TW202246309A - Synthetic degrader system for targeted protein degradation - Google Patents

Abstract

A fusion protein is provided having a binding element and a degradation initiator, where the binding element selectively binds a target molecule, and the degradation initiator has a sequence isolated or derived from an E3 ligase. A composition is provided comprising: (a) a first fusion protein comprising a first binding element; and (b) a second fusion protein comprising a second binding element; wherein: (1) the first fusion protein further comprises a degradation initiator or a functional variant thereof and the second fusion protein further comprises a target molecule; or (2) the first fusion protein further comprises a target molecule and the second fusion protein further comprises a degradation initiator or a functional variant thereof. The fusion proteins and compositions may be used for the targeted degradation of endogenous and exogenous proteins, optionally, in a cell or in vivo, for the treatment or prevention of a disease or disorder.

Description

Synthetic degradation system for targeted protein degradation

CROSS REFERENCE TO RELATED APPLICATIONS: This application claims priority to U.S. Provisional Patent Applications US 63/144,895, filed February 2, 2021, and U.S. Provisional Patent Applications US 63/248,516, filed September 26, 2021, and 2022 Priority of International Patent Application No. PCT/US22/14998 filed February 2, 2009. The entire contents of these applications are incorporated herein by reference.

Incorporation of sequence listing: The sequence listing submitted here is a text file named "016-TNP023TW_SeqList_v2", created on February 2, 2022, with a size of 52,267 KB, which is incorporated herein by reference.

The present disclosure relates to a synthetic degradation system for targeted degradation of related molecules. The degradation system includes a degradation initiator fused to a binding element for concentrating the degradation initiator to the relevant molecule to initiate degradation.

Protein degradation is an important cellular process that provides a mechanism for protein quality control and a rapid response to a multitude of cellular signals. In cells, proper coordination and synchronization of protein functions is governed by their spatially and temporally sequential degradation. To develop effective and safe next-generation cell and gene therapies, technologies that enable precise control of protein stability and half-life to regulate cellular processes are increasingly required.

The present disclosure provides a fusion protein comprising a binding element and a degradation initiator, wherein the binding element selectively binds a target molecule, and wherein the degradation initiator comprises a sequence isolated or derived from an E3 ligase. In some embodiments, (a) the E3 ligase comprises LNX1, RNF4, RNF43, RNF128, XNRF3, MARCH8, LRG1, NEDD4, SOCS2, CHIP, SPOP, FBXW7, FBXW1A, ELOC, TRAF6, VHL, or any functional fragment thereof; or (b) the E3 ligase comprises LNX1, RNF4, RNF43 or any functional fragment thereof; or (c) the E3 ligase comprises LNX1 or any functional fragment thereof; or (d) the E3 ligase comprises RNF4 or any functional fragment thereof; or (e) E3 ligase comprises RNF43 or any functional fragment thereof; Or (f) is isolated or is derived from the sequence of E3 ligase, comprises the sequence of Table 10, its functional fragment; A sequence of 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% homology; or (g) a sequence isolated or derived from an E3 ligase comprising SEQ ID NO: 74- or (h) a sequence isolated or derived from an E3 ligase comprising any one of SEQ ID NO: 159-163; or (i) an E3 ligase comprising a motif of Table 14. In some embodiments, the fusion protein further includes a DAP10 sequence, optionally including a tag or the sequence of SEQ ID NO:157. In some embodiments, the fusion protein further comprises a CD8 transmembrane domain, optionally comprising the sequence of SEQ ID NO:158. In some embodiments, the molecule of interest is an endogenous molecule in the cell capable of expressing the fusion protein. In some embodiments, the molecule of interest is a naturally occurring molecule. In some embodiments, a binding element includes a DNA sequence, RNA sequence, amino acid sequence, or any combination thereof. In some embodiments, the sequence of the binding element forms a nucleic acid duplex with the sequence of the target molecule. In some embodiments, the sequence of the binding element forms a nucleic acid duplex with the sequence of the target molecule by hybridization of the DNA or RNA sequence of the binding element to the DNA or RNA sequence of the target molecule. In some embodiments, a binding element selectively binds to an epitope of a molecule of interest. In some embodiments, binding elements include antibodies or functional fragments thereof, antibody mimetics, fibronectin domains, protein scaffolds, or aptamers. In some embodiments, the target molecule comprises a sequence in the sequence listing file "016-TNP023PCT_SeqList_v2", which was created on February 2, 2022 and is 52,267 KB in size, which is hereby incorporated by reference in its entirety, or includes the sequence encoding the target molecule nucleic acid sequence.

In some embodiments of the fusion proteins of the disclosure, the fusion protein or binding element includes a dimerization domain. In some embodiments, the dimerization domain comprises a designed heterodimer (DHD) polypeptide. In some embodiments, the molecule of interest or the sequence encoding the molecule of interest is modified (a) to include a binding element capable of forming a heterodimer with the dimerization domain of the fusion protein; or (b) operably linked to a Binding element that forms a heterodimer with the dimerization domain of a fusion protein. In some embodiments, a binding element includes a dimerization domain. In some embodiments, the dimerization domain comprises a designed heterodimer (DHD) polypeptide. In some embodiments, the binding element of the fusion protein is a first binding element and the binding element of the target molecule is a second binding element, and (a) wherein the first binding element or the second binding element comprises a single helix; or (b) wherein the first binding element or the second binding element comprises at least a double helix; or (c) wherein the first binding element or the second binding element comprises 3, 4, 5, 6, 7 or 8 helices; or (d) the first The binding element comprises a single helix and the second binding element comprises three helices; or (e) the first binding element comprises three helices and the second binding element comprises a single helix. In some embodiments, (a) the first binding element comprises DHD-A and the second binding element comprises DHD-B; or (b) the first binding element comprises DHD-B and the second binding element comprises DHD-A. In some embodiments, DHD-B comprises the sequence DHD37-short-B-KtoR (SEQ ID NO: 16). In some embodiments, the first binding element or the second binding element comprises a non-helical element. In some embodiments, the heterodimer includes non-helical elements. In some embodiments, non-helical elements include small molecules.

The present disclosure provides a composition comprising: (a) a first fusion protein comprising a first binding element; (b) a second fusion protein comprising a second binding element;

Wherein, (1) the first fusion protein further includes a degradation initiator or a functional variant thereof, and the second fusion protein further includes a target molecule; or (2) the first fusion protein further includes a target molecule, and the second fusion protein further includes a degradation trigger agents or their functional variants.

In some embodiments of the compositions of the present disclosure, the first binding element and the second binding element are capable of forming a heterodimer. In some embodiments, the first binding element or the second binding element comprises a single helix. In some embodiments, the first binding element or the second binding element comprises at least a double helix. In some embodiments, the first binding element or the second binding element comprises 3, 4, 5, 6, 7 or 8 helices. In some embodiments, (a) the first binding element comprises a single helix and the second binding element comprises three helices, or (b) the first binding element comprises three helices and the second binding element comprises a single helix. In some embodiments, (a) the first binding element comprises DHD-A and the second binding element comprises DHD-B; or (b) the first binding element comprises DHD-B and the second binding element comprises DHD-A. In some embodiments, the first binding element or the second binding element comprises a non-helical element. In some embodiments, the heterodimer includes non-helical elements. In some embodiments, non-helical elements include small molecules.

In some embodiments of the compositions of the present disclosure, the first binding element comprises the sequence of DHD37-short-B-KtoR (SEQ ID NO: 16). In some embodiments, the first binding element further comprises a sequence isolated or derived from an E3 ligase. In some embodiments, (a) the E3 ligase comprises LNX1, RNF4, RNF43, RNF128, XNRF3, MARCH8, LRG1, NEDD4, SOCS2, CHIP, SPOP, FBXW7, FBXW1A, ELOC, TRAF6, VHL, or any functional fragment thereof; or (b) the E3 ligase comprises LNX1, RNF4, RNF43 or any functional fragment thereof; or (c) the E3 ligase comprises LNX1 or any functional fragment thereof; or (d) the E3 ligase comprises RNF4 or any functional fragment thereof; or (e) an E3 ligase comprising RNF43 or any functional fragment thereof; or (f) a sequence isolated or derived from an E3 ligase comprising a sequence of Table 10, a functional fragment thereof, or at least 70% of a sequence of Table 10 or a functional fragment thereof %, 75%, 80%, 85%, 90%, 95%, 97% or 99% homologous sequence; or (g) a sequence isolated or derived from E3 ligase comprising SEQ ID NO: 74-114 (h) the sequence isolated or derived from the E3 ligase comprises any one of SEQ ID NO: 159-163; or (i) the E3 ligase comprising the motif of Table 14. In some embodiments, the first fusion protein further comprises a DAP10 sequence, optionally comprising a tag sequence or the sequence of SEQ ID NO:157. In some embodiments, the first fusion protein further comprises a CD8 transmembrane domain, optionally comprising the sequence of SEQ ID NO:158. In some embodiments, the first binding element comprises the sequence of SEQ ID NO:174.

In some embodiments of the compositions of the present disclosure, the first binding element comprises the sequence of any one of SEQ ID NOs: 1-73 or 121-125.

In some embodiments of the compositions of the present disclosure, the second binding element comprises the sequence of DHD37-short-B-KtoR (SEQ ID NO: 16). In some embodiments, the first binding element further comprises a sequence isolated or derived from an E3 ligase. In some embodiments, (a) the E3 ligase comprises LNX1, RNF4, RNF43, RNF128, XNRF3, MARCH8, LRG1, NEDD4, SOCS2, CHIP, SPOP, FBXW7, FBXW1A, ELOC, TRAF6, VHL, or any functional fragment thereof; or (b) the E3 ligase comprises LNX1, RNF4, RNF43 or any functional fragment thereof; or (c) the E3 ligase comprises LNX1 or any functional fragment thereof; or (d) the E3 ligase comprises RNF4 or any functional fragment thereof; or (e) the E3 ligase comprises RNF43 or any functional fragment thereof; or (f) a sequence isolated or derived from the E3 ligase comprising the sequence of Table 10, a functional fragment thereof, or at least 70% of the sequence of Table 10 or a functional fragment thereof %, 75%, 80%, 85%, 90%, 95%, 97% or 99% homologous sequence; or (g) a sequence isolated or derived from E3 ligase comprising SEQ ID NO: 74-114 or (h) a sequence isolated or derived from an E3 ligase comprising any one of SEQ ID NO: 159-163; or (i) an E3 ligase comprising a motif of Table 14. In some embodiments, the second fusion protein further comprises a DAP10 sequence, optionally comprising a tag or the sequence of SEQ ID NO:157. In some embodiments, the second fusion protein further comprises a CD8 transmembrane domain, optionally comprising the sequence of SEQ ID NO:158. In some embodiments, the first binding element comprises the sequence of SEQ ID NO:174.

In some embodiments of the compositions of the present disclosure, the second binding element comprises the sequence of any one of SEQ ID NOs: 1-73 or 121-125.

In some embodiments of the compositions of the present disclosure, a small molecule mediates the formation of the heterodimer. In some embodiments, the first binding element or the second binding element binds the small molecule. In some embodiments, the first binding element and the second binding element bind the small molecule. In some embodiments, the first binding element and the second binding element are not directly bound to each other. In some embodiments, small molecules increase heterodimer formation. In some embodiments, small molecules reduce heterodimer formation. In some embodiments, the first small molecule and the second small molecule regulate the formation of a heterodimer, and wherein the second small molecule reduces the interaction with the first binding element or the second binding element by competing with the first small molecule for binding to the first binding element or the second binding element. The first small molecule forms a heterodimer. In some embodiments, (a) the first binding element comprises an NS3a sequence and the second binding element comprises a DNCR2 sequence or a GNCR1 sequence; or (b) the first binding element comprises a DNCR2 sequence or a GNCR1 sequence and the second binding element comprises an NS3a sequence . In some embodiments, the small molecule comprises danoprevir or an analog thereof. In some embodiments, the first small molecule or the second small molecule comprises danoprevir or an analog thereof. In some embodiments, the small molecule comprises glazoprevir or an analog thereof. In some embodiments, the first small molecule or the second small molecule includes glazoprevir or an analog thereof.

In some embodiments of the compositions of the present disclosure, including those comprising a first fusion protein and a second fusion protein, the target molecule comprises a synthetic molecule or an exogenous molecule. In some embodiments, the synthetic or exogenous molecule comprises a protein. In some embodiments, the synthetic or exogenous molecule comprises a chimeric protein, chimeric receptor or chimeric antigen receptor. In some embodiments, the chimeric receptor comprises an extracellular domain comprising an antigen sensing domain, a transmembrane domain and an intracellular domain. In some embodiments, the antigen sensing domain comprises one or more of: (a) a set of three complementarity determining regions (CDRs) of the heavy chain variable region; (b) a set of three complementarity determining regions (CDRs) of the heavy chain variable region Three complementarity-determining regions (CDRs) and a set of three complementarity-determining regions (CDRs) for the light chain variable region; (c) a fibronectin-based scaffold; where the antigen-sensing domain specifically binds the target antigen. In some embodiments, the antigen sensing region comprises one or more sequences isolated or derived from mammalian sequences. In some embodiments, the antigen sensing region comprises one or more sequences isolated or derived from human sequences. In some embodiments, the antigen sensing region comprises humanized or fully human antibodies. In some embodiments, the antigen sensing region comprises a single chain variable fragment (scFv).

In some embodiments of the compositions of the present disclosure, the extracellular domain further comprises one or more of a hinge region, a spacer sequence, or a safety switch. In some embodiments, the hinge region comprises a sequence isolated or derived from a CD4 (cluster of differentiation 4) polypeptide, a CD8 (cluster of differentiation 8) polypeptide, or a CD28 (cluster of differentiation 28) polypeptide. In some embodiments, the hinge region comprises a sequence isolated or derived from a human sequence. In some embodiments, the hinge region and the spacer region are the same region. In some embodiments, the hinge region and the spacer region are different regions.

In some embodiments of the compositions of the present disclosure, the spacer sequence comprises a sequence isolated or derived from a CD4 polypeptide, a CD8 polypeptide, a CD28 polypeptide. In some embodiments, the spacer sequence comprises a sequence isolated or derived from a human sequence.

In some embodiments of the compositions of the present disclosure, the safety switch comprises a sequence isolated or derived from an epidermal growth factor receptor (EGFR) polypeptide. In some embodiments, the safety switch comprises a truncated EGFR (EGFRt) polypeptide. In some embodiments, the safety switch comprises a sequence isolated or derived from a human sequence.

In some embodiments of the compositions of the present disclosure, the transmembrane domain comprises a sequence isolated or derived from a CD4 polypeptide, a CD8 polypeptide, a CD28 polypeptide. In some embodiments, the transmembrane domain comprises a sequence isolated or derived from a human sequence.

In some embodiments of the compositions of the present disclosure, the intracellular domain comprises one or more co-stimulatory domains. In some embodiments, the one or more co-stimulatory domains comprise a sequence isolated or derived from a CD3ζ (cluster of differentiation 3zeta) polypeptide. In some embodiments, the one or more co-stimulatory domains comprise a sequence isolated or derived from a CD28 polypeptide, a 4-1BB (cluster of differentiation 137) polypeptide, an ICOS (inducible T cell co-stimulator) polypeptide, an OX40 polypeptide, or CD27 (cluster of differentiation 27) polypeptide. In some embodiments, the one or more co-stimulatory domains comprise (a) a first co-stimulatory domain comprising a sequence isolated or derived from a CD28 polypeptide, a 4-1BB polypeptide or an ICOS polypeptide; (b) comprising a sequence derived from 4 - a second co-stimulatory domain of an isolated or derived sequence of a 1BB polypeptide, OX40 polypeptide or CD27 polypeptide. In some embodiments, one or more co-stimulatory domains comprise sequences isolated or derived from human sequences.

