPs; also known as alarmins) released by normal tissues in response to localized damage caused by tumors. Exemplary DAMPs to which the antigen-binding domain of the CAR described herein may bind include, but are not limited to, heat shock proteins, chromatin-associated protein high mobility group box 1 (HMGBl), S100A8 (MRP8, calgranulin A), S100A9 (MRP14, calgranulin B), serum amyloid A (SAA), deoxyribonucleic acid, adenosine triphosphate, uric acid, and heparin sulfate.

[0047] Representative transmembrane domains As used herein, "transmembrane domain" includes transmembrane domains, where a polypeptide that comprises a transmembrane domain includes both an intracellular and an extracellular domain, and membrane anchor domains, where a polypeptide that comprises a transmembrane domain includes an intracellular domain but no extracellular domain.

[0048] The transmembrane domain of the modified polypeptides described herein can include any molecule known in the art to function as a transmembrane domain, e.g., known by a person skilled in the art to function in the context in which it is used (e.g., in CAR, etc.). The transmembrane domain of the modified polypeptides described herein can be obtained or derived from the transmembrane domain of any membrane-spanning protein and can include all or a portion of such a transmembrane domain.

[0049] In some embodiments, the transmembrane domain of an engineered polypeptide described herein (such as a CAR) is obtained or derived from a T cell receptor, e.g., the transmembrane domain of an engineered polypeptide described herein is obtained or derived from the alpha chain of the T cell receptor, the beta chain of the T cell receptor, or the zeta chain of the T cell receptor.

[0050] In some embodiments, the transmembrane domain of the modified polypeptides described herein is obtained or derived from CD28, CD3s, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS, TIM3, LAB3, TIGIT, PD1, or CTLA4, a cytokine receptor, an interleukin receptor, or a growth factor receptor.

[0051] Representative signaling domains The primary cell signaling domain of the CARs described herein can include any molecule known in the art to function as a cell signaling domain, e.g., known by a person skilled in the art to function in the context of a CAR. In some embodiments, the cell signaling domain of the CARs described herein includes a primary T cell signaling domain.

[0052] In some embodiments, the primary cell signaling domain of a CAR described herein is or comprises ZAP-70, or a signaling variant thereof.

[0053] In some embodiments, the primary cell signaling domain of a CAR described herein is or comprises an ITAM, hi some embodiments, the ITAM is an ITAM of CD3ε, CD3ζ, CD3η, FcRγ, FcRβ, CD3δ, CD3γ, CD5, CD22, CD20, CD79a, CD79b, CD278 (ICOS), FcERI, CD66d, DAP10, or DAP12.

[0054] Representative costimulatory domains In certain embodiments, the CARs described herein comprise a costimulatory domain. The costimulatory domain of the CARs described herein can comprise any molecule known in the art to function as a costimulatory domain, e.g., known by a person skilled in the art to function in the context of a CAR.

[0055] In some embodiments, the costimulatory domain of a CAR described herein is obtained or derived from a costimulatory CD27 polypeptide sequence, a costimulatory CD28 polypeptide sequence, a costimulatory OX40 (CD134) polypeptide sequence, a costimulatory 4-1BB (CD137) polypeptide sequence, or a costimulatory-inducible T cell costimulatory (ICOS) polypeptide sequence.

[0056] In some embodiments, the costimulatory domain of a CAR described herein is selected from the group consisting of 4-1BB (CD137), CD28, OX40, K cell activating receptor, BTLA, Toll ligand receptor, CD2, CD7, CD27, CD30, CD40, CDS, ICAM-L LFA-1 (CD1la / CD18), B7-H3, CDS, ICAM-1, ICOS (CD278), RANK, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, Kp80 (KLRF1), Kp44, Kp30, Kp46, CD19, CD4, CD8a, CD8p, IL2Rp, IL2Ry, IL7Ra, ITGA4, VLAl, CD49a , ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, LFA-1, ITGAM, CDllb , ITGAX, CDllc, ITGB1, CD29, ITGB2, IL15Ra, IL7R, CD18, CD132, LFA-1, ITGB7, KG2D, KG2C, TFR2, TRANCE / RA is or comprises KL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, DAP10, DAP12, a ligand for CD83, an MHC class I molecule, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, and / or a signaling lymphocyte activation molecule.

[0057] Other typical components In certain embodiments, the modified polypeptides, such as the CARs described herein, further comprise a T cell survival motif. The T cell survival motif can be any amino acid sequence or motif that promotes the survival of T lymphocytes after stimulation with an antigen. In certain embodiments, the T cell survival motif is or is derived from CD3, CD28, the intracellular signaling domain of the IL-7 receptor (IL-7R), the intracellular signaling domain of the IL-12 receptor, the intracellular signaling domain of the IL-15 receptor, the intracellular signaling domain of the IL-21 receptor, or the intracellular signaling domain of the transforming growth factor beta (TGFB) receptor.

[0058] Representative modifications In certain embodiments, the altered polypeptides provided herein are modified, e.g., by acylation, amidation, glycosylation, methylation, phosphorylation, sulfation, sumoylation, and / or ubiquitination (or other protein modifications).

[0059] In certain embodiments, the modified polypeptides provided herein are labeled with a label capable of providing a detectable signal, such as a radioisotope or a fluorescent compound.

[0060] In certain embodiments, one or more side chains of the modified polypeptides provided herein are derivatized, for example, by derivatization of lysinyl and amino terminal residues with succinic or other carboxylic acid anhydrides, or by transaminase-catalyzed reaction with, for example, imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and glyoxylic acid. In certain embodiments, carboxyl side chains (aspartyl or glutamyl) may be selectively modified by reaction with a carbodiimide (RN=C=N-), such as 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide.

[0061] Representative nucleic acids Nucleic acids encoding the modified polypeptides described herein are provided herein. Nucleic acids useful for producing the modified polypeptides described herein include DNA, RNA, and nucleic acid analogs. Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone, and can include deoxyuridine substitution for deoxythymidine, 5-methyl-2'-deoxycytidine or 5-bromo-2'-deoxycytidine substitution for deoxycytidine. Modifications of the sugar moiety include modification of the 2' hydroxyl of the ribose sugar to form 2'-O-methyl or 2'-O-allyl sugars. The deoxyribose phosphate backbone can be modified to become morpholino nucleic acid, in which each base moiety is linked to a six-membered morpholino ring, or peptide nucleic acid, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and four bases are retained. See, e.g., Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev. 7: 187-195; and Hyrup et al. (1996) Bioorgan. Med. Chain. 4:5-23. Additionally, the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoramidite, or an alkyl phosphotriester backbone.

[0062] In certain embodiments, the modified polypeptide-encoding nucleic acid described herein is contained within a nucleic acid vector. For example, cells of interest (e.g., T lymphocytes) can be transformed with synthetic vectors, lentiviruses or retroviruses, self-replicating plasmids, viruses (e.g., retroviruses, lentiviruses, adenoviruses, or herpes viruses), or the like, that contain the modified polypeptide-encoding nucleic acid (polynucleotide) described herein. In some embodiments, the vector that contains the modified polypeptide described herein is a retrovirus vector. In some embodiments, the vector that contains the modified polypeptide-encoding nucleic acid described herein is a lentivirus vector. Lentivirus vectors suitable for transforming cells (e.g., T lymphocytes) include, but are not limited to, the lentivirus vectors described in U.S. Patent Nos. 5,994,136; 6,165,782; 6,428,953; 7,083,981; and 7,250,299. Suitable HIV vectors for transforming cells (eg, T lymphocytes) include, but are not limited to, those described in US Pat. No. 5,665,577.

[0063] In certain embodiments, the modified polypeptide-encoding nucleic acid described herein is operably linked to a promoter. In some embodiments, the promoter is a T cell-specific promoter, a natural killer (NK) cell-specific promoter, an inducible promoter that functions in T cells or NK cells, or a constitutive promoter.

[0064] Representative cells The modified polypeptides provided herein can be expressed in cells in which expression of the modified polypeptide (such as a CAR) is useful, i.e., the cells are modified to contain the modified polypeptide-encoding nucleic acid provided herein, such that upon expression of the nucleic acid in the cell, the cell expresses the modified polypeptide described herein. For example, the modified polypeptides described herein can be expressed in T lymphocytes or natural killer cells. The cells provided herein that express the CARs described herein may be referred to as "CAR cells."

[0065] In certain embodiments, provided herein are cells (e.g., T lymphocytes or natural killer cells) modified to express modified polypeptides comprising the degradation domains provided herein. In some embodiments, the cells are modified to express modified polypeptides that are CARs, comprising (a) components of the CAR, such as antigen-binding domains, transmembrane domains, cell signaling domains, and / or costimulatory domains, and (b) a degradation domain. In some embodiments, the cells are modified to express modified polypeptides that comprise a degradation domain fused to or inserted into another protein. Contacting the modified cells with the degradation agents provided herein causes ubiquitination and degradation of the modified polypeptides.

[0066] In some embodiments, the modified polypeptides provided herein are expressed in T lymphocytes. The T lymphocytes can be naive T lymphocytes or MHC-restricted T lymphocytes. In certain embodiments, the T lymphocytes are tumor-infiltrating lymphocytes (TILs). In certain embodiments, the T lymphocytes are isolated from a tumor biopsy or expanded from T lymphocytes isolated from a tumor biopsy. In certain other embodiments, the T lymphocytes are expanded from T lymphocytes isolated from or expanded from peripheral blood, umbilical cord blood, or lymph.

[0067] In some embodiments, the cells (e.g., T lymphocytes) modified to contain / express modified polypeptides described herein are autologous to the individual to whom the cells (e.g., T lymphocytes) are administered as part of the therapeutic methods described herein. In other embodiments, the cells (e.g., T lymphocytes) modified to contain / express modified polypeptides described herein are allogeneic to the individual to whom the cells (e.g., T lymphocytes) are administered. When allogeneic cells (e.g., T lymphocytes) are used to prepare modified cells such as CAR cells, it is preferable to select cells (e.g., T lymphocytes) that reduce the likelihood of graft-versus-host disease (GVHD) in the individual. For example, in certain embodiments, virus-specific T lymphocytes are selected for the preparation of CAR T lymphocytes, and such lymphocytes are expected to have a greatly reduced natural ability to bind and thereby activate any recipient antigen. In certain embodiments, recipient-mediated rejection of allogeneic cells (e.g., T lymphocytes) can be mitigated by co-administration of one or more immunosuppressants to the host, such as cyclosporine, tacrolimus, sirolimus, cyclophosphamide, or the like.

[0068] In some embodiments, T lymphocytes are obtained from an individual, appropriately expanded, then transformed with a vector encoding a modified polypeptide provided herein, then appropriately expanded. In some embodiments, T lymphocytes are obtained from an individual, appropriately expanded, then transformed with a vector encoding a modified polypeptide that is a CAR described herein, then appropriately expanded. In some embodiments, cells containing a vector can be obtained using a selectable marker. In some embodiments, T lymphocytes are obtained from an individual, appropriately expanded, then modified to insert a degradation domain into an endogenous protein gene of interest to express a modified polypeptide that includes a degradation domain that is fused or inserted into the endogenous protein. The modified T lymphocytes may be further expanded as appropriate.

[0069] In certain embodiments, the T lymphocytes used to express the modified polypeptides provided herein contain native TCR proteins (e.g., TCR-α and TCR-β) capable of forming a native TCR complex. In certain other embodiments, either or both of the native genes encoding TCR-α and TCR-β in the T lymphocytes are modified to be non-functional, e.g., deleted in part or in whole or with an inserted mutation.

[0070] In certain embodiments, the signaling domain of the CAR described herein can be used to promote the proliferation and expansion of cells (e.g., T lymphocytes) that contain / express the CAR. For example, unmodified T lymphocytes and T lymphocytes that contain a polypeptide that includes a CD3 zeta signaling domain and a CD28 costimulatory domain can be expanded using antibodies against CD3 and CD28, such as antibodies bound to beads. See, for example, U.S. Patent Nos. 5,948,893; 6,534,055; 6,352,694; 6,692,964; 6,887,466; and 6,905,681. Similarly, antibodies against signaling motifs can be used to stimulate the proliferation of cells (e.g., T lymphocytes) that contain the CAR described herein.

[0071] In certain embodiments, the modified polypeptide may be used as a "suicide gene" or "safety switch" that allows for the death of substantially all cells expressing the modified polypeptide when desired. For example, a degradation domain may be inserted into a gene expressing an endogenous protein required for survival and / or a particular activity of the cell. Contacting a cell with a degradation drug causes ubiquitination and degradation of the endogenous protein (i.e., the modified polypeptide comprising the degradation domain and the endogenous protein), disabling the activity of the cell or killing the cell.

[0072] Representative decomposition agents The term "degradation agent" as used herein refers to a molecule (e.g., a small molecule) that can bind to a degradation domain and a ubiquitin ligase, such as an E3 ligase, as provided herein. In some embodiments, the degradation agent binds to the degradation domain and cereblon. In some embodiments, the degradation agent binds to the degradation domain and the ubiquitin ligase, resulting in the association of the E3 ligase with the degradation domain. In some such embodiments, the modified polypeptide comprising the degradation domain is ubiquitinated by the ubiquitin ligase following the association mediated by the degradation agent.

[0073] In some embodiments, the degrading agent is a cereblon-binding compound. In some embodiments, the degrading agent is 3-(5-(6,7-dihydro-5H-pyrrolo[3,4-b]pyridine-6-carbonyl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (Compound B), 3-(5-((4-(2-methylpyridin-3-yl)piperazin-1-yl)methyl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (Compound C), or 3-[5-[1-(1,3-benzothiazol-6-ylmethyl)-4-piperidyl]-1-oxo-isoindolin-2-yl]piperidine-2,6-dione (Compound D). [Table 1]

[0074] In some embodiments, the decomposition agent is a compound described in WO2019 / 038717A1, the entirety of which is incorporated herein by reference.

[0075] The disintegrants used in accordance with the methods described herein, or an enantiomer or a mixture of enantiomers thereof; or a pharma-ceutically acceptable salt, solvate, hydrate, cocrystal, clathrate, or polymorph thereof, may be delivered as a single dose (e.g., a single bolus injection, or an oral tablet or pill, etc.) or over time (e.g., by continuous infusion over a long period of time or by divided bolus administration over a long period of time).

[0076] The degradants used in accordance with the methods described herein may be formulated for intravenous, intraarterial, parenteral, intramuscular, subcutaneous, intrathecal, or intraocular administration, or for administration into a specific organ or tissue.

[0077] Representative methods In some embodiments, a method is provided for decreasing the level of a modified polypeptide comprising a degradation domain, the method comprising contacting the modified polypeptide with a degradation agent. In some embodiments, the contact occurs intracellularly, and the degradation agent binds to the degradation domain and a ubiquitin ligase, causing ubiquitination and degradation of the modified polypeptide. In some embodiments, degradation of the modified polypeptide results in a decrease in at least one cellular activity and / or an increase in at least one cellular activity and / or cell death. Non-limiting representative effects include lowering the threshold for cell (such as T cells) activation, increasing the functional persistence of cells (such as T cells), promoting cell survival, and increasing cell proliferation. In some embodiments, the degradation agent is Compound B, Compound C, or Compound D. In some embodiments, the method comprises administering a degradation agent to a subject, wherein the subject comprises cells comprising the modified polypeptide.

[0078] In some embodiments, the modified polypeptide is degraded in the presence of a degradation agent. In some embodiments, the degradation agent interacts with a degradation domain and a ubiquitin ligase, such as cereblon. In some embodiments, the degradation agent mediates a complex comprising the degradation domain, the degradation agent, and a ubiquitin ligase, causing ubiquitination of the modified polypeptide.

[0079] The engineered cells provided herein (e.g., CAR cells), such as T lymphocytes (i.e., T cells) engineered to contain / express an engineered polypeptide, can be used to treat individuals who would benefit from the engineered cells, for example, because they have a cancer that expresses the target of the CAR. In some embodiments, the cells are effector T cells. In some embodiments, the cells are CD4+ T cells or CD8+ T cells. In some embodiments, either the T cells, effector T cells, CD4+ T cells, or CD8+ T cells comprise the engineered polypeptide.

[0080] In some embodiments, provided herein are methods of killing a target cell that expresses an antigen bound by an antigen binding domain of a CAR described herein, wherein the method comprises contacting the target cell with a modified cell provided herein (e.g., a T cell or an NK cell) that comprises / expresses a CAR described herein. In some embodiments, the target cell is a cancer cell, e.g., a hematological cancer cell or a solid tumor cell. In some embodiments, provided herein are methods of treating cancer, comprising administering to a subject a population of modified cells described herein (e.g., a T cell or an NK cell) that comprise / express a CAR described herein, wherein the CAR comprises an antigen binding domain specific for a cancer antigen (e.g., a TSA or a TAA).