In some embodiments of the compositions of the present disclosure, the chimeric receptor comprises an intracellular domain further comprising an inducible cytokine domain. In some embodiments, the inducible cytokine domain comprises a nuclear factor of activated T cell (NFAT) polypeptide capable of inducing expression of the IL-12 cytokine.

In some embodiments of the compositions of the present disclosure, the chimeric receptor comprises an intracellular domain, further comprising an intracellular domain of a cytokine receptor. In some embodiments, the intracellular domain of the cytokine receptor comprises an IL-2 receptor beta (IL-2Rβ) chain fragment. In some embodiments, the chimeric receptor comprises an intracellular domain that further comprises a Signal Transducer and Initiator of Transcription (STAT3/5) binding motif.

In some embodiments of the compositions of the present disclosure, the chimeric receptor comprises an intracellular domain further comprising at least one immunoreceptor tyrosine-based initiation motif (ITAM) sequence.

The disclosure also provides a nucleic acid sequence, comprising: encoding: (a) the fusion protein of the disclosure; or (b) one or more elements of (a). In some embodiments, the fusion protein comprises a binding element and a degradation initiator, wherein the binding element selectively binds a target molecule, and wherein the degradation initiator comprises a sequence isolated or derived from an E3 ligase. In some embodiments, the molecule of interest is an endogenous molecule in the cell capable of expressing the fusion protein. In some embodiments, the molecule of interest is a naturally occurring molecule.

The present disclosure also provides a nucleic acid sequence, comprising: encoding: (a) the first fusion protein of the present disclosure; (b) the second fusion protein of the present disclosure; (c) one or more elements of (a); (d ) the nucleic acid sequence of one or more elements of (b).

The disclosure also provides a nucleic acid sequence, including: a nucleic acid sequence encoding the first fusion protein disclosed herein and the second fusion protein disclosed herein.

In some embodiments of the nucleic acid sequences of the disclosure, the nucleic acid sequence further includes one or more of non-coding sequences, untranslated regions, regulatory elements, segregating elements, polycistronic elements, or post-translational elements.

In some embodiments of the nucleic acid sequences disclosed herein, the nucleic acid sequences further comprise at least one promoter capable of driving expression of the nucleic acid sequences in mammalian cells. In some embodiments, the nucleic acid sequence further comprises at least one promoter capable of driving expression of the nucleic acid sequence in human cells. In some embodiments, at least one promoter includes a constitutive promoter. In some embodiments, the constitutive promoter comprises one of MND promoter, hPGK promoter, CMV promoter, CAG promoter, SFFV promoter, EF1α promoter, UBC promoter and CD43 promoter isolated or derived from or Various sequences. In some embodiments, the at least one promoter comprises an inducible promoter. In some embodiments, the inducible promoter comprises a sequence isolated or derived from one or more of the YB_TATA promoter, the human beta globin (huBG) promoter, the minIL2 promoter, the minimalCMV (minCMV) promoter, and the TRE3G promoter . In some embodiments, an inducible promoter comprises minimal sequence. In some embodiments, the inducible promoter further comprises a transcription factor specific recognition sequence. In some embodiments, the transcription factor specific recognition sequence comprises a transcription factor specific response element. In some embodiments, the transcription factor response element comprises a sequence isolated or derived from an NFAT sequence. In some embodiments, the transcription factor-specific recognition sequence comprises at least one repeat of a transcription factor-specific response element. In some embodiments, the transcription factor-specific recognition sequence comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats of a transcription factor-specific response element.

In some embodiments of the nucleic acid sequences of the disclosure, (a) the first promoter drives expression of the first fusion protein; or (b) the second promoter drives expression of the second fusion protein. In some embodiments, the first promoter and the second promoter are the same. In some embodiments, the first promoter and the second promoter are not the same.

In some embodiments of the nucleic acid sequences of the disclosure, the separation element comprises a ribosomal skipping sequence. In some embodiments, the separation element comprises at least two ribosomal skipping sequences. In some embodiments, the ribosomal skipping sequence comprises a P2a sequence or a T2a sequence. In some embodiments, the ribosomal skipping sequence comprises a T2a-RFP-P2a sequence, a P2a-T2a sequence, or a T2a-P2a sequence.

In some embodiments of the nucleic acid sequences of the disclosure, the polycistronic element comprises an internal ribosome entry site (IRES) sequence.

The present disclosure provides a vector, which includes: the vector of the nucleic acid sequence disclosed herein.

The present disclosure provides a first vector comprising a nucleic acid sequence encoding a first fusion protein of the present disclosure and a second vector comprising a nucleic acid sequence encoding a second fusion protein of the present disclosure.

In some embodiments of the disclosed vectors, the vector is an expression vector capable of expressing a nucleic acid in a mammalian cell. In some embodiments, the mammalian cells are human cells. In some embodiments, vectors comprise plasmids.

In some embodiments of the vectors of the present disclosure, the vector comprises a delivery vehicle capable of introducing nucleic acid into a mammalian cell. In some embodiments, the mammalian cells are human cells. In some embodiments, delivery vehicles include one or more of viral vectors, non-viral vectors, liposomes, micelles, polymers, and nanoparticles. In some embodiments, a viral vector comprises sequences isolated or derived from a virus or viral vector. In some embodiments, the viral vector comprises an adenoviral vector, a lentiviral vector, a baculoviral vector, an Epstein-Barr virus vector, a Babeicavirus (parvovirus) vector, a vaccinia virus vector, a herpes simplex virus vector, One or more isolated or derived sequences from adeno-associated virus (AAV) vectors and hepatitis B virus vectors. In some embodiments, vectors include sequences isolated or derived from transposon systems. In some embodiments, the vector comprises one or more of the piggyBAC transposition system, Sleeping Beauty transposition system, Tc1/mariner transposition system, Tol2 transposition system, helraiser transposition system and Tn7 transposition system isolated or derived from the sequence of.

In some embodiments of the disclosed vectors, the vector comprises one or more sequences that mediate homology-directed repair

The present disclosure provides a cell comprising: a cell comprising the fusion protein of the present disclosure. In some embodiments, the fusion protein comprises a binding element and a degradation initiator, wherein the binding element selectively binds a target molecule, and wherein the degradation initiator comprises a sequence isolated or derived from an E3 ligase. In some embodiments, the molecule of interest is an endogenous molecule in the cell capable of expressing the fusion protein. In some embodiments, the molecule of interest is a naturally occurring molecule.

The disclosure provides a cell comprising a composition of the disclosure. In some embodiments, the composition comprises a first fusion protein and a second fusion protein.

The disclosure provides a cell comprising the nucleic acid sequence of the disclosure.

The disclosure provides a cell comprising a vector of the disclosure. In some embodiments, cells stably express fusion proteins of the disclosure. In some embodiments, the fusion protein comprises a binding element and a degradation initiator, wherein the binding element selectively binds a target molecule, and wherein the degradation initiator comprises a sequence isolated or derived from an E3 ligase. In some embodiments, the molecule of interest is an endogenous molecule in the cell capable of expressing the fusion protein. In some embodiments, the molecule of interest is a naturally occurring molecule.

The disclosure provides a cell comprising a vector of the disclosure. In some embodiments, the cell stably expresses the first fusion protein or the second fusion protein of the present disclosure.

In some embodiments of the cells of the present disclosure, the cells are eukaryotic cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human cells. In some embodiments, the cells are stem cells. In some embodiments, the stem cells are hematopoietic cells. In some embodiments, the stem cells are mesenchymal cells. In some embodiments, the cells are immune cells. In some embodiments, the immune cells are T cells, natural killer (NK) cells, or innate lymphoid cells (ILCs).

The disclosure provides a composition comprising (a) the fusion protein disclosed herein; or (b) the composition of any one of the disclosures; or (c) the nucleic acid sequence of any one of the disclosures; or (d) A vector of any of the disclosures; or (e) a cell of any of the disclosures.

The present disclosure provides a pharmaceutical composition, which comprises the composition of the present disclosure and a pharmaceutically acceptable carrier.

This disclosure provides any fusion protein of this disclosure; the use of any composition of this disclosure; any nucleic acid sequence of this disclosure; any vector of this disclosure; any cell of this disclosure; or the drug combination of this disclosure A substance for the preparation of a medicament for treating a disease or a condition.

This disclosure provides any fusion protein of this disclosure; the use of any composition of this disclosure; any nucleic acid sequence of this disclosure; any vector of this disclosure; any cell of this disclosure; or the drug combination of this disclosure substances for the treatment of diseases or conditions.

In some embodiments of the disclosed uses, the disease or condition comprises one or more of an autoimmune disease or condition; an inflammatory disease or condition; an immunodeficiency disease or disorder; an ischemic disease or condition; diseases or conditions of the nervous system; diseases or conditions of the heart; diseases or conditions of the blood vessels; diseases or conditions of the metabolic system; diseases or conditions of the digestive system; diseases or conditions of the kidney disease or disorder; hearing disease or disorder; ophthalmic disease or disorder; and proliferative disease or disorder.

In some embodiments of the disclosed uses, the disease comprises cancer. In some embodiments, the cancer comprises adult acute lymphoblastic leukemia (ALL), adult acute myeloid leukemia (AML), adrenal cancer, anal cancer, basal cell and squamous cell skin cancer, cholangiocarcinoma selected from one or more of the following , bladder cancer, bone cancer, adult brain and spinal cord tumors, childhood brain and spinal cord tumors, breast cancer, male breast cancer, adolescent cancer, childhood cancer, young adult cancer, cancer of unknown primary, cervical cancer, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), colorectal cancer, endometrial cancer, esophageal cancer, Ewing tumor family, eye cancer (ocular melanoma), gallbladder cancer , neuroendocrine (carcinoid) tumors of the gastrointestinal tract, gastrointestinal stromal tumors (GIST), head and neck cancers, Hodgkin's lymphoma, Kaposi's sarcoma, renal cancer, laryngeal and hypopharyngeal cancers, leukemia, childhood leukemia, Liver cancer, lung cancer, lung cancer, lymphoma, cutaneous lymphoma, malignant mesothelioma, melanoma skin cancer, Merkel cell skin cancer, multiple myeloma, myelodysplastic syndrome, nasal cavity and sinus cancer, nasopharyngeal cancer, Neuroblastoma, Non-Hodgkin's Lymphoma, Pediatric Non-Hodgkin's Lymphoma, Oral (mouth) and Oropharyngeal (Throat) Cancers, Osteosarcoma, Ovarian Cancer, Pancreatic Cancer, Pancreatic Neuroendocrine Tumors (NET), Penis Carcinoma, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, small bowel cancer, soft tissue sarcoma, gastric cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's giant Gammaglobulinemia and Wilms tumor.

In some embodiments of the disclosed uses, the disease or condition comprises an infection or a disease or condition caused by an infectious disease.

In some embodiments of the disclosed uses, the disease or condition comprises a genetic disease or condition.

The present disclosure further provides a method for treating a disease or condition, comprising administering to a subject an effective dose of any fusion protein disclosed herein, any composition disclosed herein, any nucleic acid sequence disclosed herein, any nucleic acid sequence disclosed herein, any A vector of any of the disclosures, a cell of any of the disclosures, or a pharmaceutical composition of the disclosures, wherein the severity of the signs or symptoms of the disease or disorder is reduced, thereby treating the disease or disorder.

The disclosure provides a method for treating a disease or condition, comprising administering to a subject an effective dose of any fusion protein of the disclosure, any composition of the disclosure, any nucleic acid sequence of the disclosure, any carrier of the disclosure, Any cell or pharmaceutical composition disclosed herein is used to delay or inhibit the appearance or recurrence of signs or symptoms of a disease or disorder, thereby preventing the disease or disorder.

In some embodiments of the methods of the present disclosure, the disease or disorder comprises one or more of an autoimmune disease or disorder; an inflammatory disease or disorder; an immunodeficiency disease or disorder; an ischemic disease or disorder; Diseases or conditions of the nervous system; Diseases or conditions of the nervous system; Diseases or conditions of the nervous system; Diseases or conditions of the blood vessels; renal disease or disorder; hearing disease or disorder; ophthalmic disease or disorder; and proliferative disease or disorder.

In some embodiments of the disclosed methods, the disease comprises cancer. In some embodiments, the cancer includes adult acute lymphoblastic leukemia (ALL), adult acute myeloid leukemia (AML), adrenal cancer, anal cancer, basal cell and squamous cell skin cancer selected from one or more of the following, Cholangiocarcinoma, bladder cancer, bone cancer, adult brain and spinal cord tumors, childhood brain and spinal cord tumors, breast cancer, male breast cancer, adolescent cancer, childhood cancer, young adult cancer, cancer of unknown primary, cervical cancer, chronic lymphatic Cellular leukemia (CLL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), colorectal cancer, endometrial cancer, esophageal cancer, Ewing tumor family, eye cancer (ocular melanoma), Gallbladder cancer, Gastrointestinal neuroendocrine (carcinoid) tumors, Gastrointestinal stromal tumor (GIST), Head and neck cancers, Hodgkin lymphoma, Kaposi's sarcoma, Kidney cancer, Laryngeal and hypopharyngeal cancers, Leukemia, Children Leukemia, liver cancer, lung cancer, lung cancer, lymphoma, cutaneous lymphoma, malignant mesothelioma, melanoma skin cancer, Merkel cell skin cancer, multiple myeloma, myelodysplastic syndrome, nasal cavity and sinus cancer, nasopharynx Carcinoma, Neuroblastoma, Non-Hodgkin's Lymphoma, Pediatric Non-Hodgkin's Lymphoma, Oral (Mouth) and Oropharyngeal (Throat) Cancers, Osteosarcoma, Ovarian Cancer, Pancreatic Cancer, Pancreatic Neuroendocrine Tumors (NET) , penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, small intestine cancer, soft tissue sarcoma, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia and Wilms tumor.

In some embodiments of the methods of the present disclosure, the disease or condition comprises an infection or a disease or condition caused by an infectious disease.

In some embodiments of the methods of the present disclosure, the disease or condition comprises a genetic disease or condition.

The disclosure provides a set of polynucleotides comprising: (a) a first polynucleotide encoding a first fusion protein comprising a first binding element; and (b) a second fusion protein encoding a second binding element A second polynucleotide; wherein: (i) the first fusion protein or the second fusion protein further comprises a degradation initiator or a functional variant thereof; and (ii) the other of the first fusion protein or the second fusion protein further comprising a target molecule of interest; (iii) the interaction of the first binding element and the second binding element mediates the concentration of the degradation initiator to the target molecule of interest to initiate degradation.

In some embodiments of the disclosed set of polynucleotides, the first and second binding elements comprise a pair of heterodimeric proteins. In some embodiments, the pair of heterodimeric proteins comprises a dimer having one or two subunits consisting of single helical subunits. In some embodiments, the pair of heterodimeric proteins comprises a dimer having one or two subunits consisting of double helical subunits. In some embodiments, the pair of heterodimeric proteins comprises a dimer with one or two subunits consisting of multihelical subunits. In some embodiments, the pair of heterodimeric proteins comprises a triple helical subunit and a single helical subunit. In some embodiments, the pair of heterodimeric proteins comprises non-helical subunits. In some embodiments, the first or second binding element is selected from: DHD37-short-A (SEQ ID NO: 11) and DHD37-short-B (SEQ ID NO: 13), DHD37-short-B-Ntrunc ( SEQ ID NO: 15), or the group consisting of DHD37-short-B-KtoR (SEQ ID NO: 16).

In some embodiments of the disclosed set of polynucleotides, the first binding element and the second binding element are selected such that the interaction of the first binding element and the second binding element is mediated by the presence of the small molecule.

In some embodiments of the set of polynucleotides disclosed herein, the set of polynucleotides described above, wherein: (a) the first binding element or the second binding element comprises NS3a; (b) the first binding element or the second binding element Another one of the binding elements is selected from the group consisting of DNCR2 and GNCR1; and (c) wherein the first binding element and the second binding element are selected such that the interaction of the first binding element and the second binding element is achieved by The presence of small molecules to regulate. In some embodiments, the small molecule modulates the binding of the first binding element and the second binding element. In some embodiments, the small molecule disrupts the binding of the first binding element and the second binding element. In some embodiments, the second small molecule disrupts the binding of the first binding element and the second binding element by competing with the first small molecule. In some embodiments, the interaction of the first binding member and the second binding member in the cell is titratable relative to administration of the small molecule to the cell. In some embodiments, the small molecule is selected from: danoprevir and glazoprevir and analogs thereof.

In some embodiments of the disclosed polynucleotide sets, the molecules of interest include synthetic molecules or exogenous molecules.

In some embodiments of the sets of polynucleotides disclosed herein, the synthetic or exogenous molecules comprise proteins.

In some embodiments of the set of polynucleotides of the present disclosure, the first polynucleotide and the second polynucleotide are on a single vector.

In some embodiments of the set of polynucleotides disclosed herein, the set of polynucleotides comprises at least two vectors comprising: (a) a first vector comprising a first polynucleotide; (b) a second vector comprising A secondary vector for the polynucleotide.

In some embodiments of the set of polynucleotides disclosed herein, the set of polynucleotides is incorporated into a vector backbone. In some embodiments, the vector backbone is selected from adenoviral vectors, lentiviral vectors, baculoviral vectors, Epstein Barr viral vectors, polyvacuum viral vectors, vaccinia viral vectors, herpes simplex viral vectors, adeno-associated viral (AAV) vectors and the backbone of the transposon vector. In some embodiments, the vector backbone comprises a homology directed repair vector.

In some embodiments of the set of polynucleotides disclosed herein, the set of polynucleotides is integrated into a chromosome.

In some embodiments of the disclosed set of polynucleotides, the first polynucleotide encoding the first fusion protein or the second fusion protein and/or the second polynucleotide is operably linked to a polynucleotide comprising one or more The polynucleotide component of the promoter sequence.

In some embodiments of the set of polynucleotides of the present disclosure, (a) a first polynucleotide or a second polynucleoside encoding a first fusion protein or a second fusion protein comprising a degradation initiator or a functional variant thereof The acid is operably linked to a polynucleotide component encoding a sequence comprising an inducible promoter; and (b) a first polynucleotide or a second polynucleotide encoding a first fusion protein or a second fusion protein comprising a molecule of interest Nucleotides are operably linked to elements encoding polynucleotides that include constitutive promoter sequences.

In some embodiments of the set of polynucleotides of the present disclosure, (a) a first polynucleotide or a second polynucleoside encoding a first fusion protein or a second fusion protein comprising a degradation initiator or a functional variant thereof Acid is operably linked to a polynucleotide component encoding a first inducible promoter sequence; and (b) a first polynucleotide or a second polynucleotide encoding a first fusion protein or a second fusion protein comprising a molecule of interest The nucleotide is operably linked to the polynucleotide component encoding the second inducible promoter sequence.

In some embodiments of the set of polynucleotides of the present disclosure, the first polynucleotide encoding the first fusion protein and the second polynucleotide encoding the second fusion protein comprise a set of two or more polynucleotides Element.

The present disclosure provides a set of polynucleotides wherein a first polynucleotide encoding a first fusion protein and a second polynucleotide encoding a second fusion protein comprise a single polynucleotide component. In some embodiments, the polynucleotide component encoding the first fusion protein and the polynucleotide encoding the second fusion protein are separated by a separation element comprising a polynucleotide sequence that prevents fusion of the first fusion protein and the second fusion protein. In some embodiments, the separation element comprises a polynucleotide sequence comprising a ribosomal skipping sequence. In some embodiments, the separation element comprises a polynucleotide sequence comprising at least two ribosomal skipping sequences. In some embodiments, the ribosomal skipping sequence comprises a polynucleotide sequence comprising P2a and/or T2a. In some embodiments, the isolated element comprises a polynucleotide sequence selected from the group consisting of: P2a, T2a, T2a-RFP-P2a, P2a-T2a, T2a-P2a, and IRES. In some embodiments, the isolated element comprises a polynucleotide sequence comprising a second constitutive promoter.

In some embodiments of the disclosed polynucleotide sets, the constitutive promoter sequence is selected from the group consisting of: MND, hPGK, CMV, CAG, SFFV, EF1α, UBC, and CD43.

In some embodiments of the disclosed polynucleotide sets, the inducible promoter sequence comprises a minimal promoter sequence selected from the group consisting of: YB_TATA, human beta globin (huBG), minIL2, minimalCMV (minCMV) and TRE3G.

In some embodiments of the disclosed set of polynucleotides, the inducible promoter sequence comprises a transcription factor specific recognition sequence comprising a transcription factor specific response element. In some embodiments, the transcription factor specific response element comprises a polynucleotide selected from the group consisting of: NFAT-AP1. In some embodiments, the transcription factor response elements are repeats. In some embodiments, the transcription factor specific response element has 2, 3, 4, 5, 6, 7, 8, 9, 10 or more repeats.

The present disclosure provides a set of polynucleotides comprising a single polynucleotide component encoding a fusion protein comprising: (a) a binding element specific for a native motif on a molecule of interest; and (b) a degradation trigger agent; and wherein binding of the fusion protein to the target molecule modulates reassembly of the degradation initiator to the target molecule to initiate degradation. In some embodiments, molecules of interest include endogenous molecules. In some embodiments, endogenous molecules comprise proteins. In some embodiments, binding of the fusion protein to the molecule of interest involves conformation-specific interactions. In some embodiments, the binding of the fusion protein to the molecule of interest corresponds to specific modifications on the molecule. In some embodiments, specific modifications to molecules include post-translational modifications. In some embodiments, post-translational modifications include phosphorylation. In some embodiments, the binding element comprises a domain specific for a protein-protein interaction domain. In some embodiments, the binding element comprises a single chain variable fragment (scFVf). In some embodiments, the binding element comprises a monomeric variable antibody domain. In some embodiments, the binding element comprises a designed ankyrin repeat protein domain (DARPin). In some embodiments, the binding element comprises a variable lymphocyte receptor (VLR) domain.

In some embodiments of the disclosed polynucleotide sets, the degradation initiator comprises an E3 ubiquitin ligase domain or any functional variant thereof.

The disclosure further provides a cell comprising the set of polynucleotides of the disclosure. In some embodiments, the cells are prokaryotic cells. In some embodiments, the cells are yeast cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human cells. In some embodiments, the cells are human cells in vivo. In some embodiments, the cells are ex vivo human cells. In some embodiments, the cells are stem cells. In some embodiments, the cells are pluripotent stem cells. In some embodiments, the cells are pluripotent stem cells. In some embodiments, the cells are hematopoietic stem cells. In some embodiments, the cells are mesenchymal stromal cells. In some embodiments, the cells are mesenchymal cells. In some embodiments, the cells are autologous cells selected for cell therapy or progeny of autologous cells selected for cell therapy. In some embodiments, the cells are allogeneic cells selected for cell therapy or progeny of allogeneic cells selected for cell therapy.

The present disclosure provides a method of achieving differentiation of stem cells comprising modifying the stem cells using the polypeptide set of the polynucleotide set of the present disclosure. In some embodiments, the cells are cancer cells. In some embodiments, the cells are non-cancerous cells from a human subject diagnosed with cancer. In some embodiments, the cells are immune cells. In some embodiments, the cells are selected from the group consisting of leukocytes, lymphocytes, T cells, regulatory T cells, effector T cells, CD4+ effector T cells, CD8+ effector T cells, memory T cells, autoreactive T cells, exhausted T cells, Natural killer T cells, B cells, dendritic cells and macrophages. In some embodiments, the cells are selected from cardiomyocytes, lung cells, myocytes, epithelial cells, pancreatic cells, skin cells, CNS cells, neurons, myocytes, skeletal muscle cells, smooth muscle cells, liver cells, kidney cells, and glia cell.

The disclosure provides a cell genetically modified to express a CAR comprising the set of polynucleotides of the disclosure. In some embodiments, the cells are T cells, natural killer (NK) cells, natural killer T (NKT) cells, or ILC cells.

The disclosure provides a capsid comprising the set of polynucleotides of the disclosure. In some embodiments, the viral capsid is selected from capsids of adenovirus, lentivirus, baculovirus, Epstein-Barr virus, Barr virus, vaccinia virus, herpes virus, herpes simplex virus, and adeno-associated virus .

The disclosure provides a cell that produces capsids of the disclosure. In some embodiments, the viral capsid is selected from capsids of adenovirus, lentivirus, baculovirus, Epstein-Barr virus, Barr virus, vaccinia virus, herpes virus, herpes simplex virus, and adeno-associated virus .

The present disclosure provides a composition comprising the set of polynucleotides of the present disclosure.

The present disclosure provides a use comprising a composition of the present disclosure, including a composition comprising a polynucleotide set of the present disclosure, for treating a subject in need of CAR therapy.

The disclosure provides a kit comprising the set of polynucleotides of the disclosure.

The present disclosure also provides a method of making an engineered cell comprising introducing a polynucleotide of any set of polynucleotides of the present disclosure into the cell. In some embodiments, the polypeptide is expressed in a cell. In some embodiments, the method further comprises administering the cells to the subject in need thereof. In some embodiments, the method further comprises administering the small molecule to the subject.

The present disclosure also provides a method of controlling a T cell-mediated immune response in a subject in need thereof, comprising administering to the subject an effective dose of the cells of the present disclosure.

The present disclosure also provides a method of providing anti-tumor immunity in a subject in need thereof, the method comprising administering to the subject an effective dose of the cells of the present disclosure. In some embodiments, the cells are T cells. In some embodiments, the cells are autologous T cells. In some embodiments, the cells are allogeneic T cells. In some embodiments, the method further comprises administering the small molecule to the subject.

The present disclosure also provides a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective dose of the cells of the present disclosure. In some embodiments, the cells are T cells. In some embodiments, the cells are autologous T cells. In some embodiments, the cells are allogeneic T cells. In some embodiments, the method further comprises administering the small molecule to the subject.

The present disclosure also provides a gene therapy method comprising: (a) a set of polynucleotides, including a degradation initiator, operably linked to a small molecule regulated promoter; and (b) a therapeutic molecule; the method comprising adding A subject in need thereof is administered a therapeutically effective dose of a panel of polynucleotides of the present disclosure. In some embodiments, the method further comprises administering the small molecule to the subject. In some embodiments, the method further comprises adjusting the dose of the small molecule to adjust the amount of the therapeutic molecule in the subject. In some embodiments, the method further comprises (a) monitoring production of the therapeutic molecule in the subject; and (b) adjusting the dose of the small molecule to adjust the level of the therapeutic molecule in the subject to a desired level. In some embodiments, the subject has a condition selected from the group consisting of cancer, cystic fibrosis, heart disease, diabetes, hemophilia, and AIDS.

The present disclosure also provides a use of the polynucleotide set of the present disclosure for the manufacture of a medicament for treating cancer in a subject in need thereof.

Nucleic Acids and Related Terms

In some embodiments, the terms "nucleic acid", "nucleic acid molecule", "nucleotide", "nucleotide sequence", "polynucleotide" and grammatical variants thereof are used interchangeably herein and refer to ribonucleoside (adenosine, guanosine, uridine, or cytidine; "RNA molecule") or the phosphate polymerized form of deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecule") or any of its phosphate analogues, such as single-stranded or double-stranded helical phosphorothioates and thioesters. Single-stranded nucleic acid sequence refers to single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA). Double-stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible.

In some embodiments, the term "nucleic acid" as used herein, particularly a DNA or RNA molecule, may refer only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary form. Thus, the term includes double-stranded DNA found inter alia in linear or circular DNA molecules (such as restriction fragments), plasmids, supercoiled DNA, and chromosomes. When discussing the structure of a particular double-stranded DNA molecule, it is normal convention to follow the 5' to 3' direction from left to right along the non-transcribed strand of DNA (that is, the strand with sequence homology to messenger RNA or mRNA) Write the sequence. Unless otherwise indicated, all nucleic acids and nucleotide sequences are written left to right in 5' to 3' orientation.

Nucleotides are denoted by the well-known single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Thus, "A" stands for adenine, "C" for cytosine, "G" for guanine, "T" for thymine, and "U" for uracil.

In some embodiments, the term "polynucleotide" as used herein refers to a polymer of nucleotides of any length or type, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. The term refers to the primary structure of a molecule. Accordingly, the term includes triple-, double-, and single-stranded deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA"). It also includes modified, for example by alkylation and/or by capping, and unmodified polynucleotide forms. More specifically, "polynucleotide" includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose) and polyribonucleotides (containing D-ribose), including mRNA, whether spliced or Unspliced, any other type of polynucleotide that is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing a nucleotide backbone, for example, polyamide (e.g., peptide nucleic acid "PNA" ) and polymorpholine polymers, and other synthetic sequence-specific nucleic acid polymers, provided that these polymers contain bases in configurations that allow base pairing and base stacking, such as those in deoxyribonucleic acid and ribonucleic acid base.

In some embodiments, a vector of the disclosure comprises a nucleic acid sequence and a backbone sequence(s) of the disclosure.

In some embodiments, a polynucleotide comprises DNA, eg, inserted into a vector. In other embodiments, the polynucleotide comprises mRNA. In some embodiments, the mRNA is synthetic mRNA. In some embodiments, the synthetic mRNA includes at least one non-natural nucleobase. In some embodiments, all nucleobases of a class have been replaced with non-natural nucleobases (e.g., all uridines in a polynucleotide may be replaced with non-natural nucleobases, such as 5-methoxyuridine ).

In some embodiments, the term "expression" as used herein refers to the transcription and/or translation of a specific nucleotide sequence driven by a promoter.

In some embodiments, the term "expression vector" as used herein refers to a plasmid, virus or other nucleic acid designed to express a polypeptide in a cell. A vector or construct is used to introduce a gene into a host cell whereby the vector will interact with the polymerase in the cell to express the protein encoded in the vector/construct. Expression vectors can be present in the cell extrachromosomally or can be integrated into the chromosome. Expression vectors may include additional sequences that render the vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (eg, shuttle vectors). The polynucleotides of the present disclosure may be provided as components of expression vectors.

In some embodiments, the term "cloning vector" as used herein refers to a plasmid, virus or other nucleic acid designed to produce copies of a polynucleotide. Cloning vectors can contain transcriptional and translational initiation sequences, transcriptional and translational termination sequences, and polyadenylation signals. Such constructs will typically include a 5' LTR, a tRNA binding site, a packaging signal, the origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof. The polynucleotides of the disclosure can be provided as components of cloning vectors, which can be used to generate the polynucleotides of the disclosure.

In some embodiments, the term "promoter" as used herein refers to a nucleotide sequence that indicates where transcription of a gene begins and the direction in which transcription will continue.

In some embodiments, the term "encoding" as used herein refers to a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA or mRNA) as a template for use in a specific nucleotide sequence (such as rRNA, tRNA and mRNA) or the ability to synthesize other polymers and macromolecules in biological processes that specify amino acid sequences. Thus, a gene, cDNA or RNA encodes a protein if mRNA transcription and translation of that gene, cDNA or RNA produces the protein in a cell or other biological system. Both the coding strand (which has the same nucleotide sequence as the mRNA sequence and is usually given in a sequence listing) and the noncoding strand (used as a template for gene or cDNA transcription) can be referred to as encoding the protein or other product of that gene or cDNA.

Unless otherwise stated, a nucleotide sequence "encoding an amino acid sequence" (including a polynucleotide "encoding" a chimeric polypeptide of the disclosure) includes all nucleotide sequences that are degenerate forms of each other and that encode the same amino acid sequence.