[0081] In some embodiments, the target cells or cancer cells express one or more of the following antigens, or fragments thereof: 4-IBB, 5T4, 8H9, B7-H6, adenocarcinoma antigen, a-fetoprotein, B cell maturation antigen (BCMA), BAFF, B lymphoma cells, C242 antigen, CA9, carcinoembryonic antigen, CA-125, carbonic anhydrase 9 (CA-IX), CCR4, CD3, CD4, CD19, CD20, CD22, CD23 (IgE receptor), CD28, CD30 (T FRSF8), CD33, CD38, CD40, CD44v6, CD44v7 / 8, CD51, CD52, CD56, CD74, CD80, CD123, CD152, CD171, CD200, CD221, CE7, CEA, C-MET, CNT0888, CTLA-4, DRS, EpCAM, ErbB2, ErbB3 / 4, EGFR, EGFRvIII, EphA2, EGP2, EGP40, FAP, Fetal AchR, Fibronectin extra domain B, Folate receptor a, Folate receptor 1, G250 / CAIX, GD2, GD3, Glycoprotein 75, GP MB, HER2 / neu, HGF, HLA-AI MAGE Al, HLA-A2 NY-ESO-1, HMW-MAA, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgGl, IL-6, IL-13, IL-13 receptor a2, IL-11 receptor a, insulin-like growth factor I receptor, integrin a5I31, integrin avI33, kappa light chain, Ll-CAM, lambda light chain, Lewis Y, mesothelin, MORAb-009, MS4A1, MUCl, MUCl 6, mucin CanAg, NCAM, N-glycolylneuraminic acid, NKG2D ligand, NPC-IC, PDGF-R a, PDL192, phosphatidylserine, prostate specific cancer antigen (PSCA), prostate cancer cells, PSMA, PSC1, RANKL, RON, ROR1, SCH 900105, SDC1, SLAMF7, spl7, TAG72, tenascin C, TGF β2, TGF-I3, TL1A, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGF receptor, VEGFR-1, VEGFR2, TEM1, TEM8, and / or vimentin.

[0082] In some embodiments, after administration of modified cells provided herein, such as CAR cells, it may be desirable to reduce or eliminate the expression of CAR, thereby reducing or eliminating the killing of target cells. In some such embodiments, the method may further comprise administering to the subject a degrading agent provided herein. The administration of the degrading agent causes the degradation of the modified polypeptide (e.g., CAR), reducing or eliminating the targeting of the modified cells to cells expressing the antigen bound by the antigen-binding domain of the CAR. In this manner, the activity of CAR cell therapy may be modulated, and safety may be improved.

[0083] In some embodiments, the modified cell population is first administered to a subject, followed by administration of a degrading agent at a period of time after administration of the modified cell population, for example, 30 minutes, 1 hour, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week after administration of the cell population.

[0084] In some embodiments, the degrading agent is Compound B, Compound C, or Compound D.

[0085] A non-limiting list of cancers that may be treated according to the therapeutic methods described herein include lymphoma, leukemia, lung cancer, breast cancer, prostate cancer, adrenocortical carcinoma, thyroid cancer, nasopharyngeal carcinoma, melanoma, skin cancer, colorectal cancer, desmoid tumor, desmoplastic small round cell tumor, endocrine tumor, Ewing's sarcoma, peripheral primitive neuroectodermal tumor, solid germ cell tumor, hepatoblastoma, neuroblastoma, non-rhabdomyosarcoma soft tissue sarcoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, Wilms' tumor, glioma, glioblastoma, myxoma, fibroma, and lipoma. Representative lymphomas and leukemias include, but are not limited to, chronic lymphocytic leukemia (small lymphocytic lymphoma), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, Waldenstrom's macroglobulinemia, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, extranodal marginal zone B-cell lymphoma, MALT lymphoma, nodal marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary body cavity effusion lymphoma, Burkitt's lymphoma, T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, aggressive NK-cell leukemia, adult T-cell leukemia / lymphoma, extranodal NK / T-cell lymphoma, nasal type, enteropathy type T-cell lymphoma, hepatosplenic T-cell lymphoma, blastic NK-cell lymphoma, mycosis fungoides, Sezary syndrome, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T-cell lymphoma, peripheral T-cell lymphoma (not specified), anaplastic large cell lymphoma, Hodgkin's lymphoma, or non-Hodgkin's lymphoma.

[0086] The effectiveness of modified cells described herein, such as CAR cells, in treating a disease or disorder, for example, in treating an individual with cancer, can be evaluated by one or more criteria specific to the particular disease or disorder that are known to those skilled in the art to indicate the progression of the disease or disorder. Generally, administration of CAR cells (e.g., CAR T lymphocytes) to an individual with a disease / disorder (e.g., cancer) is effective when one or more of the above criteria detectably, e.g., significantly, move from a disease state value or range to or toward a normal value or range.

[0087] The modified cells described herein can be formulated in any pharma- ceutically acceptable solution, preferably a solution suitable for delivery of live cells (e.g., saline (such as Ringer's solution), gelatin, carbohydrates (e.g., lactose, amylose, starch, or the like), fatty acid esters, hydroxymethylcellulose, polyvinylpyrolidine, etc.). Such formulations are preferably sterilized prior to addition of the CAR cells and may be mixed with auxiliary agents (e.g., lubricants, preservatives, stabilizers, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, etc.). Pharmaceutical carriers suitable for use in formulating CAR cells are known in the art and are described, for example, in WO96 / 05309.

[0088] In certain embodiments, the engineered cells (e.g., CAR cells) described herein are formulated into an individual dose, wherein the individual dose is at least, at most, or about 1×10 4 , 5xl0 4 , lx10 5 , 5xl0 5 , lxl0 6 , 5xl0 6 , lxl0 7 , 5xl0 7 , lxl0 8 , 5xl0 8 , lxl0 9 , 5xl0 9 , lxl0 10 , 5xl0 10 , or lxl0 11 Contains cells.

[0089] In certain embodiments, the modified cells (e.g., CAR cells) described herein are formulated for intravenous, intraarterial, parenteral, intramuscular, subcutaneous, intrathecal, or intraocular administration, or for administration into a particular organ or tissue. EXAMPLES

[0090] Example 1: IKZF1 ZNF2 G motif tagged CAR retains function The function of IKZF1 ZNF2-labeled CD19 CAR T cells (Figure 2, ZNF2 amino acid sequence in SEQ ID NO: 15) was evaluated in a reporter assay. Here, Jurkat cells have been modified to express tdTomato when the Nur77 gene, which is associated with CAR and TCR activation, is actively transcribed (Figure 3). Lentiviral vectors encoding ZNF-labeled CD19 CAR, unlabeled CD19 CAR, or unlabeled BCMA CAR negative control were transduced into Jurkat reporter cell lines. Transduced cells were then co-cultured with CD19-expressing K562 target cell lines at 37 °C. Levels of tdTomato, both as mean fluorescence intensity (MFI) and overall percentage of positive cells, were assessed by flow cytometry every 2 h for 12 h. Results showed that both the timing and level of CAR activation were the same between labeled and unlabeled CD19 CARs, but BCMA CAR T did not activate in the presence of the CD19 target cell line (Figure 4A).

[0091] To assess the level of CAR degradation achieved with different concentrations of compound A, titrations of compound A (Figure 4B) were performed on Jurkats transduced with IKZF1 ZNF2-tagged or untagged CD19 CAR. Compound A is shown in U.S. Patent Publication 2019 / 0008852A1, page 42, Table 4 (Compound A). Cells were incubated with compound A for 24 hours at 37°C, after which CAR levels were assessed by flow cytometry. The data show that the IKZF1 ZNF2 G motif degron mediated approximately 55% CAR degradation at high compound A concentrations (Figure 4C). This level of CAR degradation may not be sufficient to suppress CAR activity when challenged with K562 expressing the antigen recognized by CAR (data not shown). This result prompted studies to modify the degron protein sequence to more potently respond to compound A.

[0092] Example 2: Evaluation of alternative sequences for the IKZF1 zinc finger The IKZF1-ZNF2 degron was further modified to promote improved CAR degradation. Unlabeled CARs and CARs labeled with the original IKZF1-ZNF2 degron were tested side-by-side with alternative orientations and combinations of IKZF1 ZNF1, ZNF2, and ZNF3 (Figure 5A). Each CAR contained a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ (CD3z) signaling domain. The IKZF1 degron was fused to the C-terminus. See SEQ ID NOs: 53-57. Jurkat cell lines were transduced with lentiviral vectors encoding unlabeled CD19 CARs or CARs labeled with various sequences of IKZF1-ZNFs. Transduced cells were first co-cultured with a CD19-expressing K562 target cell line at 37°C for 7.5 hours, and normalized levels of tdTomato reporter were assessed by flow cytometry. The results showed that none of the degron tags reduced CAR activity in the presence of antigen compared to unlabeled CAR. Next, CAR-transduced Jurkat cells were cultured in the presence of compound A (titration) at 37°C for 24 hours, and the Y-max and EC50 values ​​were determined for each degron. This experiment showed that ZNF2_3 tagging enhanced degradation at the lowest concentration of compound A (Figure 5C). ZNF1_2 tagging was excluded because ZNF1 contains a G motif, which adds additional complexity to the tandem degron. Therefore, this sequence was selected for further testing.

[0093] To demonstrate that this degradation is dependent on a functional degron, CAR tagged with WT ZNF2_3 (SEQ ID NO: 13) was titrated with Compound A as described above to ZNF2_3 containing a G6N mutation in the G motif (QCNQC N ASF (SEQ ID NO: 17)-labeled CAR was tested alongside the G6N mutant-labeled CAR (Figure 5D). While the WT degron strongly degraded CAR at high compound A concentrations, the G6N mutant-labeled CAR did not respond to any dose of compound A. These results suggest that the G6 residue of the G motif contributes to the interaction of compound A with the optimized ZNF2_3 degron.

[0094] Example 3: Degradation of IKZF1 ZNF2_3-tagged CAR suppresses activity The ability of IKZF1 to suppress CAR T signaling by degrading ZNF2_3-labeled CD19 CAR T cells was evaluated in a Jurkat reporter assay. A lentiviral vector (see Example 2) encoding ZNF2_3-labeled or unlabeled CD19 CAR was transduced into a reporter cell line. The transduced cells were then pretreated with 100 nM compound A for 48 hours and then co-cultured with a CD19-expressing K562 target cell line at 37°C for 8 hours. The levels of tdTomato in the form of mean fluorescence intensity (MFI) (Figure 6B) and the overall percentage of positive cells (Figure 6C), as well as normalized CAR levels, were assessed by flow cytometry (Figure 6A). At this concentration of compound A, the degradation of CAR containing ZNF2_3 degron was strong enough to strongly suppress CAR activity when challenged with K562 cells expressing the cognate antigen.

[0095] Inhibition of signaling by CAR degradation was assessed by measuring activation of downstream MAPK signaling pathways. Jurkat cells were transduced with unlabeled, WT degron-labeled, or G6N degron-labeled CAR as described above. Cells were pretreated with 1 mM compound A for 12 h at 37 °C and then cocultured with either parental or CD19-expressing K562 cells for 30 min. Cells were then pelleted, lysed, run on a denaturing protein gel, transferred to a membrane, probed with antibodies, and visualized on film (Figure 7). These results show that administration of compound A to cells containing WT IKZF1 ZNF2_3-tagged CAR attenuates the endogenous signaling pathway activated by CAR, represented here as Erk phosphorylation (pErk), in the presence of antigen.

[0096] Example 4: Degron mutagenesis to identify novel degron-compound pairs Structural studies reveal that position 1 of the G motif is in close proximity to the compound when the substrate binds to the cereblon / compound complex (not shown). Alignment of G motif degron sequences in the IKZF family (Figure 8) and predicted degrons in other C2H2 zinc fingers (Figure 9) reveal the presence of diverse amino acids at this position. Therefore, to identify additional compound:degron pairs, we made mutations at position Q1 of IKZF1 ZNF2 and screened promising compounds for activity against these mutants. We constructed a plasmid containing Ikaros MBP-ZNF2 (aa 141-196) with a different amino acid at position Q1 and performed an in vitro ubiquitination screen with various compounds, including compound B (Figure 10A). After treatment, cells were pelleted, lysed, run on a denaturing protein gel, transferred to a membrane, probed with antibodies, and visualized on film (Figure 10B). As shown in FIG. 10B, IKZF1 ZNF2 Q1F was strongly ubiquitinated in the presence of Compound B.

[0097] Example 5: Small molecule screen to identify compounds with potent activity against the Q1F degron A series of promising degron-targeting compounds were screened against the Q1F degron, resulting in a series of potent degron / small molecule pairs. Jurkat cells were transduced with lentiviral vectors containing CD19 CAR tagged with IKZF1 ZNF2_ZNF3 Q1F Nluc. This CD19 CAR is similar to CD19 in Example 2, but contains ZNF2_ZNF3 Q1F degron (SEQ ID NO: 19). Transduced cells were treated with titrations of each small molecule or without drug, followed by incubation at 37°C for 18 hours. Cells were washed, stained with appropriate staining reagent, and CAR levels were measured. Cells were incubated at 4°C in staining reagent for 20 minutes, followed by washing three times, and then read on a flow cytometer. CAR levels were normalized to cells not treated with drug. The resulting titration curves were used to calculate EC50 and Ymin values. This identified small molecules that potently degraded Q1F degron-tagged CAR (Table 1). [Table 2]

[0098] Example 6: IKZF1 ZNF2_3 Q1F-tagged CAR is degraded in a CRBN- and ubiquitin-proteasome pathway-dependent manner in the presence of Compound C The dependency of Q1F degron-tagged CAR degradation on CRBN and the ubiquitin proteolytic pathway was assessed. CD19-targeted CARs were tagged with either IKZF1 ZNF2_3 Q1F (SEQ ID NO: 19) or Q1F / G6N (SEQ ID NO: 59). Lentiviral vectors containing these tagged CARs were then transduced into wild-type (WT) or cereblon (CRBN) knockout (KO) Jurkat cells.

[0099] To determine whether Q1F-labeled CAR can be degraded by compound C in a CRBN-dependent manner, transduced Jurkat WT or Jurkat CRBN KO cells were treated with DMSO or 1 mM compound C (Figure 11A) for 1 hour at 37 °C, and then CAR levels were evaluated by flow cytometry. At this concentration of compound C, labeled CAR was degraded to less than 20% remaining in WT Jurkat cells, whereas the level of labeled CAR remained the same as unlabeled CAR in Jurkat CRBN KO cells (Figure 11B). Next, to test the dependency of CAR degradation on the ubiquitin-proteosome pathway, WT Jurkat transduced with labeled CAR was treated for 2 hours with DMSO, 20 nM or 200 nM compound C alone, or with either 2 μM of the NEDD8 E1 enzyme inhibitor MLN4924 or 2 μM of the proteasome inhibitor bortezomib (Figure 11C). Compound C did not degrade labeled CAR in the presence of any inhibitor, indicating that a functional ubiquitin proteasome pathway (UPP) is essential for the degradation of Q1F-labeled CAR.

[0100] Example 7: Degradation of IKZF1 ZNF2_3 Q1F-tagged CAR suppresses activity The ability of IKZF1 to suppress CAR T signaling by degrading ZNF2_3 Q1F-tagged CD19 CAR T cells was assessed in a Jurkat reporter assay (Figures 2 and 3). Lentiviral vectors encoding ZNF2_3 Q1F or Q1F / G6N-tagged CD19 CARs were transduced into reporter cell lines. Transduced cells were then pretreated with 1 mM Compound C or Compound B for 12 hours and then co-cultured with parental or CD19-expressing K562 target cell lines for 8 hours at 37°C. The levels of tdTomato in the form of the overall percentage of positive cells (Figure 12A) as well as mean fluorescence intensity (MFI) (Figure 12B) were assessed by flow cytometry. Degradation of CARs containing the ZNF2_3 Q1F degron reduced signaling by approximately 30% with 1 mM Compound C and approximately 75% with Compound B. This relative difference correlates with the strength of degradation seen with each. Furthermore, the reduction in activity is dependent on having an intact G motif, such that the G6N mutation prevents repression.

[0101] Example 8: Degradation of IKZF1 ZNF2_3 Q1F-tagged CAR suppresses the activity of primary T cells The ability of the compounds to modulate primary CAR T effector function by degrading IKZF1 ZNF2_3 Q1F-labeled anti-ROR1 CAR (Figure 13A) was evaluated. The CAR contained transmembrane, costimulatory, and signaling domains similar to the CD19 CAR, but with an anti-ROR1 scFv. Activated primary T cells were transduced with lentiviral vectors encoding ZNF2_3 Q1F-labeled or unlabeled anti-ROR1 CAR. Transduced cells were grown in medium supplemented with IL2, IL7, and IL15 for 10 days and then frozen. Prior to the experiment, cells were thawed and left in medium containing compound C at escalating doses or without drug. CAR levels were measured by flow cytometry (Figure 13B), showing that the surface levels of ZNF2_3 Q1F-labeled CAR could be modulated over a range of compound C concentrations. To determine the effect of CAR degradation on CAR T activity, cells were next co-cultured with Nuclight Red-labeled H-1975 ROR1-expressing target cell line at a 1:4 effector:target cell ratio. Co-cultures were placed in an Incucyte for 24 h to monitor tumor killing via Nuclight Red signal (Figure 14A), and supernatants were collected to measure inflammatory cytokine production by MSD (Figure 14B-14D; IL-2, TNFα, and IFNγ). These experiments demonstrated that effector function of primary CAR T cells, as measured by both killing and cytokine production, could be tuned by targeting and degrading ZNF2_3 Q1F-labeled CAR with different concentrations of Compound C.