Peptides and related terms

Amino acids may be referred to by their common three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Amino acid residues are abbreviated as follows, where the abbreviation is shown in parentheses: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), Cysteine (Cys; C), Glutamic acid (Glu; E), Glutamine (Gln; Q), Glycine (Gly; G), Histidine (His; H), Isoleucine ( Ile; I), Leucine (Leu; L), Lysine (Lys; K), Methionine (Met; M), Phenylalanine (Phe; F), Proline (Pro; P), Serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V).

Amino acid sequences are written left to right in amino to carboxyl orientation.

In some embodiments, the term "polypeptide" as used herein refers to a sequence of amino acid subunits. In some embodiments, a "peptide" may comprise up to 50 amino acids. In some embodiments, a "peptide" may comprise about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids. The term "polypeptide" as used herein may refer to proteins, polypeptides and peptides of any length, size, structure or function. As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably.

In some embodiments, polypeptides of the present disclosure comprise amino acids that are naturally or synthetically produced or modified, e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, e.g., with labeling Combination of components. In some embodiments, a polypeptide of the present disclosure comprises one or more amino acid residues that are the corresponding natural amino acids (including, for example, synthetic amino acids, such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acid and creatine), and other modifications known in the art. In some embodiments, polypeptides of the present disclosure include gene products, homologues, orthologs, paralogs, fragments, and other equivalents, variants, and analogs of the foregoing. In some embodiments, polypeptides of the present disclosure comprise a single polypeptide or may be a multimolecular complex, such as a dimer, trimer, or tetramer. In some embodiments, polypeptides of the present disclosure include single-chain or multi-chain polypeptides. In some embodiments, polypeptides of the present disclosure comprise disulfide bonds, which may be present in multi-chain polypeptides.

In some embodiments, polypeptides of the present disclosure include L-amino acids+glycine, D-amino acids+glycine (which are resistant to L-amino acid-specific proteases in vivo), or a combination of D- and L-amino acids+glycine. The polypeptides of the present disclosure can be chemically synthesized or expressed recombinantly.

A polypeptide of the disclosure may include additional residues at the N-terminus, C-terminus, internal to the polypeptide, or a combination thereof; these additional residues are not included in determining the percent identity of a polypeptide of the disclosure relative to a reference polypeptide. Such residues may be any suitable for the intended use, including but not limited to tags.

In some embodiments, the term "tag" as used herein refers to a detectable moiety including, but not limited to, a fluorescent protein, an antibody epitope tag, a purification tag, a histidine tag, or a linker. In some embodiments, a labeled therapeutic agent of the disclosure or a labeled ligand of the disclosure comprises a detectable moiety suitable for purification purposes, to drive localization of the polypeptide, and to add functionality to the polypeptide.

In some embodiments, the term "chimeric polypeptide" as used herein refers to any polypeptide comprising a first amino acid sequence derived from a first source operably with a second amino acid sequence derived from a second source. Covalently or non-covalently linked, where the first source and the second source are not the same (two different sources). The non-identical first and second sources may comprise two different biological entities, or two different proteins from the same biological entity, or a biological entity and a non-biological entity. Chimeric proteins of the present disclosure can include proteins derived from at least 2 different biological sources. A biological source can include any non-synthetically produced nucleic acid or amino acid sequence (eg, a genomic or cDNA sequence, a plasmid or viral vector, a natural virion, or a mutant or analog of any of the foregoing). Synthetic sources can include protein or nucleic acid sequences that are produced chemically rather than by biological systems (eg, solid-phase synthesis of amino acid sequences). Chimeric proteins of the present disclosure can include proteins derived from at least two different synthetic sources or proteins derived from at least one biological source and at least one synthetic source. A chimeric protein of the present disclosure may comprise a first amino acid sequence derived from a first source, covalently or non-covalently linked to a nucleic acid derived from any source or to a small organic or inorganic molecule derived from any source. A chimeric protein may include a linker molecule between a first amino acid sequence and a second amino acid sequence or between a first amino acid sequence and a nucleic acid, or between a first amino acid sequence and a small organic or inorganic molecule.

In some embodiments, the term "fragment" of a polypeptide or "truncated polypeptide" as used herein may refer to an amino acid sequence of a polypeptide that is shorter than a reference sequence. In some embodiments, the reference sequence comprises or consists of a naturally occurring sequence. This fragment may contain N-terminal or C-terminal deletions (optionally referred to as truncations) compared to the reference sequence. Alternatively or additionally, the fragment may comprise an internal deletion at any one or more amino acid positions of the polypeptide compared to the reference sequence. Polypeptides of the disclosure may be provided as fragments or truncated forms of a reference polypeptide. Furthermore, all possible fragments and truncated variants of the polypeptides of the disclosure are encompassed by the examples provided in the disclosure.

In some embodiments, the term "functional fragment" as used herein refers to a fragment of a polypeptide that retains the function of the polypeptide. Thus, in some embodiments, a functional fragment such as a biologically active peptide of an enzyme retains the ability to catalyze a biological action because the functional fragment comprises the catalytic domain of the enzyme. Polypeptides of the disclosure may be provided as fragments or truncated forms of a reference polypeptide. Furthermore, all possible fragments and truncated variants of the polypeptides of the disclosure are encompassed by the examples provided in the disclosure. In some embodiments, the functional fragments of the disclosure retain the function of the polypeptide even though the activity of the functional fragment or the efficacy of the functional fragment is modified compared to the full-length polypeptide. In some embodiments, the functional fragments of the present disclosure retain the function of the polypeptide even though the activity of the functional fragment or the efficacy of the functional fragment is reduced compared to the full-length polypeptide.

In some embodiments, the term "functional variant" as used herein refers to a modified form of a polypeptide, fragment, or member of a class of polypeptides that retains the function of the polypeptide. In some embodiments, the functional variants of the disclosure retain the function of the polypeptide even though the activity of the functional variant or the efficacy of the functional variant is modified compared to the unmodified polypeptide. In some embodiments, the functional variants of the disclosure retain the function of the polypeptide even though the activity of the functional variant or the efficacy of the functional variant is reduced compared to the unmodified polypeptide.

In some embodiments, the term "amino acid substitution" as used herein refers to the substitution of an amino acid residue present in a parental or reference sequence (eg, a wild-type sequence) with another amino acid residue. Amino acids may be substituted, for example, by chemical peptide synthesis or by recombinant methods known in the art. For example, substitution of an amino acid residue with a substitute amino acid residue is performed by substituting a codon encoding a first amino acid with a codon encoding a second amino acid. Various polypeptide compositions of the present disclosure may have amino acid substitutions.

In some embodiments, the term "conservative amino acid substitution" as used herein is one in which an amino acid residue is replaced with an amino acid residue having a chemically similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine ), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, Valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine). Thus, if an amino acid in a polypeptide is substituted with another amino acid from the same side chain family, the substitution is considered conservative. In another aspect, a string of amino acids can be conservatively substituted with chemically similar strings that differ in the order and/or composition of side chain family members. Various polypeptide components of the disclosure may be provided with conservative amino acid substitutions.

In some embodiments, the term "non-conservative amino acid substitution" as used herein may refer to (i) a residue with a positively charged side chain (such as Arg, His, or Lys) being replaced or a negatively charged residue (such as Glu or Asp) Substitutions, (ii) those in which a hydrophilic residue (such as Ser or Thr) is substituted or replaced by a hydrophobic residue (such as Ala, Leu, Ile, Phe, or Val), (iii) cysteine or proline is replaced by Any other residue is substituted, or substituted by any other residue, or (iv) a residue with a large number of hydrophobic or aromatic side chains (e.g., Val, His, Ile, or Trp) is replaced by a residue with a smaller side chain (e.g., Ala or Ser) or no side chain (e.g., Gly), or by a residue with a smaller side chain (e.g., Ala or Ser) or no side chain (e.g., Gly). Various polypeptide components of the disclosure may provide non-conservative amino acid substitutions. The likelihood that one of the above non-conservative substitutions will alter the functional properties of the protein is also related to the position of the substitution relative to regions important for protein function: thus, some non-conservative substitutions will have little or no effect on the biological properties. Various polypeptide components of the disclosure can have non-conservative amino acid substitutions that do not significantly alter the function of the altered component.

In some embodiments, the term "transmembrane element" or "transmembrane domain" as used herein may refer to a polypeptide element between an extracellular element and an intracellular element. A portion of transmembrane elements reside within the cell membrane. The chimeric antigen receptors (CARs) of the present disclosure include transmembrane elements.

In some embodiments, the term "intracellular element" or "intracellular domain" as used herein may refer to a polypeptide element that is located on the cytoplasmic side of the plasma membrane of a cell and transmits signals into eukaryotic cells. The CARs of the present disclosure include intracellular elements. In some embodiments, the cells are eukaryotic cells.

In some embodiments, the term "intracellular signaling element" or "intracellular signaling domain" as used herein refers to a portion of an intracellular element that transduces effector function signals and subsequently directs the cell to perform a specific function. In some embodiments, the cells are eukaryotic cells.

In some embodiments, the term "extracellular element" or "extracellular element" as used herein may refer to a polypeptide element located outside the plasma membrane of a cell. In a CAR-expressing cell, the extracellular element can include the antigen-binding element of the CAR. In some embodiments, the cells are eukaryotic cells.

Sequence Identity and Related Terms

In some embodiments, two or more sequences are said to be "identical" if they are 100% identical to each other.

In some embodiments, "homology" refers to overall monomeric conservation between polymeric molecules, such as between polypeptide molecules or polynucleotide molecules. Without any additional qualification "identical", for example, protein A is identical to protein B, meaning that the sequences are 100% identical (100% sequence identity). Describing two sequences as "70% identical" is equivalent to describing them as having "70% sequence identity".

In some embodiments, when a position in the first sequence is occupied by the same amino acid as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and the determination of percent identity between two sequences can be accomplished using a mathematical algorithm.

In certain aspects, the percent identity (%ID) of a first amino acid (or nucleic acid) sequence to a second amino acid (or nucleic acid) sequence is calculated as %ID = 100 (Y/Z), where Y is the difference between the first sequence and The number of amino acid (or nucleobase) residues scored as identical matches in an alignment of the second sequence (by visual inspection or by a specific sequence alignment program), where Z is the residue in the second sequence total. If the first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be higher than the percent identity of the second sequence to the first sequence.

In some embodiments, the percent identity of two polypeptide sequences can be calculated by aligning the two sequences, eg, for optimal alignment purposes. For example, gaps can be introduced in one or both of the first and second polypeptide sequences for optimal alignment, and differing sequences can be ignored for comparison purposes. In certain aspects, the length of the sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 90% of the length of the reference sequence. At least 95% or 100%. The amino acids at corresponding amino acid positions are then compared.

In some embodiments, the generation of sequence alignments for calculating percent sequence identity is not limited to binary sequence-sequence comparisons driven only by primary sequence data. It should also be appreciated that sequence alignments can be generated by integrating sequence data with data from heterogeneous sources such as structural data (eg, crystal protein structures), functional data (eg, mutation positions), or phylogenetic data. A suitable program for integrating heterogeneous data to generate multiple sequence alignments is T-Coffee, available at www.tcoffee.org, or from, for example, the European Bioinformatics Institute (EBI) website ebi.ac.uk/Tools/psa . It is also understood that the final alignment used to calculate percent sequence identity can be managed automatically or manually.

Suitable software programs are available from various sources for the alignment of protein and nucleotide sequences. One suitable program for determining percent sequence identity is bl2seq, which is part of the BLAST suite of programs available from the US Government's National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.gov). B12seq uses the BLASTN or BLASTP algorithms to make a comparison between two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water or Matcher, which are part of the EMBOSS suite of bioinformatics programs, also available from EBI. Sequence alignments can be performed using methods known in the art, such as MAFFT, Clustal (ClustalW, Clustal X, or Clustal Omega), MUSCLE, and the like. Different regions within a single polynucleotide or polypeptide target sequence aligned to a polynucleotide or polypeptide reference sequence may each have their own percent sequence identity. Note that percent sequence identity values are rounded to the nearest tenth. For example, values from 80.11 to 80.14 are rounded down to 80.1, and values from 80.15 to 80.19 are rounded up to 80.2. It should also be noted that the length value will always be an integer.

In some embodiments, the term "similarity" as used herein refers to the overall relatedness between polymer molecules, eg, between polynucleotide molecules and/or between polypeptide molecules. Calculations of percent similarity of polymer molecules to one another can be performed in the same manner as percent identity calculations, except that percent similarity calculations take into account conservative substitutions as understood in the art. It will be appreciated that the percent similarity depends on the comparison scale used, e.g. whether amino acids are compared e.g. according to their evolutionary proximity, charge, volume, flexibility, polarity, hydrophobicity, aromaticity, isoelectric point, antigenicity or their combination.

In some embodiments, the term "linked" as used herein may refer to the fusion of a first moiety to a second moiety at the C-terminus or N-terminus. In some embodiments, the term "linking" as used herein may refer to the insertion of the entire first part (or the second part) at any two points, such as amino acids, in the second part (or the first part, respectively). In some aspects, the first moiety is linked to the second moiety by a peptide bond or a linker. The first part can be linked to the second part by a phosphodiester bond or a linker. Linkers can be peptides, polypeptides, nucleotides, chains of nucleotides, or any chemical moiety.

In some embodiments, the term "operably linked" as used herein may refer to two or more elements that are functionally linked within a sequence. In some embodiments, two elements can be functionally linked in a folded protein that is discontinuous with respect to a linear sequence. In some embodiments, two elements may be functionally linked, following expression from a polycistronic sequence. In some embodiments, two elements can be functionally linked in that one element can directly or indirectly induce or inhibit the expression or function of the other element.

In some embodiments, the term "non-naturally occurring" as used herein may refer to a polypeptide or polynucleotide sequence that does not occur in nature. In some embodiments, a non-naturally occurring sequence does not occur in nature because the sequence is relative to A naturally occurring sequence is altered. In some embodiments, a non-naturally occurring sequence does not occur in nature because it is a combination of two naturally occurring sequences that do not occur together in nature (eg, a chimeric polypeptide). In some embodiments, the non-naturally occurring polypeptide is a chimeric polypeptide. In some embodiments, the polypeptide or polynucleotide is not naturally occurring because the sequence contains portions (e.g., fragments) that cannot be found in nature, i.e., novel Sequence. Any polynucleotide described herein may be provided as a non-naturally occurring sequence, for example, having a sequence altered relative to a native sequence or provided as a polynucleotide linked to other polynucleotides in a manner that does not occur in nature. Any of the polypeptides described may be provided as a non-naturally occurring sequence, eg, having a sequence altered relative to a native sequence or as a polypeptide linked to other polypeptides in a manner not found in nature.

Antibodies and related terms

In some embodiments, the term "antibody" as used herein may refer to various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and antibody fragments, so long as they exhibit the desired antigen-binding activity.

In some embodiments, the term "antibody fragment" as used herein may refer to a molecule other than an intact antibody, which includes a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, scFv antibody fragments, linear antibodies, single domain antibodies such as sdAb (VL or VH), camelid VHH domains, and antibodies formed from antibody fragments multispecific antibodies. For example, a gene of interest in the present disclosure can include an antibody fragment.

In some embodiments, the term "single chain antibody" (scFv) as used herein may refer to an antibody fragment comprising heavy (VH) and light (VL) chain variable regions linked by a flexible peptide linker.

In some embodiments, the term "antigen-binding molecule" as used herein may refer to a molecule that specifically binds an antigenic determinant. A gene of interest of the present disclosure may, for example, include an antigen binding molecule.

In some embodiments, the term "antigen" as used herein may refer to a molecule that elicits an immune response.

In some embodiments, the term "chimeric antigen receptor" or "CAR" as used herein may refer to a fusion protein comprising an antigen recognition portion and a cell activation element. A polynucleotide of the present disclosure may include a gene of interest for the production of a CAR.

In some embodiments, the term "CAR T cell" or "CAR T lymphocyte" as used herein may refer to a T cell having the ability to produce a CAR polypeptide. For example, a cell capable of expressing a CAR is a T cell that contains a nucleic acid sequence for expressing a CAR in the cell. The cells of the present disclosure may be CAR T cells.

In some embodiments, the term "costimulatory element" or "costimulatory signaling domain" or "costimulatory polypeptide" as used herein may refer to the intracellular portion of a costimulatory polypeptide. In some embodiments, a CAR comprising a co-stimulatory domain exhibits increased or enhanced T cell expansion, function, persistence, and anti-tumor activity when expressed in a T cell compared to a CAR lacking a co-stimulatory domain active. Costimulatory domains can be provided in CARs of the present disclosure by incorporating intracellular signaling domains from one or more T cell costimulatory molecules (eg, CD28 or 4-1BB).

In some embodiments, costimulatory polypeptides may comprise sequences isolated or derived from one or more of: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins) and activation of natural killer cell receptors. In some embodiments, the co-stimulatory polypeptide may comprise a protein selected from CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen 1 (LFA-1), CD2 , CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3 and MyD88 one or more isolated or derived sequences.

Effective treatment and related technologies

In some embodiments, the term "therapeutically effective" as used herein may refer to providing a beneficial effect to a recipient, for example, providing some relief, relief or reduction of a subject's clinical symptoms or an improvement in a subject's clinical state. The therapeutic effect need not be complete or curative, just provide some benefit to the subject.

In some embodiments, the term "therapeutically effective dose" as used herein may refer to a dose sufficient to confer a therapeutically effective benefit in a recipient. For example, a fusion protein, nucleic acid, vector, cell, composition or pharmaceutical composition of the present disclosure can be administered in a therapeutically effective dose. A subject who has been administered a fusion protein, nucleic acid, vector, cell, composition or pharmaceutical composition of the disclosure may then be administered a therapeutically effective amount of a small molecule of the disclosure to induce or disrupt dimer formation of the fusion protein of the disclosure, to achieve the desired cellular outcome.

cell term

In some embodiments, the term "stem cell" as used herein may refer to an undifferentiated or partially differentiated cell that can differentiate into various types of cells and immortalize to produce more of the same stem cell.

In some embodiments, the term "pluripotent stem cell" (PSC) as used herein may refer to a cell that is capable of maintaining an undifferentiated state indefinitely and that can differentiate into most, if not all, cells of the body.

In some embodiments, the term "induced pluripotent stem cells" (iPS or iPSCs) as used herein may refer to pluripotent stem cells that can be generated directly from somatic cells. This includes, but is not limited to, specialized cells from adults, such as skin or blood cells.

In some embodiments, the term "pluripotent" as used herein may refer to cells that can develop into more than one cell type but are more restricted than pluripotent cells. For example, adult stem cells and cord blood stem cells can be considered multipotent.

In some embodiments, the term "hematopoietic cell" as used herein may refer to a cell derived from a hematopoietic stem cell. These include, but are not limited to, myeloid progenitors, lymphoid progenitors, megakaryocytes, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, macrophages, platelets, Nuclear cells, natural killer cells, T lymphocytes, B lymphocytes and plasma cells .

In some embodiments, the term "T-lymphocyte" or "T-cell" as used herein may refer to a hematopoietic cell that normally develops in the thymus. T lymphocytes or T cells include, but are not limited to, natural killer T cells, regulatory T cells, helper T cells, cytotoxic T cells, memory T cells, γδ T cells, and mucosal invariant T cells.

In some embodiments, the term "mesenchymal" as used herein may refer to an animal tissue comprising loose cells embedded in a network of proteins and fluid, ie, an extracellular matrix. Mesenchyme directly produces most of the body's connective tissue, including bone, cartilage, lymphatic system, and circulatory system.

In some embodiments, the term "mesenchymal cells" as used herein may refer to cells derived from mesenchymal tissue. In some cases, cells of the present disclosure can be mesenchymal cells.

In some embodiments, the term "mesenchymal stromal cells" (MSCs) as used herein may refer to spindle-shaped plastic adherent cells isolated from bone marrow, adipose, and other tissue sources, having pluripotent differentiation capacity in vitro. For example, mesenchymal stromal cells can differentiate into osteoblasts (bone cells), chondrocytes (chondrocytes), myocytes (muscle cells), and adipocytes (fat cells that give rise to the fatty tissue of the bone marrow). The use of the term mesenchymal stromal cells has been suggested in the scientific literature instead of the term "mesenchymal stem cells". In some cases, the cells of the present disclosure can be mesenchymal stromal cells.

In some embodiments of the present disclosure, an "autologous cell" is a cell obtained from the same individual to which it can be administered as therapy (the cell is autologous to the subject). Autologous cells of the present disclosure include, but are not limited to, hematopoietic cells and stem cells, eg, hematopoietic stem cells.

In some embodiments of the present disclosure, allogeneic cells are cells obtained from an individual who is not the intended recipient of the treated cells (the cells are allogeneic to the subject). The allogeneic cells of the present disclosure can be selected from immunologically compatible donors relevant to the subject matter of the methods of the present disclosure. The allogeneic cells of the disclosure can be modified to generate "universal" allogeneic cells, suitable for administration to any subject without unintended immunogenicity. Allogeneic cells of the present disclosure include, but are not limited to, hematopoietic cells and stem cells, eg, hematopoietic stem cells.

In some embodiments, the term "transfection" or "transformation" or "transduction" as used herein may refer to the process of transferring or introducing exogenous nucleic acid into a cell or host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed or transduced with exogenous nucleic acid or progeny of the cell.

In some embodiments, the term "cell therapy" as used herein may refer to the delivery of one or more cells into a recipient for therapeutic purposes.

Small Molecule Terminology

In some embodiments, the term "analog" as used herein may refer to a chemically modified form of a compound or member of a class of compounds described herein that retains the binding properties of the compound or class. For example, an analog of danoprevir would include a chemically modified form of danoprevir that retains the ability to bind DNCR2 and NS3a as described herein.

In some embodiments, the term "prodrug" as used herein may include any covalently bound carrier that releases the disclosed small molecule in vivo when such prodrug is administered to a patient. Prodrugs of the present disclosure can be prepared by modifying functional groups present on the compounds in such a way that the modifications are cleaved to the parent compound during routine manipulation or in vivo. For example, in vivo transformations may be the result of certain metabolic processes, such as chemical or enzymatic hydrolysis of carboxylate, phosphate, or sulfate esters, or reduction or oxidation of sensitive functional groups. Prodrugs within the scope of the present disclosure include compounds wherein a hydroxy, amino or thiol group is bonded to any group that, when the prodrugs of the present disclosure are administered to a mammalian subject, cleaves to form a free hydroxy, free amino or free thiol, respectively. Functional groups that can be rapidly transformed in vivo by metabolic cleavage form a class of groups reactive with the carboxyl group of the disclosed compounds. They include, but are not limited to, alkylyl groups (such as acetyl, propionyl, butyryl, etc.), unsubstituted and substituted aryl groups (such as benzoyl and substituted benzoyl), alkoxycarbonyl groups (such as ethoxy carbonyl), trialkyl groups such as trimethyl and triethyl, monoesters formed from dicarboxylic acids such as succinyl, etc. Small molecules of the present disclosure can be administered as prodrugs. Small molecules of the present disclosure can be administered to a subject as prodrugs. A therapeutically effective amount of such prodrugs of the present disclosure may be administered. Prodrugs can be administered concurrently with or subsequent to administration of the polynucleotides, gene therapy vectors or cells of the present disclosure.

Formula and related technology

In some embodiments, the term "pharmaceutically acceptable" as used herein refers to a compound, material, composition and/or dosage form suitable for use in contact with human and animal tissues within the scope of reasonable medical judgment, without excessive toxicity, irritation sex, allergic reactions, or other problems or complications commensurate with a reasonable benefit/risk ratio. For example, the small molecules, polynucleotides, polypeptides, gene therapy vectors or cells of the disclosure can be administered as part of a composition with other pharmaceutically acceptable ingredients, including a pharmaceutically acceptable carrier.

In some embodiments, the term "pharmaceutically acceptable salt" as used herein may refer to a derivative of a small molecule of the present disclosure, wherein the specified compound is converted to its acid or base salt. Such pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; alkali metal or organic salts of acidic residues such as carboxylic acids, and the like. Pharmaceutically acceptable salts include, for example, conventional non-toxic or quaternary ammonium salts of the parent compound formed from non-toxic inorganic or organic acids. Such conventional nontoxic salts include, for example, those derived from inorganic acids, such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and those prepared from organic acids, such as acetic, propionic, succinic, Glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfonamide, 2 -Acetyloxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isosulfide, etc. For example, small molecules of the present disclosure can be provided as pharmaceutically acceptable salts.

In some embodiments, the term "controlled release" as used herein may refer to part or all of a dosage form that releases one or more active agents over an extended period of time (ie, over a period of more than 1 hour). The controlled release (CR) profile may also be referred to as sustained release (SR), extended release (PR) or extended release (ER). When used in conjunction with the dissolution profiles discussed herein, the term "controlled release" as used herein refers to the portion of a dosage form according to the present disclosure that releases the active agent over a period of more than 1 hour. For example, small molecules of the present disclosure can be administered in controlled release compositions.

In some embodiments, the term "immediate release" as used herein may refer to part or all of a dosage form that releases the active agent substantially immediately upon contact with gastric juices and results in substantially complete dissolution within about 1 hour. Immediate release (IR) properties may also be referred to as immediate release (IR). When used in conjunction with the dissolution profiles discussed herein, the term "immediate release" refers to that portion of the dosage form according to the present disclosure that delivers the active agent over a period of less than 1 hour. The small molecules of the present disclosure can be administered as an immediate release composition.

In some embodiments, the term "excipient" as used herein may refer to a pharmacologically inert ingredient that has no activity in vivo. See, eg, Hancock, B. C., Moss, G. P., & Goldfarb, D. J. (2020), Handbook of Pharmaceutical Excipients, London: Pharmaceutical Press, the entire disclosure of which is incorporated herein by reference. Small molecules of the present disclosure may be mixed with pharmaceutically acceptable carriers, diluents, adjuvants, excipients or vehicles, such as preservatives, fillers, polymers, disintegrants, glidants, wetting agents, emulsifying Agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, lubricants, acidifying agents and distributing agents, depending on the mode of administration and the nature of the dosage form. Such ingredients include pharmaceutically acceptable carriers and excipients useful for formulating oral dosage forms. Pharmaceutically acceptable carriers include water, alcohols, polyols, vegetable oils, fats, waxes, polymers, including gel-forming and non-gel-forming polymers, and suitable mixtures thereof. Examples of excipients include starch, pregelatinized starch, microcrystalline cellulose, lactose, milk sugar, sodium citrate, calcium carbonate, dicalcium phosphate, and colored starch mixtures. Examples of disintegrants include starch, alginic acid, and certain complex silicates. Examples of lubricants include magnesium stearate, sodium lauryl sulfate, talc, and high molecular weight polyethylene glycols. For example, the small molecules, polynucleotides, polypeptides, gene therapy vectors or cells of the present disclosure can be provided and administered in compositions comprising pharmaceutically acceptable excipients.

general terms

The term "subject" as used throughout this document includes any mammal, including but not limited to a human. In some embodiments, the subject is a human. In some embodiments, the subject is a neonate, infant, toddler, child, adolescent, adult, elderly, centenarian. In some embodiments, the subject has at least 1, 2 or 3 X chromosomes. In some embodiments, the subject has at least 1 or 2 Y chromosomes. In some embodiments, the subject is diagnosed with a disease or condition disclosed herein, or is otherwise in need of a treatment disclosed herein. In some embodiments, the subject is at risk of developing, or in need of prevention of, a disease or disorder of the present disclosure.

In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a mammal, including but not limited to livestock, horses, dogs, cats, pigs, rabbits, guinea pigs, rodents, rats, gerbils, and mice. In some embodiments, the subject is a non-primate mammal and the subject is genetically modified.

"A", "an" and "the" include plural forms unless the context clearly dictates otherwise.

"And" is used interchangeably with "or" unless expressly stated otherwise.

"And/or" is to be considered as specific disclosure of each of the two specified features or elements, with or without the other. Thus, "and/or" used in terms such as "A and/or B" includes "A and B", "A or B", "A" (alone) and "B" (alone). Likewise, the use of "and/or" in terms such as "A, B and/or C" is intended to cover each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

In some embodiments, the term "about" as used herein means roughly, roughly, around, or within the region thereof. When "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth.

In some embodiments, numerical ranges include numbers defining the range. Where a range of values is stated, each intervening integer value and each fraction thereof between the stated upper and lower limits of that range, and each subrange between such values, is also specifically disclosed. The upper and lower limits of any range may independently be included or excluded from that range, and every range where either, neither, or both limits are included is also encompassed within the disclosure. Accordingly, range is understood to be shorthand for all values within the range, including the enumerated endpoints. For example, a range of 1 to 10 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

Where a value is explicitly stated, it is to be understood that approximately the same number or amount of the stated value is also within the scope of the disclosure. Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of the disclosure. Conversely, where different elements or groups of elements are disclosed individually, their combinations are also disclosed. Where any element of the disclosure is disclosed as having multiple alternatives, examples of the disclosure are also disclosed herein in which each alternative is excluded alone or in any combination with other alternatives; a disclosure More than one element of may have such exclusion, and all combinations of elements with such exclusion are disclosed herein.

Unless the context requires otherwise, throughout the specification and claims, the words "comprise", "comprise", etc. are to be interpreted in an inclusive sense rather than an exclusive or exhaustive sense; that is, in the " including but not limited to ".

Words that are singular or plural also include plural and singular respectively. Thus, for example, where the specification describes a gene of interest, the disclosure includes polynucleotides having a single gene of interest or a plurality of genes of interest.

"Above" and "below" and words of similar import refer to the text as a whole and not to any particular parts of the text.

A "group" includes a collection of one or more elements or objects.

Units, prefixes and symbols are indicated in their International System of Units (SI) accepted form.

This article includes headings for reference and to help define the various sections. These headings are not intended to limit the scope of the concepts described with respect to the headings. Such concepts may have applicability throughout the specification herein.

Although the disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications will be practiced by those skilled in the art. References to "the present disclosure" and the like are intended as references to any of various embodiments or aspects of the disclosure and not to limit the disclosure to a single embodiment or object. As used in this disclosure, the terms "aspect" and "embodiment" are interchangeable. A feature discussed in the context of "some", "some" or "other" aspects or embodiments of the disclosure may be found in any embodiments of the disclosure, however, in those embodiments the feature may be considered are preferred features in these highlighted embodiments.

The descriptions and examples should not be construed to limit the scope of the disclosure to the embodiments and examples described herein, but include all modifications and substitutions falling within the true scope and spirit of the disclosure.

Synthetic degradation system for directed degradation

The present disclosure provides a synthetic degradation system for the degradation of target molecules. The system typically includes a polynucleotide encoding a degradation initiator fused to a binding element, wherein the binding element concentrates the degradation initiator to the molecule of interest to initiate degradation.

In some embodiments, the system comprises a set of polynucleotides comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide or the second polynucleotide encodes a fusion The degradation initiator to the first binding element, and the other of the first polynucleotide or the second polynucleotide encodes a synthetic or exogenous target molecule fused to the second binding element. For example, a first polynucleotide can encode a degradation initiator fused to a first binding element, and a second polynucleotide can encode a synthetic or exogenous target molecule, wherein the interaction of the first binding element and the second binding element Action Modulated Degradation Initiators concentrate to target molecules to initiate degradation. The first and second polynucleotides may be provided as a single polynucleotide or as a set of two or more polynucleotides.