[0102] Example 9: Q1F degron-mediated degradation of CAR preserves CAR function during chronic antigen exposure in vitro The functional impact of providing transient rest to primary T cells during chronic antigen exposure by degradation of ZNF2_3 Q1F-labeled CD19 CAR was tested in vitro. Stimulation was provided by plates coated with antibodies against the CAR scFv, and various periods of rest were evaluated (Figure 15). Cells were treated with 3.9 nM compound D (Figure 16A), followed by washing and leaving in media to allow the CAR to fully rebound before rechallenge. Left cells were analyzed by flow cytometry to assess CD27 and CD28 levels as a surrogate for naïve populations (Figures 16B and 16C). CAR T cells were then challenged with Nuclight red-labeled tumor cell lines in spheroid (3D) format, and supernatants were harvested to measure inflammatory cytokine production by Meso Scale Discovery ELISA assays. These experiments show that providing transient rest to CAR T cells reduces activation and maintains a more naïve population compared to CAR T cells undergoing continuous antigen exposure. This transient rest also provides functional benefits in terms of the production of inflammatory cytokines such as IL-2, TNFα, and IFNγ (Figure 17A) and antitumor function (Figure 17B). The gradual decline of these cytokines and cytotoxicity is a hallmark of T cell exhaustion.

[0103] Example 10: Q1F degron-mediated degradation of CAR preserves CAR function during chronic antigen exposure in vitro The ability of ZNF2_3 Q1F-tagged CAR to be degraded in vivo was evaluated. A lentiviral vector encoding ZNF2_3 Q1F-tagged CD19 CAR was transduced into activated primary T cells. Transduced cells were grown in medium supplemented with IL2, IL7, and IL15 for 10 days and then frozen.

[0104] To determine the ability to degrade degron-tagged CARs in vivo in the absence of tumors, CAR T cells were thawed, left for 24 hours, and then injected into tumor-free female NSG™ immunodeficient mice at 2x10 per mouse. 6Mice were matched and transplanted with a dose of 0.85 or 8.5 mg / kg Compound D (Figure 18B) after 24 hours. Blood was collected at 8, 24, 48, and 72 hours (Figure 18A) and the percentage of CAR+ T cells was assessed by flow cytometry as determined by staining with anti-scFv and anti-CD3 antibodies. At both doses, less than 5% of CD3+ CAR+ cells remained at 8 hours, and the CAR remained significantly degraded at 24 hours (Figure 18C), indicating that Q1F degron-tagged CAR can be efficiently degraded in vivo. By 48 hours after administration, CAR expression was substantially restored (Figure 18C).

[0105] To study the in vivo downregulation of degron-tagged CARs and the impact on CAR T function in xenograft cancer models, 5x10 mice stably expressing Renilla luciferase were cultured in female NSG™ mice. 5 Six days later, CAR T cells were thawed and left for 24 hours, and mice were injected with 2x10 Raji tumor cells per mouse. 6 Mice were orally dosed with vehicle or 6.85 mg / kg compound D BID on days 0 and 1 (Figure 19A). Blood was collected on D1, D3, and D10, tumor fluorescence was measured (Figure 19A), and the percentage of CAR+ T cells, as determined by staining with anti-scFv and anti-CD3 antibodies, was assessed by flow cytometry. Degradation of the CAR reduced the proliferation and cytotoxic function of degron-labeled CAR T cells (Figure 19B-D), demonstrating the ability of CAR cycling to functionally rest CAR T cells.

[0106] Example 11: In-frame degron tagged knock-in allows compound-mediated control of endogenous protein levels We studied the ability to knock-in degron tags in-frame into gene loci to allow compound-mediated regulation of endogenous protein levels. Adeno-associated viral vectors were designed to deliver IKZF1 ZNF2_3-V5 tag-T2A-muThy1.1 tag knocked-in in-frame into the AURA (Figure 20A-10B) or TOX locus (Figure 20C-20D) in both N- and C-terminal orientations in Jurkat cells. Jurkats were then electroporated and transduced with Cas9 / guide RNA ribonucleoproteins. After 5 days, knock-in cells were incubated with 1 μM compound A or DMSO for 16 hours. Cells were then pelleted, lysed, run on a denaturing protein gel, transferred to a membrane, probed with antibodies, and visualized on film. Western blotting showed that the levels of both Aurora A (Figures 20A-20B) and labeled TOX (Figure 20D) were reduced in the presence of compound A, indicating that tagging endogenous proteins with degrons allows for small molecule control of protein levels.

[0107] Example 12: Synthesis of 3-(5-(6,7-dihydro-5H-pyrrolo[3,4-b]pyridine-6-carbonyl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (Compound B) [ka] To a solution of 2-(2,6-dioxo-3-piperidyl)-1-oxo-isoindoline-5-carboxylic acid (400 mg, 1.4 mmol) in CH3CN (ACN) (5 mL) was added N,N,N',N'-tetramethylchloroformamidinium hexafluorophosphate (TCFH) (779 mg, 2.8 mmol), N-methylimidazole (NMI) (1.2 g, 14 mmol), and 6,7-dihydro-5H-pyrrolo[3,4-b]pyridine; dihydrochloride (349 mg, 1.8 mmol). The mixture was then stirred at room temperature for 1 h. The reaction was monitored by LCMS. The precipitated solid was collected by filtration. The crude was purified by Prep-HPLC to give 3-[5-(5,7-dihydropyrrolo[3,4-b]pyridine-6-carbonyl)-1-oxo-isoindolin-2-yl]piperidine-2,6-dione (438.7 mg, 79% yield) as a grey solid. Analytical LC / MS (Method 5): MS[M+H + ] 391.1. 1 H NMR (300 MHz, DMSO-d6): δ 11.03 (s, 1H), 8.54 - 8.43 (m, 1H), 7.94 - 7.65 (m, 4H), 7.41 - 7.23 (m, 1H), 5.23 - 5.11 (m, 1H), 5.00 - 4.75 (m, 4H), 4.62 - 4.36 (m, 2H), 3.04 - 2.84 (m, 1H), 2.69 - 2.56 (m, 1H), 2.50 - 2.36 (m, 1H), 2.12 - 2.00 (m, 1H).

[0108] Example 13: Synthesis of 3-[5-(bromomethyl)-1-oxo-isoindolin-2-yl]piperidine-2,6-dione (Intermediate 1c) [ka]

[0109] Synthesis of compound 2 [ka] To a solution of 4-bromo-2-methylbenzoic acid (120 g, 558 mmol) in methanol (1.0 L) at 20-40 °C, concentrated sulfuric acid (109 g, 1.12 mol, 60 mL) was added, and the mixture was then heated to 65 °C for 18 h. TLC (petroleum ether / ethyl acetate = 3:1, R f (reactant) = 0.1, R f (product) = 0.4) indicated the reaction was complete. The reaction mixture was concentrated and the residue was partitioned into an aqueous phase and an organic phase. The aqueous phase was extracted with ethyl acetate (1 L x 3). The combined organic layers were washed with saturated aqueous sodium bicarbonate (500 mL), brine (500 mL) and dried over anhydrous sodium sulfate. The mixture was filtered and the filtrate was concentrated to give methyl 4-bromo-2-methylbenzoate (122 g, 95% yield) as a yellow oil. 1 H NMR (400 MHz, CDCl3): δ 7.76 (d, J = 8.4 Hz, 1H), 7.40 (s, 1H), 7.35 (dd, J = 8.4 Hz, 1.6 Hz, 1H), 3.87 (s, 3H), 2.56 (s, 3H)

[0110] Synthesis of compound 3 [ka] To a solution of methyl 4-bromo-2-methylbenzoate (122 g, 533 mmol) in acetonitrile (1.20 L) was added 2,2'-azobis(2-methylpropionitrile) (6.12 g, 37.3 mmol), the reaction mixture was heated to 82°C, N-bromosuccinimide (142 g, 799 mmol) was added in portions, and stirred for 3 h. LCMS showed the reaction was complete. The reaction was concentrated in vacuo (50°C). The residue was suspended in petroleum ether / dichloromethane (20:1, 100 mL), filtered, and the filtrate was concentrated to give 150 g of crude material, which was used directly in the next step without further purification.

[0111] Synthesis of compound 5 [ka] To a solution of methyl 4-bromo-2-(bromomethyl)benzoate (115 g, 375 mmol) and 3-aminopiperidine-2,6-dione (61.7 g, 375 mmol, HCl salt) in N,N-dimethylformamide (80 mL) was added N,N-diisopropylethylamine (145 g, 1.13 mol). The mixture was stirred at 50° C. for 16 h. HPLC showed that most of the starting material was consumed. Acetic acid (150 mL) was added to the reaction mixture and stirred at 50° C. for 1 h. HPLC showed that the intermediate was completely consumed. The reaction was cooled to 20° C., filtered, and the filter cake was washed with water (200 mL) and ether acetate (200 mL) to give 3-(5-bromo-1-oxo-isoindolin-2-yl)piperidine-2,6-dione (91 g, 75.1% yield) as an off-blue solid. 1 H NMR (400 MHz, DMSO-d6): δ 11.01 (s, 1H), 7.89 (s, 1H), 7.73-7.66 (m, 2H), 5.14-5.09 (m, 1H), 4.47 (d, J = 16 Hz, 1H), 4.34 (d, J = 16 Hz, 1H), 2.92-2.89 (m, 1H), 2.73-2.58 (m, 1H), 2.41-2.37 (m, 1H), 2.03-1.99 (m, 1H)

[0112] Synthesis of compound 6 [ka] To a solution of 3-(5-bromo-1-oxo-isoindolin-2-yl)piperidine-2,6-dione (80 g, 248 mmol) in dry dioxane (20 mL) was added tributylstannylmethanol (87 g, 272 mmol), the mixture was stirred at 25° C., and tetrakis(triphenylphosphine)palladium(0) (28.6 g, 24.8 mmol) was added to the reaction under nitrogen. The reaction mixture was stirred at 100° C. for 16 hours. LCMS showed the reaction was complete. The reaction was concentrated and washed with dichloromethane / methanol (10:1, 250 mL x 2) to give 3-[5-(hydroxymethyl)-1-oxo-isoindolin-2-yl]piperidine-2,6-dione (62 g, 91.3% yield) as a brown solid. 1 H NMR (400 MHz, DMSO-d6): δ 10.96 (s, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.52 (s, 1H), 7.42 (d, J = 7.6 Hz, 1H), 5.73 (s, 1H), 5.10-5.06 (m, 1H), 4.59 (d, J = 6.0 Hz, 2H), 4.35 (dd, J = 17.2 Hz, 54 Hz, 2H), 2.91-2.85 (m, 1H), 2.59-2.55 (m, 1H), 2.38-2.35 (m, 1H), 2.02-1.99 (m, 1H)

[0113] Synthesis of compound 1c [ka] To a suspension of 3-[5-(hydroxymethyl)-1-oxo-isoindolin-2-yl]piperidine-2,6-dione (62 g, 226 mmol) in dichloromethane (1.5 L) was added sulfinyl bromide (70.5 g, 339 mmol). The reaction mixture was stirred at 18 °C for 16 h. HPLC showed the reaction was complete. The reaction mixture was filtered and the solid was washed with methanol (150 mL x 2) to give 3-[5-(bromomethyl)-1-oxo-isoindolin-2-yl]piperidine-2,6-dione (45.0 g, 59.0% yield) as an off-brown solid. 1 H NMR (400 MHz, DMSO-d6): δ 10.97 (s, 1H), 7.70-7.66 (m, 2H), 7.56 (d, J = 8.0 Hz, 1H), 5.10-5.06 (m, 1H), 4.80 (s, 2H), 4.36 (dd, J = 54 Hz, 17.2 Hz, 2H), 2.91-2.85 (m, 1H), 2.59-2.49 (m, 1H), 2.47-2.35 (m, 1H), 2.00-1.98 (m, 1H)

[0114] Example 14: Synthesis of 3-(5-((4-(2-methylpyridin-3-yl)piperazin-1-yl)methyl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (Compound C) [ka] To a stirred solution of 3-[5-(bromomethyl)-1-oxo-isoindolin-2-yl]piperidine-2,6-dione (400 mg, 1.2 mmol) in MeCN (ACN) (5 mL) was added 1-(2-methyl-3-pyridyl)piperazine (315 mg, 1.8 mmol) and DIEA (460 mg, 3.6 mmol). The mixture was stirred at 50° C. for 3 h. The reaction was monitored by LCMS. The mixture was concentrated and the residue was purified by Prep-HPLC to give 3-[5-[[4-(2-methyl-3-pyridyl)piperazin-1-yl]methyl]-1-oxo-isoindolin-2-yl]piperidine-2,6-dione (187.9 mg, 34% yield) as a grey solid. Analytical LC / MS (Method 1): MS[M+H + ] 434.2. 1H NMR (400 MHz, methanol-d4): δ 8.40 (d, J = 5.5 Hz, 1H), 8.18 (d, J = 8.2 Hz, 1H), 7.93 (d, J = 7.8 Hz, 1H), 7.83 - 7.76 (m, 2H), 7.72 (d, J = 7.6 Hz, 1H), 5.23 - 5.12 (m, 1H), 4.90 - 4.80 (m, 4H), 4.65 - 4.50 (m, 4H), 3.54 (s, 4H), 2.98 - 2.85 (m, 1H), 2.84 - 2.75 (m, 1H), 2.74 (s, 3H), 2.60 - 2.44 (m, 1H), 2.24 - 2.14 (m, 1H)

[0115] Example 15: Synthesis of 3-[5-[1-(1,3-benzothiazol-6-ylmethyl)-4-piperidyl]-1-oxo-isoindolin-2-yl]piperidine-2,6-dione (Compound D) [ka] To a stirred solution of 3-[1-oxo-5-(4-piperidyl)isoindolin-2-yl]piperidine-2,6-dione; hydrochloride (1.5 g, 4.12 mmol) in DCM (20 mL) was added 1,3-benzothiazole-6-carbaldehyde (1.35 g, 8.25 mmol) and NaBH(OAc)3 (2.62 g, 12.37 mmol). The mixture was stirred at room temperature overnight. The mixture was concentrated and purified by prep-HPLC to give 3-[5-[1-(1,3-benzothiazol-6-ylmethyl)-4-piperidyl]-1-oxo-isoindolin-2-yl]piperidine-2,6-dione (968.9 mg, 2.0233 mmol, 49.076% yield) as a white solid. LCMS: 475.1 [M+1] + . 1H NMR (300 MHz, メタノール-d4): δ 9.40 (s, 1H), 8.33 (s, 1H), 8.22 (d, J = 8.4 Hz, 1H), 7.81-7.71 (m, 2H), 7.55-7.36 (m, 2H), 5.24-5.09 (m, 1H), 4.62-4.39 (m, 4H), 3.76-3.62 (m, 2H), 3.33-3.19 (m, 2H), 3.13-2.99 (m, 1H), 2.95-2.73 (m, 2H), 2.60-2.38 (m, 1H), 2.24-1.95 (m, 5H).

[0116] Example 16: Selectivity of Compound A, Compound B, Compound C, and Compound D DF15 multiple myeloma cells stably expressing ePL-labeled Aiolos, Ikaros, or GSPT1 and MDS-L cells stably expressing ePL-labeled CK1a were generated via infection with lentivirus carrying pLOC-ePL-Aiolos (or Ikaros, GSPT1, or CK1a). DF15 multiple myeloma cells expressing Ikaros, Aiolos, and GSPT1 fused to ePL label (DiscoverX) and MDS-L cells expressing CK1a fused to ePL label were dispensed into 384-well plates (Corning no. 3570) pre-loaded with compounds (compound A, compound B, compound C, and compound D). Compounds were dispensed into 384-well plates in a 10-point dose-response curve using an acoustic dispenser (ATS acoustic transfer system from EDC Biosystems) and diluted from 10 μM to 0.0005 μM in 3-fold dilutions. 25 μL of medium (RPMI-1640 + 10% heat-inactivated FBS + 25 mM Hepes + 1 mM Na Pyruvate + 1x NEAA + 1x Pen Strep Glutamine) containing 5000 DF15 or MSD-L cells were then dispensed per well. The assay plates were incubated at 37°C, 5% CO2 for 4 hours (GSPT1 for 20 hours). After incubation, 25 μL of InCELL Hunter detection reagent working solution (DiscoverX, Fremont, CA, Cat. No. 96-0002) was added to each well and incubated at room temperature for 60 minutes, protected from light. After 60 minutes, the emission was read on an Envision or PHERAstar luminescence reader.

[0117] For Helios, a stable Jurkat cell line was modified using CRISPR / Cas9 to insert an in-frame HiBit tag into the carboxy-terminal reading frame of the IKZF2 gene. Test compounds were transferred to 1536-well plates using an acoustic dispenser and Jurkat / Helios / HiBit cells were seeded at 10,000 cells / well in a final volume of 5ul in DMEM / 10% FCS. Cells were incubated at 37°C, 95% RH for 18 hours. Luciferase activity was measured by adding Nano-Glo reagent (Promega) at 2ul / well, incubating at RT for 30 minutes, and reading luminescence on a microtiter plate reader.