In some embodiments, the system comprises a set of polynucleotides comprising a polynucleotide encoding an initiator of degradation fused to a binding element specific for a native motif on an endogenous target molecule . For example, a binding domain can recognize and bind to a specific region on a target molecule or a specific modification on a target molecule. The interaction of the binding element with the native motif on the molecule concentrates the degradation initiator to the molecule to initiate degradation.

In some embodiments, a pair of designer heterodimeric (DHD) proteins can be used as binding elements, where one DHD binding element is fused to a degradation initiator and the second DHD binding element is fused to a synthetic or exogenous target molecule fusion. Dimerization of the DHD-binding element brings the degradation initiator closer to the target molecule, thereby promoting the degradation of the target molecule.

In some embodiments, a pair of small molecule regulated polypeptides can be used as binding elements, where one binding element is fused to a degradation initiator and the second polypeptide is fused to a synthetic or exogenous protein molecule of interest. The polypeptides modulated by the first and second small molecules can be selected such that the interaction of the first and second polypeptides is modulated by the presence of the small molecules. For example, first and second small molecule modulatory polypeptides can assemble with the small molecule to form a dimeric complex. The formation of the dimerization complex brings the degradation initiator close to the target molecule, thereby promoting the degradation of the target molecule.

In some embodiments, the system comprises a set of polynucleotides comprising polynucleotides encoding a degradation initiator fused to a binding element specific for a native motif on an endogenous target molecule . For example, a binding element can recognize and bind to a specific region on a target molecule or a specific modification on a target molecule. The interaction of the binding element with the native motif on the molecule effectively concentrates the degradation initiator to the molecule of interest to initiate degradation.

In some embodiments, the present disclosure provides a synthetic degradation system for targeted degradation of a synthetic or exogenous protein of interest. The system typically includes a polynucleotide encoding an E3 ubiquitin ligase ("E3 ligase") domain (or functional variant thereof) fused to a binding element for centralizing the E3 ligase domain to a sensory protein of interest for targeted degradation through the cellular ubiquitin pathway.

In some embodiments, a pair of designed heterodimeric (DHD) proteins are used as binding elements, wherein one DHD binding element is fused to an E3 ligase domain (or a functional variant thereof) and the second DHD binding element is fused to a synthetic target fusion with native or exogenous protein of interest. Dimerization of the DHD-binding element brings the modified E3 ligase into proximity with the target protein, thereby promoting the ubiquitination and subsequent degradation of the target protein.

In some embodiments, a pair of small molecule regulatory polypeptides serve as binding elements, one of which is fused to an E3 ligase domain (or a functional variant thereof) and the second polypeptide is fused to a synthetic or exogenous protein-of-interest target fusion. The polypeptides modulated by the first and second small molecules can be selected such that the interaction of the first and second polypeptides is modulated by the presence of the small molecules. For example, first and second small molecule modulatory polypeptides can assemble with the small molecule to form a dimeric complex. The formation of the dimeric complex brings the E3 ligase into proximity with the target protein, thereby promoting the degradation of the target protein.

In some embodiments, the system comprises a set of polynucleotides comprising a polynucleotide encoding an E3 ligase domain (or a functional variant thereof) that is specific for an endogenous enzyme of interest. Binding elements fused to native motifs on sex proteins. For example, a binding element can recognize and bind a specific region on a protein of interest or a specific post-translational modification on a protein of interest. The interaction of the binding element with native motifs on the protein effectively centers the E3 ligase domain to the protein for targeted degradation.

The synthetic degradation systems of the present disclosure can be used to modulate the activity of a range of target molecules (eg, proteins) in various synthetic biology applications, such as therapeutic applications.

In some embodiments, the synthetic degradation systems of the present disclosure can be used in cell therapy applications to program cell populations to perform and/or modulate therapeutic functions.

In one aspect, the synthetic degradation systems of the present disclosure can be used to modulate (degrade) T cell receptor (TCR) and/or chimeric antigen receptor (CAR) cells for cancer therapy. More specifically, the disclosed synthetic degradation systems can be used to degrade CARs and/or TCRs and inhibit excessive CAR/TCR signaling that may lead to the undesirable exhaustion phenotype observed in many existing T cell therapies.

In one embodiment, the synthetic degradation systems of the present disclosure can be used in gene therapy applications.

binding element

The present disclosure provides a binding element designed to focus a fused degradation initiator to a molecule of interest for targeted degradation. In some embodiments, binding elements are designed to specifically and/or selectively bind to target molecules targeted for degradation. In some embodiments, binding elements can be selected based on target molecules targeted for degradation.

A de novo design strategy can be used to design a binding element or pair of binding elements for targeted degradation of a protein of interest. In some embodiments, binding elements can be designed for targeted degradation of specific protein targets. In some embodiments, binding elements can be designed for targeted degradation of various proteins. For example, binding elements can be designed that are specific to conserved motifs present in multiple proteins (eg, protein families).

In some embodiments, library-based binder panning methods can be used to identify recombinant binding elements specific for a protein of interest. For example, recombinant binding elements may be immunoglobulin derivatives or non-immunoglobulin binding agents based on natural or engineered protein scaffolds. Examples of immunoglobulin-derived conjugate formats that may be used include, but are not limited to, single chain variable fragments (scFv) and Nanobodies. Examples of non-immunoglobulin binder formats that may be used include, but are not limited to, monomers, designed ankyrin repeat protein domains (DARPins), and variable lymphocyte receptor (VLR) domains.

Designed heterodimers (DHD)

A pair of designed heterodimeric (DHD) proteins can be used as binding elements to focus a fused degradation initiator to a synthetic or exogenous molecule of interest. For example, one DHD binding element can be fused to a degradation initiator, and the second DHD binding element of the binding pair can be fused to a synthetic or exogenous molecule of interest. Dimerization of a pair of DHD-binding elements brings the degradation initiator close to the target molecule for targeted degradation.

For example, the de novo Rosetta protein design suite and previously published methods (Chen, Z. et al., Nature, (2019) doi:10.1038/s41586-018-0802-y, the entire contents of which are incorporated by reference paper) to design a pair of designed DHD binding elements. Table 1 provides the amino acid sequence of the dimerization domain, including the designed DHD sequence.

For example, a DHD binding pair can include a triple helix protein bundle binding element and a single helix protein binding element, ie a 3+1 heterodimeric binding pair. An example of a 3+1 heterodimeric binding pair is shown in FIG. 1 .

Figure 1 is a schematic diagram 100 of a pair of 3+1 heterodimeric binding elements. In this example, the binding pair includes a triple helix protein bundle DHD-A and a single helix protein DHD-B. One binding element (e.g., "A") can be fused to a molecule of interest (e.g., protein; not shown), and the second binding element of the binding pair (e.g., "B") can be fused to a degradation initiator ( For example, the E3 ligase domain; not shown) to provide a "protein-only" dimerization system for targeted degradation of molecules of interest.

A pair of DHD binding elements with alternative topologies can also serve as binding elements. Alternative topologies include, but are not limited to, dimers with one or two subunits consisting of single helical subunits, double helical subunits, multihelical subunits, or subunits with non-helical topologies.

For example, a pair of binding elements can include SYNZIP. SYNZIP is a synthetic heterodimer-binding coiled-coil that forms a diverse set of interacting junctions (Thompson, K.E., ACS Synth. Biol. (2012) 1:118-129, which is hereby incorporated by reference in its entirety).

For example, a pair of binding elements can comprise a duplex binding coiled coil (Ghosh, I. et al., J. Am. Chem. Soc. (2000) 122:5658-5659, which is hereby incorporated by reference in its entirety).

In some embodiments, surface lysine residues in the designed binding element sequence can be mutated to arginine to prevent self-ubiquitination of the binding element without altering the structure and function of the binding element. The surface residues of a designed binding element can be altered to alter charge and other properties that may affect performance levels (e.g. "surface residue" variants in Table 1). Core mutations of designed binding elements can be used to alter the binding affinity of a given heterodimer (eg DHD37-short-B-Y8A; SEQ ID NO: 14).

Small Molecule Modulatory Peptides

In some embodiments, a pair of small molecule regulated polypeptides can serve as binding elements to focus the fused degradation initiator to the molecule of interest. The use of small molecule regulatory peptides extends the functionality of the degradation system and provides an extrinsic control mechanism (ie, chemical control mechanism) for modulating the degradation of target molecules.

For example, a first small molecule modulating polypeptide can be fused to a degradation initiator and a second small molecule modulating polypeptide can be fused to a synthetic or exogenous target molecule. The first and second small molecule modulating polypeptides can be selected such that the interaction of the first and second small molecule modulating polypeptides to form the dimeric complex is mediated by the presence of the small molecule. In some cases, small molecules can mediate the assembly of dimeric complexes. In other cases, small molecules may mediate the disassembly of dimeric complexes. In other cases, a first small molecule can mediate the assembly of the dimeric complex, while a second small molecule can displace the first small molecule and thereby regulate the disassembly of the dimeric complex.

Figure 2 shows a schematic diagram 200 of an embodiment of a pair of small molecule-regulated polypeptide binding elements. One binding element (e.g., "A") can be fused to a molecule of interest (e.g., a protein; not shown), and a second binding element (e.g., "B") can be fused to a degradation initiator (e.g., an E3 ligase domain; not shown). Schematic) fusion to provide a small-molecule-regulated dimerization system for targeted degradation of target molecules.

For example, a pair of small molecule-regulated binding elements may include the hepatitis C virus protease NS3a/4a protein (hereafter referred to as NS3a) or a functional variant thereof as a first binding element, and a "reader" as a second binding element. "protein. For example, a reader protein can be selected to recognize a specific drug-bound state of the NS3a protein. NS3a proteins and NS3a reader proteins have been described in International Patent Publication No. WO2020117778, Baker et al., published June 11, 2020, entitled "Reagents and Methods for Controlling Protein Function and Interactions," which is incorporated by reference in its entirety Incorporated into this article.

NS3a can integrate multiple drug inputs and convert drug inputs into different outputs using different engineered reader proteins as binding elements. The NS3a protein and pleiotropic response outputs of the danoprevir/NS3a complex reader, danoprevir/NS3a complex reader, and ANR/NS3a complex reader have been described in Nature Biotechnology by Foight, G.W. et al. (2019) 37:1209–1216; Cunningham Bryant, D. et al., J. American Chemical Society (2019) 141:3352-3355; and Kügler, J. et al., J. Biochemistry (2012 ) 287:39224–39232, which is hereby incorporated by reference in its entirety.

Interactions between NS3a and reader binding partners may be controlled by small molecule drugs. Readers can be selected to recognize and bind specific NS3a/drug complexes.

In some embodiments, the reader selected for the dimer is a danoprevir/NS3 composite reader (DNCR) polypeptide (or a functional variant thereof), designed for use in the presence of the small molecule drug danoprevir Down recognition and binding of NS3a, thus providing a drug-induced transcription system. In some embodiments, the DNCR polypeptide is DNCR2. See Foight, G.W. et al., Nature Biotechnology, (2019) 37:1209–1216.

In some embodiments, the reader selected for the pair of binding elements is a grazoprevir/NS3 composite reader (GNCR) polypeptide (or a functional variant thereof) designed to recognize and bind NS3a in the presence of the small molecule drug grazoprevir, A drug-induced transcription system is thereby provided. In some embodiments, the GNCR protein is GNCR1. See Foight, G.W. et al., Nature Biotechnology (2019) 37:1209–1216.

native motif target-specific binding element

Endogenous molecules of interest can be degraded using degradation initiators fused to binding elements that interact with native motifs on the molecule.

In one example, binding elements can be designed to bind target molecules in a conformation-specific manner.

In one example, binding elements can be designed to correspond to specific modifications of the binding molecule on the molecule. For example, binding elements can be designed to bind a protein and/or protein complex of interest in response to a post-translational modification of the protein. In one example, binding elements can be designed to bind a specific phosphorylation state of a protein of interest.

Figure 3 shows a schematic diagram 300 of an embodiment of the modulation of an endogenous molecule of interest using a binding element ("B") specific to a native motif ("A") on the molecule of interest. An endogenous target molecule can be modulated using a binding element B specific for a native motif A on the target molecule, including conformation-specific binding or binding corresponding to a specific modification on the molecule.

Targeted degradation of proteins

The present disclosure provides a synthetic degradation system for targeted degradation of a protein of interest. The system typically comprises a polynucleotide encoding an E3 ligase domain (or a functional variant thereof) fused to a binding element for targeting the E3 ligase domain to a protein of interest to initiate degradation.

E3 ubiquitin ligase

E3 ligases control substrate specificity and ubiquitination topology. For example, in the cellular ubiquitin-proteasome system, E3 ligase proteins concentrate ubiquitin-loaded E2 ubiquitin-conjugating enzymes, recognize protein substrates, and assist or directly catalyze the transfer of ubiquitin from E2 to protein substrates, thereby targeting proteins for degradation. E3 ligase domains and/or variants thereof can be selected based on the desired protein targeted for degradation. For example, an E3 ligase and/or group of E3 ligases can be selected to modulate the stability and/or half-life of a targeted transmembrane protein. In another example, an E3 ligase and/or a group of E3 ligases can be selected to modulate the stability and/or half-life of a cytoplasmic protein (eg, a transcription factor).

A screening panel strategy can be used to identify functional E3 ligases (and functional variants thereof) for efficient degradation of a protein of interest. For example, a screening panel may include constructs encoding E3-ligase domains or ubiquitin variants fused to binding elements (eg, DHD-B or DHD-A binding elements). Examples of screening panels used to identify proteins of interest for degradation by functional E3 ligases are shown in Table 2. In this example, the set of E3 ligase domains and ubiquitin variants includes several published ubiquitin and E3 ligase domains: 3xUb, LNX1, NEDD4, VHL, SPOP, SOCS2, Elongin C, CHIP, FBW1A, FBXW7 and RFN4 (Zhu, L. et al., Elife (2017) doi: 10.7554/eLife.26403; Hatakeyama, E., et al., Cancer Res. (2005) doi: 10.1158/0008-5472.CAN-05-1581 ; Lim, S. et al, Proc. Natl. Acad. Sci. U. S. A. (2020) doi: 10.1073/pnas; 1920251117; Ibrahim, A.F.M. et al., Mol. Cell (2020) doi: 10.1016/j.molcel.2020.04. 032; and Deng, W. et al., Nat. Commun. (2020) doi: 10.1038/s41467-019-14160-8; incorporated herein by reference in its entirety). The E3 ligase domain and ubiquitin variant panel also includes several new designs containing the following domains: RNF43, RNF128, ZNRF3, MARCH8, TRAF6 and tandem ubiquitin mutants (K48R and K63R). This interface includes the design of cytoplasmic and membrane-associated DHD-E3 degraders.

Screening strategies can use, for example, cotransduction of cells with a DHD-E3 ligase construct or a DHD-ubiquitin variant construct in combination with a model protein of interest fused to the corresponding DHD binding element partner. For example, to screen for DHD-E3 ligase constructs or DHD-ubiquitin variant constructs for efficient degradation of transmembrane proteins, transmembrane receptor domains fused to the corresponding DHD binding element partners can be used. In one example, a ROR1-specific chimeric antigen receptor fused to a DHD-A binding element can be used as a model target protein to screen for degradation of transmembrane proteins.

Similarly, to screen DHD-E3 ligase constructs or DHD-ubiquitin variant constructs for efficient degradation of cytoplasmic proteins, model transcription factor domains fused to corresponding DHD-binding elements can be used. In one example, the FLAG-tagged transcription factor BACH2 fused to a DHD-B binding element serves as a model target protein for screening cytoplasmic protein degradation.