[0118] To determine the EC50 value of a compound for degradation of a given substrate (the concentration of the compound that achieves half of the maximum degradation observed), a four-parameter logistic model (sigmoidal dose-response model) (FIT=(A+{(BA) / 1+[(C / x)D]})) was used, where C is the inflection point (EC50), D is the correlation coefficient, and A and B are the lower and upper limits of the goodness of fit, respectively. All substrate degradation curves were processed and evaluated using the data analysis software package ActivityBase (IDBS). Ymin is the minimum value of protein remaining.

[0119] The results are shown in Tables 2 to 5. [Table 3] [Table 4] [Table 5] [Table 6]

[0120] As shown in Table 2, compound A degraded Helios, Aiolos, and Ikaros with an EC50 of 0.022 μM or less. Compound A also degraded CK1a to a significant extent. In contrast, compound B degraded Q1F degron with an EC50 of less than 5 nM in the Jurkat assay described in Example 5. Compound B showed some degradation activity against Helios, but the EC50 was >10 μM, which was more than three orders of magnitude higher than the EC50 for Q1F degron (Table 3). Compound B also showed some degradation activity against GSPT1 (Table 3). Compound C showed high selectivity against Q1F degron in the Jurkat assay described in Example 5, with an EC50 of less than 25 nM (Table 4). Compound D also demonstrated selectivity for the Q1F degron in the Jurkat assay described in Example 5 with an EC50 of less than 1 nM (Table 5), but also showed some degradation of Helios with an EC50 of 0.46 μM (Table 5).

[0121] Equivalent The present disclosure is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the subject matter of the patented inventions provided herein, in addition to those described, will become apparent to those skilled in the art from this specification. Such modifications are intended to be encompassed within the scope of the appended claims.

[0122] Various publications, patents, and patent applications are cited herein, the disclosures of which are incorporated by reference in their entireties.

[0123] [Table 7] [Table 8] [Table 9] [Table 10] [Table 11]

Table 12

Table 13

Table 14

Claims

1. 1. A modified polypeptide comprising a degradation domain, said degradation domain having the amino acid sequence FCX １ X ２ CGX ３ X ４ (SEQ ID NO: 1), X １ is selected from asparagine, aspartic acid, glycine, glutamine, methionine, histidine, tryptophan, isoleucine, arginine, leucine, valine, threonine, and phenylalanine; X ２ is selected from glutamine, arginine, histidine, leucine, phenylalanine, tyrosine, tryptophan, isoleucine, valine, and methionine; X ３ is selected from alanine, serine, cysteine, arginine, leucine, isoleucine, methionine, and glycine; X ４ is selected from serine, methionine, lysine, isoleucine, valine, histidine, glutamine, arginine, phenylalanine, and tryptophan; Optionally, X １ is selected from asparagine, glutamine, methionine, histidine, tryptophan, isoleucine, arginine, leucine, valine, threonine, and phenylalanine; X ２ is selected from glutamine, arginine, histidine, leucine, phenylalanine, tyrosine, tryptophan, isoleucine, and methionine; X ３ is selected from alanine, serine, cysteine, and glycine; X ４ is selected from serine, methionine, histidine, glutamine, arginine, phenylalanine, and tryptophan; Modified polypeptides.

2. The degradation domain has the amino acid sequence FCX １ X ２ CGX ３ X ４ X ５ (SEQ ID NO: 2), X ５ is selected from phenylalanine, tryptophan, methionine, arginine, histidine, leucine, tyrosine, cysteine, and glutamine; Optionally, X ５ is selected from phenylalanine, tryptophan, methionine, arginine, histidine, leucine, tyrosine, and glutamine; or X ５ is selected from phenylalanine, tryptophan, methionine, leucine, tyrosine, and glutamine; or X ５ is phenylalanine, The modified polypeptide of claim 1.

3. X １ is asparagine, and / or X ２ is glutamine, and / or X ３ is alanine or serine, and / or X ４ The modified polypeptide of claim 1 , wherein is serine.

4. 2. The modified polypeptide of claim 1, wherein the degradation domain comprises the amino acid sequence FCNQCGAS (SEQ ID NO: 3).

5. The modified polypeptide of claim 1 , wherein the degradation domain comprises at least one zinc finger domain, or two zinc finger domains.

6. 2. The modified polypeptide of claim 1, wherein at least one or each zinc finger domain independently comprises an amino acid sequence having at least 85%, at least 90%, or at least 95% identity to an amino acid sequence selected from SEQ ID NOs: 20-47 and 58.

7. 2. The modified polypeptide of claim 1, wherein the degradation domain comprises an amino acid sequence having at least 85%, at least 90%, or at least 95% identity to zinc finger 2 (ZNF2) of human Ikaros, Helios, Aiolos, or Eos, and optionally the degradation domain comprises a second zinc finger domain, wherein the second zinc finger domain has at least 85%, at least 90%, or at least 95% identity to zinc finger 1 (ZNF1) or ZNF3 of human Ikaros, Helios, Aiolos, or Eos, or the degradation domain comprises an amino acid sequence having at least 85%, at least 90%, or at least 95% identity to zinc finger 2 (ZNF2) and zinc finger 3 (ZNF3) of human Ikaros, Helios, Aiolos, or Eos.

8. the degradation domain comprises an amino acid sequence having at least 85%, at least 90%, or at least 95% identity to an amino acid sequence selected from SEQ ID NOs: 21, 27, 32, and 38; or the degradation domain comprises an amino acid sequence having at least 85%, at least 90%, or at least 95% identity to an amino acid sequence selected from SEQ ID NOs: 15, 60, 61, and 62; or The degradation domain comprises an amino acid sequence having at least 85%, at least 90%, or at least 95% identity to an amino acid sequence selected from SEQ ID NOs: 6, 63, 64, and 65; The modified polypeptide of claim 1.

9. The degradation domain may be: GERPFFCX １ X ２ CGX ３ X ４ X ５ TQKGNLLRHIKLHSGEKPFKCHLCNYACRRRDALTGHLRTHS (SEQ ID NO: 5); GERPFFCX １ X ２ CGX ３ X ４ X ５ TQKGNLLRHIKLHSGEKPFKCPFCSYACRRRDALTGHLRTHS (SEQ ID NO: 66); GERPFFCX １ X ２ CGX ３ X ４ X ５ TQKGNLLRHIKLHTGEKPFKCHLCNYACQRRDALTGHLRTHS (SEQ ID NO: 67); GERPFFCX １ X ２ CGX ３ X ４ X ５ TQKGNLLRHIKLHSGEKPFKCPFCNYACRRRDALTGHLRTHS (SEQ ID NO: 68); GERPFFCNQCGASFTQKGNLLRHIKLHSGEKPFKCHLCNYACRRRDALTGHLRTHS (SEQ ID NO: 7); GERPFFCNQCGASFTQKGNLLRHIKLHSGEKPFKCPFCSYACRRRDALTGHLRTHS (SEQ ID NO: 69); GERPFFCNQCGASFTQKGNLLRHIKLHTGEKPFKCHLCNYACQRRDALTGHLRTHS (SEQ ID NO: 70); and GERPFFCNQCGASFTQKGNLLRHIKLHSGEKPFKCPFCNYACRRRDALTGHLRTHS (SEQ ID NO: 71) 2. The modified polypeptide of claim 1, comprising an amino acid sequence selected from:

10. 10. The modified polypeptide of claim 1, which is ubiquitinated and / or degraded in the presence of a degradation agent, wherein the degradation agent is a small molecule that binds to the degradation domain; Optionally, the degrading agent is: 【Chemical 1】 and tautomers thereof, pharmaceutically acceptable salts thereof, and pharmaceutically acceptable salts of these tautomers.

11. 10. The modified polypeptide of claim 1, which is substantially intracellularly cytoplasmic or nuclear, or which contains a transmembrane domain.

12. 2. The modified polypeptide of claim 1, wherein the degradation domain is fused to or located within an endogenous protein, optionally wherein the endogenous protein is a mammalian or human protein.

13. The endogenous protein is PRDM1, TGFBR2, CASP8, CBLB, CD5, CISH, CGKA, DGKz, MAP4K1, ARID2, BACH2, CHX37, KLF2, KLF3, KLF6, MAF, SIGLEC9, TOX, ZBTB32, PTPN2, AKT1, PIK3CD, MT1E, MT2A, CSK, ITK, PAG1, PDCD4, ZC3H12A, DNMT1, DNMT3A, PRBM1, STK4, TET2, BNIP3, FAS, CBL, BGAT5, RNF12 8, STK17B, TRIB1, TXNIP, UBASH3A, BATF, FLI1, IKZF1, IKZF2, IRF4, NFATC1, NR4A1, MAP2K1, MAP2K2, MAP4K4, PPARGC1A, RELB, TMEM173, USP10, MT1A, a PP2A family member, RASA2, NR4A2, NR4A3, AHR, CD70, LHALS1, SOCS1, SOCS2, SOCS3, TAZ, USP21, or YAP1.