Examples of model target protein fusion constructs that can be used to screen binding element-E3 ligase constructs or binding element-ubiquitin variant constructs for efficient degradation of transmembrane or cytoplasmic proteins are shown in Table 3.

polynucleotide

The degradation system typically comprises a polynucleotide encoding an E3 ubiquitin ligase ("E3 ligase") domain (or functional variant thereof) fused to a binding element for funneling the E3 ligase domain to Target protein for targeted degradation.

heterodimer binding element

In various embodiments, the degradation system may comprise a set of polynucleotides comprising a first polynucleotide and a second polynucleotide, wherein the first or second polynucleotide encodes a polynucleotide fused to the first binding element. E3 ligase domain, and the other of the first or second polynucleotides encodes a synthetic or exogenous protein of interest fused to a corresponding second binding element.

In some embodiments, a polynucleotide set includes a polynucleotide composition, which may include:

(i) a first polynucleotide encoding a first fusion protein comprising a first binding element and an E3 ligase domain;

(ii) a second polynucleotide encoding a second fusion protein comprising a second binding element and a synthetic or exogenous protein of interest;

(iii) one or more promoter sequences operably linked to the first and/or second polynucleotide;

(iv) optionally including a separation element comprising a polynucleotide sequence that prevents fusion of the first fusion protein and the second fusion protein; and

(v) one or more optional regulatory sequences.

wherein the first and second polynucleotides, the promoter sequence, and optionally separate and regulatory elements are configured for expression and regulated degradation of the protein of interest.

Polynucleotides encoding the first and second fusion proteins, the promoter sequence and optionally separate elements and regulatory sequences can be configured in a vector backbone for expression and regulation of degradation of the protein of interest.

In various embodiments, the polynucleotide component encoding the fusion protein can include polynucleotide sequences encoding separate elements of the fusion protein.

In some embodiments, the separation element may comprise a ribosomal skipping sequence selected from P2a and T2a.

In some embodiments, the separation element can comprise a polynucleotide sequence comprising at least two ribosomal skipping sequences selected from T2a-RFP-P2a, P2a-T2a, and T2a-P2a.

In some embodiments, the separation element can include an internal ribosome entry site (IRES).

In some embodiments, the isolated element can include a second promoter sequence.

In some embodiments, the polynucleotide component comprising the first and second polynucleotides encodes one or more constitutive promoter sequences operably linked to the first and/or second polynucleotides.

In some embodiments, the polynucleotide component comprising the first and second polynucleotides encodes one or more inducible promoter sequences operably linked to the first and/or second polynucleotides.

In some embodiments, the first polynucleotide encoding the first fusion protein comprising the E3 ligase domain also encodes an inducible promoter, and the second polynucleotide encoding the second fusion protein comprising the protein of interest encodes different inducible promoters.

In some embodiments, the polynucleotide encoding the first fusion protein comprising the E3 ligase domain encodes an inducible promoter sequence and the polynucleotide encoding the second fusion protein comprising the protein of interest encodes a constitutive promoter sequence.

In various embodiments, the constitutive promoter sequence may comprise a constitutive promoter sequence selected from the group consisting of: MND (SEQ ID NO: 144), hPGK (SEQ ID NO : 145), CMV (SEQ ID NO: 146), CAG (SEQ ID NO: 147) ), SFFV (SEQ ID NO: 148), EF1alpha (SEQ ID NO: 149), UBC (SEQ ID NO: 150) and CD43 (SEQ ID NO: 151) (see Table 8).

In some embodiments, an inducible promoter sequence can include a minimal promoter sequence, such as a minimal core promoter. In some embodiments, the minimal promoter sequence is selected from the group consisting of: YB_TATA (SEQ ID NO: 152), human beta globulin (huBG) (SEQ ID NO: 153), minIL2 (SEQ ID NO: 154), minimalCMV ( minCMV) (SEQ ID NO: 155), and TRE3G (SEQ ID NO: 156) (see Table 9).

In some embodiments, the inducible promoter sequence includes a transcription factor specific recognition sequence.

In some embodiments, a transcription factor-specific recognition sequence can include a response element repeated 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times.

In some embodiments, a transcription factor-specific recognition sequence can include a response element selected to respond to a cell-intrinsic stimulus.

In some embodiments, the transcription factor-specific recognition sequence may include a calcium-responsive NFAT-AP1 response element (Hooijberg, E., et al., Blood Journal, (2000) 96:459-466, which is incorporated herein by reference in its entirety ).

In certain embodiments, the inducible promoter sequence includes a minimal CMV (minCMV) promoter sequence and a NFAT-AP1 response element (SEQ ID NO: 143) repeated 4 times (see Table 7).

In some embodiments, transcription factor-specific recognition sequences include response elements selected to respond to environmental stimuli.

In some embodiments, the first and/or second polynucleotide may encode one or more optional regulatory sequences selected from the group consisting of polyA, endo.

In some embodiments, the polynucleotide encoding the first and/or second fusion protein further includes a protein tag sequence (eg, FLAG-tag sequence).

In some embodiments, the first or second polynucleotide encoding the E3 ligase domain and the first binding element further comprises a flexible linker sequence separating the E3 ligase domain and the binding element. In one example, the flexible linker is a glycine-serine linker.

In some embodiments, the first or second polynucleotide encoding the protein of interest and the second binding element further comprises a flexible linker sequence separating the protein of interest and the binding element. In one embodiment, the flexible linker is a glycine-serine linker.

In some embodiments, the first or second polynucleotide encodes a DHD-A binding element and the other of the first or second polynucleotide encodes a DHD-B binding element, wherein the DHD binding pair is selected from In the group formed: DHD37-short-A (SEQ ID NO: 11) and DHD37-short-B (SEQ ID NO: 13), DHD37-short-B-Ntrunc (SEQ ID NO: 13) NO: 15 ), or DHD37-short-B-KtoR (SEQ ID NO: 16).

In some embodiments, the first or second polynucleotide encodes a binding element selected from the group consisting of Synzip1 (SEQ ID NO: 66), Synzip2 (SEQ ID NO: 67), Synzip3 (SEQ ID NO: 68), Synzip4 (SEQ ID NO: 69), Synzip5 (SEQ ID NO: 70), Synzip6 (SEQ ID NO: 71), Coil-coil-CZ-A (SEQ ID NO: 72) or Coil-coil-NZ-B (SEQ ID NO: 73).

In various embodiments, the first or second polynucleotide encodes a fusion protein comprising a binding element and an E3 ligase domain, which may comprise: ^RNF43198-783 -DHD37-short-B (SEQ ID NO: 74 ), RNF43 ^198-364 -DHD37-short-B (SEQ ID NO: 75), RNF43 ^195-325 -DHD37-short-B (SEQ ID NO: 76), RNF43 ^198-317 -DHD37-short-B (SEQ ID NO: 76) ID NO: 77), RNF128 ^208-428 -DHD37-short-B (SEQ ID NO: 78), RNF128 ^208-333 -DHD37-short-B (SEQ ID NO: 79), RNF128 ^208-325 -DHD37-short -B (SEQ ID NO: 80), DHD37-short-B-3xUb (SEQ ID NO: 81), DHD37-short-B-3xUb ^K48R (SEQ ID NO: 82), DHD37-short-B-3xUb ^K63R ( SEQ ID NO: 83), ZNRF3 ^219-936 -DHD37-short-B (SEQ ID NO: 84), ZNRF3219-528-DHD37-short-B (SEQ ID NO: 85), ZNRF3 ^219-345 -DHD37-short -B (SEQ ID NO: 86), DHD37-short-B-MARCH8 ^1-154 -KLRG1 ^37-60 (SEQ ID NO: 87), DHD37-short-B-MARCH8 ^1-228 (SEQ ID NO: 88) , MARCH8 ^1-228 -DHD37-short-B (SEQ ID NO: 89), LNX1 ^1-171 -DHD37-short-B (SEQ ID NO: 90), DHD37-short-B-VHL ^152-213 (SEQ ID NO: 91), DHD37-short-B- ^{NEDD4 921-1303} (SEQ ID NO: 92), DHD37-short-B-SPOP ^167-374 (SEQ ID NO: 93), DHD37-short-B-SOCS2 ^{143- 198} (SEQ ID NO: 94), DHD37-short-B-CHIP ^128-303 ( SEQ ID NO: 95), FBXW7 ^1-293 -DHD37-short-B (SEQ ID NO: 96), ELOC ^1-87 -DHD37-short-B (SEQ ID NO: 97), FBW1A ^1-261- DHD37- short-B (SEQ ID NO: 98), DHD37-short-B-RNF4 ^71-190 (SEQ ID NO: 99), TRAF6 ^49-237 -DHD37-short-B (SEQ ID NO: 100), LNX1 ^{1- 171} -DHD37-short-A (SEQ ID NO: 101), DHD37-short-A-VHL ^152-213 (SEQ ID NO: 102), DHD37-short-A- ^{NEDD4 921-1303} (SEQ ID NO: 103) , DHD37-short-A-SPOP ^167-374 (SEQ ID NO: 104), DHD37-short-A-SOCS2 ^143-198 (SEQ ID NO: 105), DHD37-short-A-CHIP ^128-303 (SEQ ID NO: 106), FBXW7 ^1-293 -DHD37-short-A (SEQ ID NO: 107), ELOC ^1-87 -DHD37-short-A (SEQ ID NO: 108), FBW1A ^1-261 -DHD37-short-A A (SEQ ID NO: 109), DHD37-short-A- ^RNF471-190 (SEQ ID NO: 110), Synzip2-RNF4 (SEQ ID NO: 111), Synzip4-RNF4 (SEQ ID NO: 112), Synzip6 - RNF4 (SEQ ID NO: 113), Coil-coil-NZ-RNF4 (SEQ ID NO: 114), RNF43 ^198-317 - DHD37-short-B-KtoR (SEQ ID NO: 121), RNF43 ^198-317 - DHD37-short-B-endo-var1 (SEQ ID NO: 122), RNF43 ^198-317 -DHD37-short-B-endo-var2 (SEQ ID NO: 123), RNF43 ^198-317 -DHD37-short-B- endo-var3 (SEQ ID NO: 124), or RNF43 ^198-317 -KtoR-DHD37-short-B-endo-var1 (SEQ ID NO: 125). See Table 2 and Table 4.

In various embodiments, the first or second polynucleotide encodes a fusion protein comprising a binding element and a target protein of interest.

In some embodiments, the first or second polynucleotide encodes a fusion protein comprising a binding element and a ROR1-CAR protein, which may include: ROR1-CAR-DHD37-short-A (SEQ ID NO: 115), ROR1 CAR-Synzip1 (SEQ ID NO: 116), ROR1 CAR-Synzip3 (SEQ ID NO: 117), ROR1 CAR-Synzip5 (SEQ ID NO: 118), or ROR1 CAR-coil sequence-CZ (SEQ ID NO: 119) . See Table 2.

In certain embodiments, the first or second polynucleotide encodes a fusion protein comprising a DHD polypeptide and a BACH2 polypeptide (SEQ ID NO: 120). See Table 2.

Small Molecule Regulatory Polynucleotides

A pair of small molecule regulated polypeptides can be used as binding elements to focus a fused E3 ligase domain (or a functional variant thereof) to a protein of interest.

For example, a degradation system can include a set of polynucleotides comprising a first polynucleotide and a second polynucleotide, either the first or second polynucleotide encoding a protein fused to a first small molecule modulating polypeptide. E3 ligase domain, and the other of the first or second polynucleotides encodes a synthetic or exogenous protein of interest fused to a corresponding second small molecule regulatory polypeptide.

In various embodiments, the first or second polynucleotide may encode a binding element comprising NS3a (or a functional variant thereof), and the other of the first or second polynucleotide may encode a binding element selected from the group consisting of DNCR2. (or its modification) and the binding element (or its modification) in the group consisting of GNCR1.

In some embodiments, the first or second polynucleotide encodes a binding element, which may comprise a NS3a polypeptide, comprising: NS3a.

In certain embodiments, the first or second polynucleotide encodes a ROR1-CAR-NS3a fusion protein comprising a ROR1-CAR and NS3a binding element (SEQ ID NO: 126). See Table 5.

In some embodiments, the first or second polynucleotide encodes a binding element that may comprise a DNCR2 polypeptide comprising: DNCR2.

In some embodiments, the first or second polynucleotide encodes a binding element which may comprise a GNCR1 polypeptide comprising: GNCR1.

In some embodiments, the first or second polynucleotide encodes a fusion protein comprising a DNCR2 polypeptide and an E3 ligase domain, which may include: RNF43 ^198-317- DNCR (SEQ ID NO: 127), DNCR-RNF4 ^71-190 (SEQ ID NO: 128), LNX1 ^1-171- DNCR (SEQ ID NO: 129), RNF43 ^198-317 -DNCR-endo (SEQ ID NO: 130), RNF43 ^198-317 -DNCR-KtoR ( SEQ ID NO: 131), or RNF43 ^198-317 -DNCR-KtoR-endo (SEQ ID NO: 132). See Table 5.

native motif peptide

In various embodiments, a polynucleotide encoding a fusion protein includes a binding element specific for a native motif on an endogenous protein of interest and an E3 ligase domain (or a functional variant thereof).

In some embodiments, the polynucleotide can encode a binding element comprising a domain specific for a region of a transmembrane protein and/or protein complex.

In one embodiment, the binding element comprises a domain targeting the transmembrane protein and/or the intracellular domain of the protein complex.

In one embodiment, the binding element comprises a domain targeting the transmembrane domain of a transmembrane protein and/or protein complex.

In one embodiment, the transmembrane protein is a T cell receptor (TCR) complex.

In one embodiment, the polynucleotide may encode a binding element comprising a domain specific for a transmembrane domain of a TCR complex selected from ^TMCD3z .

In some embodiments, the polynucleotide can encode a binding element comprising a domain specific for a protein-protein interaction domain on an endogenous protein of interest. Examples of protein-protein interaction domains include, but are not limited to, pleckstrin homology (PH) domains, Src homology 2 domains (SH2) and Src homology 3 domains (SH3).

In some embodiments, a polynucleotide may encode a binding element comprising a domain as a single-chain variable fragment (scFV) that recognizes and binds a specific region of a protein of interest.

In some embodiments, a polynucleotide may encode a binding element, comprising a single monomeric variable antibody domain or "nanobody" that recognizes and binds a specific region of a protein of interest.

In some embodiments, the polynucleotide can encode a binding element comprising a monomer that recognizes and binds a specific region of a protein of interest. Monomer is a synthetic binding protein constructed using the fibronectin type III domain (FN3) as a molecular scaffold.

In some embodiments, the polynucleotide can encode a binding element, including a designed ankyrin repeat protein domain (DARPin), that recognizes and binds a specific region of a protein of interest.

In some embodiments, the polynucleotide can encode a binding element comprising a variable lymphocyte receptor (VLR) domain that recognizes and binds a specific region of a protein of interest.

In some embodiments, the polynucleotide encodes a fusion protein comprising a binding element and an E3 ligase domain, which may include: TM ^CD3z -RNF43 ^226-317 (v1) (SEQ ID NO: 133), TM ^CD3z-K54 R-RNF43 ^226-317 (v2) (SEQ ID NO: 134), TM ^CD3z -RNF43 ^236-317 (v3) (SEQ ID NO: 135), TM ^CD3z-K54R- RNF43 ^236-317 (v4) (SEQ ID NO: 136), ZAP70 ^nSH2-cSH2- RNF4 ^71-190 (SEQ ID NO; 137), ZAP70 ^cSH2 -RNF4 ^71-190 (SEQ ID NO: 138), LNX1 ^1-171 -ZAP70 ^nSH2-cSH2 (SEQ ID NO : 139), LNX1 ^1-171- ZAP70 ^cSH2 (SEQ ID NO: 140), NCK1 ^SH3.1 -RNF4 ^71-19 (SEQ ID NO: 141), or LNX1 ^1-171 -NCK1 ^SH3.1 (SEQ ID NO : 142). See Table 6.

target protein

The synthetic degradation systems disclosed herein can be used to modulate the activity of a range of target molecules. The combination of binding elements and degradation initiators (eg, E3 ligase domains) used in the degradation system can be selected based on the target protein for degradation. The target protein can be a synthetic protein or an exogenous protein. The protein of interest can be an endogenous protein and/or protein complex.