14. The modified polypeptide of claim 12, wherein the biological activity of the endogenous protein is substantially retained in the modified polypeptide.

15. 2. The modified polypeptide of claim 1, comprising a transmembrane domain, optionally said transmembrane domain being a transmembrane domain of a protein selected from the alpha chain of the T cell receptor, the beta chain of the T cell receptor, the zeta chain of the T cell receptor, CD28, CD3s, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS, TIM3, LAB3, TIGIT, PD1, or CTLA4.

16. 16. The modified polypeptide of claim 15, comprising an extracellular domain, a transmembrane domain, and an intracellular domain, optionally wherein the extracellular domain comprises a ligand, a ligand-binding domain, or an antigen-binding domain.

17. The extracellular domain comprises an antigen binding domain, optionally wherein the antigen binding domain binds to a cancer antigen, optionally wherein the antigen binding domain binds to any of the following antigens: 4-1BB, 5T4, 8H9, B7-H6, adenocarcinoma antigen, α-fetoprotein, B cell maturation antigen (BCMA), BAFFR, B lymphoma cells, C242 antigen, CA9, carcinoembryonic antigen, CA-125, carbonic anhydrase 9 (CA-IX), CCR4, CD3, CD4, CD19, CD20, CD22, CD23 (IgE receptor), CD28, CD30 (TFRSF8), CD33, CD38, CD40, CD44v6, CD44v7 / 8, CD51, CD52, CD56, CD70 CD74, CD80, CD123, CD152, CD171, CD200, CD221, CE7, CEA, C-MET, CLAUDIN6, CLAUDIN18.3, CNT0888, CTLA-4, DRS, EpCAM, ErbB2, ErbB3 / 4, EGFR, EGFRγIII, EphA2, EGP2, EGP40, FAP, Fetal AchR, fibronectin extra domain B, folate receptor a, folate receptor 1, G250 / CAIX, GD2, GD3, glycoprotein 75, GP MB, HER2 / neu, HGF, HLA-AI MAGE AI, HLA-A2 NY-ESO-1, HMW-MAA, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgG1, IL-6, IL-13, IL-13 receptor a2, IL-11 receptor a, insulin-like growth factor I receptor, integrin a5I31, integrin avI33, kappa light chain, L1-CAM, lambda light chain, Lewis Y, mesothelin, MORAb-009, MS4A1, MUCl, MUCl 6, mucin CanAg, NCAM, N-glycolylneuraminic acid, NKG2D ligand, NPC-IC, PDGF-R a, PDL192, phosphatidylserine, prostate-specific cancer antigen (PSCA), prostate cancer cells, PSMA, PSC1, RANKL, RON, ROR1, SCH 900105, SDC1, SLAMF7, spl7, TAG72, tenascin-C, TGFβ2, TGF-I3, TL1A, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, UPK1B, VEGF-A, VEGF receptor, VEGFR-1, VEGFR2, TEM1, TEM8, and / or vimentin.

18. 18. The modified polypeptide of claim 17, wherein the antigen-binding domain comprises an antibody heavy chain variable region and an antibody light chain variable region or a single-domain antibody antigen-binding domain.

19. The intracellular domain comprises at least one costimulatory domain, or two costimulatory domains, optionally at least one costimulatory domain selected from the group consisting of 4-1BB (CD137), CD28, OX40, K cell activating receptor, BTLA, Toll Ligand Receptor, CD2, CD7, CD27, CD30, CD40, CDS, ICAM-L LFA-1 (CD1la / CD18), B7-H3, CDS, ICAM-1, ICOS (CD278), RANK, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2 , SLAMF7, Kp80 (KLRF1), Kp44, Kp30, Kp46, CD19, CD4, CD8a, CD8p, IL2Rp, IL2Ry, IL7Ra, ITGA4, VLA1, CD49a , ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, LFA-1, ITGAM, CDllb , ITGAX, CDllc, ITGB1, CD29, ITGB2, IL15Ra, IL7R, CD18, CD132, LFA-1, ITGB7, KG2D, KG2C, TFR2, TRANCE / RA KL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), P SGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (C 17. The modified polypeptide of claim 16, which is a costimulatory domain of a receptor protein selected from: D162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, DAP10, DAP12, a ligand for CD83, an MHC class I molecule, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, and / or a signaling lymphocyte activation molecule.

20. 17. The modified polypeptide of Claim 16, wherein the intracellular domain comprises at least one signaling domain, and optionally the at least one signaling domain is an immunoreceptor tyrosine-based activation motif (ITAM) signaling domain, or the at least one signaling domain is a signaling domain of a receptor protein selected from CD3ε, CD3ζ, CD3η, FcRγ, FcRβ, CD3δ, CD3γ, CD5, CD22, CD20, CD79a, CD79b, CD278 (ICOS), FcERI, CD66d, DAP10, and DAP12.

21. 17. The modified polypeptide of claim 16, comprising, from amino terminus to carboxy terminus, (i) extracellular domain [ECD]-transmembrane domain [TM]-costimulatory domain [CoD]-signaling domain [SigD]-degradation domain [DD]; or (2) ECD-TM-CoD-DD-SigD; or (3) ECD-TM-DD-CoD-SigD.

22. 17. The modified polypeptide of claim 16, which is a chimeric antigen receptor (CAR).

23. 2. The modified polypeptide of claim 1, wherein the modified polypeptide is contained within a cell, and optionally the cell is a human effector cell, or the cell is a T cell or an NK cell.

24. 24. An isolated nucleic acid molecule comprising a polynucleotide sequence encoding a modified polypeptide according to any one of claims 1 to 23.

25. A vector comprising the nucleic acid molecule of claim 24.

26. A cell comprising the nucleic acid molecule of claim 24.

27. A cell comprising a modified polypeptide according to any one of claims 1 to 23.

28. 28. The cell of claim 27, which is a human effector cell, optionally a T cell or an NK cell, optionally wherein the T cell is a CD4+ T cell or a CD8+ T cell.

29. A pharmaceutical composition comprising the cells of claim 26.

30. 28. A method for reducing the level of a modified polypeptide in a cell, comprising: 【Chemistry 2】 and tautomers thereof, and pharmaceutically acceptable salts thereof, and pharmaceutically acceptable salts of these tautomers.

31. 1. An agent for reducing the level of a modified polypeptide in a cell in a subject, said agent comprising: 【Chemistry 3】 and tautomers thereof, and pharmaceutically acceptable salts thereof, and pharmaceutically acceptable salts of these tautomers, wherein the cell comprises a modified polypeptide of any one of claims 1 to 23.

32. 31. The method of claim 30, wherein the cell is a human effector cell, optionally wherein the cell is a T cell or an NK cell.

33. 31. The method of claim 30, wherein the modified polypeptide is ubiquitinated and / or degraded in the presence of the degradation agent.

34. A drug for treating a disease or disorder in a subject, the drug comprising the cells of claim 27, the cells being administered to the subject.

35. The agent of claim 34, wherein the cell comprises the modified polypeptide of claim 22.

36. 35. The method of claim 34, wherein the disease or disorder is cancer, and optionally the cancer is selected from a hematological cancer or a solid cancer.

37. 37. The method of claim 36, wherein the blood cancer is acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), lymphoma, non-Hodgkin's lymphoma (NHL), Hodgkin's disease, multiple myeloma, or myelodysplastic syndrome, and the solid cancer is brain cancer, prostate cancer, breast cancer, lung cancer, colon cancer, uterine cancer, skin cancer, liver cancer, bone cancer, pancreatic cancer, ovarian cancer, testicular cancer, bladder cancer, kidney cancer, head and neck cancer, gastric cancer, cervical cancer, rectal cancer, laryngeal cancer, and esophageal cancer.

38. the cells are contacted ex vivo with a degradative agent prior to administration to the subject, wherein the degradative agent is a small molecule that binds to the degradation domain; or The cell is administered to the subject together with a degradative agent, wherein the degradative agent is a small molecule that binds to the degradation domain; or The method is characterized in that the method is used to administer a degradative agent after administering the cells to the subject, wherein the degradative agent is a small molecule that binds to the degradation domain. The agent of claim 34.

39. The decomposition agent is 【Chemistry 4】 and tautomers thereof, pharmaceutically acceptable salts thereof, and pharmaceutically acceptable salts of these tautomers.

40. 【Chemical 5】 and tautomers thereof, pharmaceutically acceptable salts thereof, and pharmaceutically acceptable salts of these tautomers.

41. 41. A pharmaceutical composition comprising the compound of claim 40 and at least one pharmaceutically acceptable carrier.

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