The degradation systems of the present disclosure can be used to provide constitutive regulation of a target protein of interest.

The degradation systems of the present disclosure can be used to provide dynamic regulation of a target protein of interest in a context-sensitive manner. For example, a degradation system can be an inducible system that can be "turned off" at one point in the cellular process, where the function of the target protein is required, and "turned on" at another point in the cellular process, where the function of the target protein is not required .

The degradation systems of the present disclosure can be used to regulate multiple proteins simultaneously. For example, a targeted degradation system can use a degradation initiator and a single binding element that recognizes and binds a conserved domain present in multiple proteins (eg, protein family members). For example, a binding element may be a design binding element with mixed target properties.

Proteins that can be targeted for degradation include, but are not limited to, metabolic enzymes such as those involved in glycolysis, oxidative phosphorylation, and fatty acid metabolism; enzymes involved in cellular signaling processes; transcription factors; scaffold and adapter proteins; and mitochondrial proteins.

A domain and/or motif in a protein can be used to target the protein for degradation.

Certain post-transcriptional modifications to proteins and/or protein domains can be used to target proteins for degradation.

transmembrane receptor

The degradation systems of the present disclosure can be used for the targeted degradation of integral (intrinsic) membrane proteins such as transmembrane receptors. Examples of transmembrane receptors that can be targeted for degradation include, but are not limited to, chimeric antigen receptors (CARs), T cell receptors (TCRs), receptor tyrosine kinases (RTKs), Notch, Notch-like receptors, growth Factor family receptors, receptor serine/threonine kinases, G protein-coupled receptors (GPCRs), inhibitory receptors such as PD-1, TIGIT, LAG3, TGFβ, FAS, and cytokine receptors.

Examples of receptor tyrosine kinases (RTKs) that can be targeted for degradation include, but are not limited to, epidermal growth factor receptor (EGFR), ErbB, ERBB2 (HER2), fibroblast growth factor receptor (FGFR), insulin-like growth Factor 1 receptor (IGF1R) and vascular endothelial growth factor receptor (VEGFR).

In some embodiments, the transmembrane protein targeted for degradation is T cell receptor (TCR).

In some embodiments, the transmembrane protein targeted for degradation is a chimeric antigen receptor (CAR). In one example, the CAR protein targeted for degradation is the ROR1-specific chimeric antigen receptor protein (ROR1-CAR).

In some embodiments, the cytokine receptor IL-7R may be targeted for degradation. Constitutive IL-7 receptor signaling has been shown to promote T cell proliferation and persistent killing of tumor cells (Shum et al., (20017) and Peng (2017), which are hereby incorporated by reference in their entireties).

transcription factor

The degradation systems of the present disclosure can be used for targeted degradation of transcription factors. Examples of transcription factors (TFs) that can be targeted for degradation include, but are not limited to, TFs that regulate T cell receptor signaling, T cell exhaustion, T cell memory formation, T cell differentiation; TFs that regulate stem cell differentiation and/or stem cells; bzip TFs in series; TOX, TOX2, NR4A1, NR4A2, NR4A3, IRF4, BATF, XBP1, c-Jun, Fos, AP1, Bach2, Tcf1/Tcf7, FoxP1 and FoxP3.

In some embodiments, the NR4A family of transcription factors NR4A1 (Nur77), NR4A2, and NR4A3 can be targeted for degradation using binding elements that recognize and bind all three family members. The NR4A transcription factor family has been shown to play a role in T cell exhaustion and knockout (or reduced) expression of three NR4A transcription factors is expected to alleviate exhaustion and improve T cell efficacy (Chen et al., 2019, the entire contents of which are incorporated herein by reference ).

In some embodiments, the transcription factors TOX and/or TOX2 can be degraded in a context-dependent manner (i.e., dynamically regulated). TOX and TOX2 have been shown to play a role in T cell exhaustion, but cannot be completely knocked out due to their important roles in T cell memory formation (Sekine et al., (2020), Seo et al., (2019), and Bordon et al. (2019), the entire contents of which are incorporated herein by reference).

In some embodiments, the transcription factor IRF4 may be targeted for degradation. IRF4 (Man et al., (2017), which is hereby incorporated by reference in its entirety).

In some embodiments, the transcription factor BATF may be targeted for degradation. BATF (Kurachi et al., (2014), the entire contents of which are incorporated herein by reference).

In some embodiments, the transcription factor XBP1 may be targeted for degradation. XBP1 (Song et al., (2018); American Association for Cancer Research, which is hereby incorporated by reference in its entirety).

In some embodiments, the transcription factor c-Jun may be targeted for degradation. c-Jun (Lynn et al., (2019), which is incorporated by reference in its entirety)

In some embodiments, the transcription factor Bach2 may be targeted for degradation. Bach2 (Utzschneider et al., (2020); Sidwell et al., (2020); Roychoudhuri et al., (2016); and Richer et al., (2016), incorporated herein by reference in their entirety).

Cytoplasmic enzymes and enzyme regulators

Proteins used for degradation may be cytoplasmic enzymes or regulators of enzymatic activity. Examples of cytoplasmic enzymes and/or their modulators that can be targeted for degradation include, but are not limited to, non-receptor tyrosine kinases (nRTKs), non-receptor serine/threonine kinases, phosphoinositide kinases, phosphatases, and E3 ubiquitin kinases. prime ligase.

Non-receptor tyrosine kinases that can be targeted for degradation include, but are not limited to, Src family tyrosine kinases such as Lck, Src, Fyn, Yes, Fgr, Hck, Blk, and Lyn; Tec family tyrosine kinases such as Tec, Itk, Btk, Rlk, and Bmx; C-terminal Src kinase (CSK) and Zap70.

In some embodiments, the non-receptor tyrosine kinase Lck may be targeted for degradation. Lck has been shown to be critical for initiating proximal T cell signaling (Salmond et al., (2009), which is hereby incorporated by reference in its entirety).

In some embodiments, the non-receptor tyrosine kinase Fyn may be targeted for degradation. Fyn has been shown to be critical for initiating proximal T cell signaling and CAR T cell signaling (Salmond et al., (2009), Salter et al., (2018), Bommhardt et al., (2019), Suryadevara et al., (2019) and Hartl et al., (2020), which are incorporated by reference in their entirety).

In some embodiments, the non-receptor tyrosine kinase Itk may be targeted for degradation. (Andreotti et al., (2010) and Andreotti et al., (2018), which are hereby incorporated by reference in their entirety).

In some embodiments, the non-receptor tyrosine kinase CSK may be targeted for degradation. (Okada, (2012), the entire contents of which are incorporated herein by reference).

In some embodiments, the non-receptor tyrosine kinase Zap70 may be targeted for degradation. (Yokosuka et al., (2005) and Wang et al., (2010)), which are hereby incorporated by reference in their entirety).

Non-receptor serine/threonine kinases that can be targeted for degradation include, but are not limited to, mitogen-activated protein kinase (MAPK), MAPK kinase (MAPKK), MAPKK kinase (MAPKKK), calcium-dependent kinase, cyclin-dependent Kinases such as CDK1 and CDK2.

Phosphoinositide kinases that can be targeted for degradation include, but are not limited to, phosphoinositide 3-kinase (PI3K or PIK3), PI3Ka p85, PI3Ka p110, and phosphoinositide 5-kinase (PI5K or PIK5).

Phosphatases that can be targeted for degradation include, but are not limited to, PTEN, SHP1, SHP2, and PTPN2/TCPTP.

In some embodiments, the phosphatase PTEN may be targeted for degradation. (Newton and Turka, (2012); Lin et al., (2021); Amaria et al., (2016); Cheng et al., (2019); and Locke et al., (2013), the entire contents of which are incorporated by reference. into this article).

In some embodiments, the phosphatase SHP1 may be targeted for degradation. (Johnson et al., (2013); and Brockdorff et al., (1999), the entire contents of which are incorporated herein by reference).

In some embodiments, the phosphatase SHP2 may be targeted for degradation. (Yokosuka et al., (2012); and Strazza et al., (2021), the entire contents of which are incorporated herein by reference).

In some embodiments, the phosphatase PTPN2/TCPTP may be targeted for degradation. PTPN2/TCPTP (LaFleur et al., (2019); and Wiede et al., (2020), which are hereby incorporated by reference in their entirety).

E3 ubiquitin ligase proteins and/or components of E3 ubiquitin ligase complexes that can be targeted for degradation include, but are not limited to, von Hippel-Lindau protein (VHL), cytokine-induced SH2 protein (CISH), Cbl-b , c-Cbl, inhibitor of cytokine signaling (SOCS) 1, SOCS2, SOCS3, SOCS4, SOCS5, SOCS6, SOCS7, Elongin B, Elongin C, and Elongin BC heterodimers.

In some embodiments, the E3 ubiquitin ligase complex protein VHL may be targeted for degradation. VHL has been shown to regulate hypoxia-inducible factors that regulate T cell differentiation and function (Cardote et al., (20177); Stebbins et al., (1999); Velixa et al., (2021); and McNamee et al., (2013) , which is incorporated herein by reference in its entirety).

In some embodiments, the E3 ubiquitin ligase complex protein CISH may be targeted for degradation. CISH has been shown to downregulate T cell signaling by targeting PLCγ (and possibly other targets). Loss of CISH in T cells increases T cell activity and cytotoxicity (Palmer et al., (2015); Palmer et al., (2014); and Guittard et al., (2018), which are hereby incorporated by reference in their entirety).

In some embodiments, the E3 ubiquitin ligase complex proteins Cbl-b and/or c-Cbl may be targeted for degradation. Cbl-b and c-Cbl (Nguyen et al., (2021); Lutz-Nicoladoni et al., (2015); and Tang et al., (2019), which are hereby incorporated by reference in their entirety).

In some embodiments, the E3 ubiquitin ligase complex protein SOCS1 may be targeted for degradation. SOCS1 (Takahashi et al., (2011); Takahashi et al., (2017); and Tamiya et al., (2011), which are incorporated herein by reference in their entirety).

In some embodiments, the E3 ubiquitin ligase complex protein SOCS2 may be targeted for degradation. SOCS2 (Tannahill et al., (2005); Greenhalgh et al., (2005); and Knosp et al., (2011), all of which are incorporated herein by reference).

In some embodiments, the E3 ubiquitin ligase complex protein SOCS3 may be targeted for degradation. (Croker et al., (2003); and Chen et al., (2006), which are incorporated herein by reference in their entirety).

In some embodiments, the E3 ubiquitin ligase complex protein SOCS4 may be targeted for degradation. (Bullock et al., (2007), the entire contents of which are incorporated herein by reference).

In some embodiments, the E3 ubiquitin ligase complex protein SOCS5 may be targeted for degradation. (Zhang et al., (2009), the entire contents of which are incorporated herein by reference).

In some embodiments, the E3 ubiquitin ligase complex protein SOCS6 may be targeted for degradation. (Choi et al., (2010), the entire contents of which are incorporated herein by reference).

In some embodiments, the E3 ubiquitin ligase complex protein SOCS7 may be targeted for degradation. (Bondar et al. (2018), which is incorporated by reference in its entirety).

In some embodiments, E3 ubiquitin ligase complex proteins Elongin B, elongin C and/or Elongin BC heterodimers can be targeted for degradation. Elongin B, Elongin C, and Elongin BC heterodimers have been shown to play key roles in regulating Cullin-Ring ubiquitin ligases (Okumura et al., (2012), which is hereby incorporated by reference in its entirety).

signal protein

The protein targeted for degradation may be a signaling protein. Examples of signaling proteins that can be targeted for degradation include, but are not limited to, CIN85, PLCγ1, PLCγ2, Smad2, Smad3, and Smad4.

In some embodiments, the signaling protein PLCγ1 may be targeted for degradation. (Su et al., (2016); Bilal and Houtman (2015); and Braiman et al., (2006), all of which are incorporated herein by reference).

In some embodiments, the signaling proteins Smad2 and/or Smad3 may be targeted for degradation. (Yang et al., (1999); Takimoto et al., (2010); Malhotra and Kang (2013); and Kashiwagi et al., (2015), the entire contents of which are incorporated herein by reference).

Scaffolds and adapter proteins

Proteins targeted for degradation can be scaffold proteins or adapter proteins. Examples of scaffolds and adapter proteins that can be targeted for degradation include, but are not limited to, SLP76, LAT, Grb2, GADS, Ste5p, KDS, HOP, PSD95, Cullin-Skpl, RACK1, and ADAP.

In some embodiments, the adapter protein LAT may be targeted for degradation. (Zhang et al., (1998); Williamson et al., (2011); and Liu et al., (1999), all of which are incorporated herein by reference).

In some embodiments, the adapter protein SLP76 may be targeted for degradation. (Yokosuka et al., (2005), which is hereby incorporated by reference in its entirety).

In some embodiments, the adapter protein Grb2 may be targeted for degradation. (Zhang et al., (2000); Gong et al., (2001); and Bilal and Houtman (2015), all of which are incorporated herein by reference).

In some embodiments, adapter protein Gads may be targeted for degradation. (Liu et al., (1999); Zhang et al., (2000); and Yoder et al., (2001), all of which are incorporated herein by reference).

protein domains as targets

Functional and/or structural domains of proteins can be used to target proteins for degradation. Examples of protein domains that can be used to target proteins for degradation include, but are not limited to, signaling domains, regulatory domains, lipid membrane binding domains, phospholipid binding domains, and DNA binding domains. Specific examples of regulatory and signaling domains that can be targeted include, but are not limited to, Src homology 2 (SH2), src homology 3 (SH3), pleckstrin homology (PH), dbl homology (DH), DHPH, Bcl-2 homology (BH), PDZ domain, PDZ binding and C2 domain. Specific examples of lipid membrane binding domains that can be targeted include, but are not limited to, DHPH, PH, and C2. Specific examples of targetable phospholipid binding domains include, but are not limited to, PH, DHPH, C2, PI(3,4,5)P3 binding, PI(3,4)P2 binding, and PI(4,5)P2. Specific examples of DNA binding domains that can be targeted include, but are not limited to, zinc fingers (ZFs), TALEs, Cas9, and Cas12a.

SOCS-box pattern

SOCS-box motifs can be used to target SOCS-box containing proteins for degradation. Examples of SOCS-box-containing proteins include, but are not limited to, suppressor of cytokines (SOC) family proteins, VHL, and CISH.

chimeric antigen receptor

Target proteins of interest may include chimeric antigen receptors (CARs). A CAR may be a fusion protein comprising an extracellular antigen binding/recognition element, a transmembrane element that anchors the receptor to the cell membrane, and at least one intracellular element. These CAR elements are known in the art, for example, as described in patent application US20140242701 published on August 28, 2014, entitled "Chimeric Antigen Receptor", which is incorporated herein by reference in its entirety. A CAR may be a recombinant polypeptide expressed from a polynucleotide comprising at least an extracellular antigen binding element, a transmembrane element, and an intracellular signaling element, including a functional signaling element derived from a stimulatory molecule.

Stimulatory molecules can be, for example, zeta chains associated with T cell receptor complexes.

A cytoplasmic signaling element may, for example, comprise one or more functional signaling elements derived from at least one co-stimulatory molecule.

Costimulatory molecules may eg be selected from 4-1BB (ie CD137), CD27 and/or CD28.

A CAR may be a chimeric fusion protein that includes an extracellular antigen recognition element, a transmembrane element, and an intracellular signaling element, including a functional signaling element derived from a stimulatory molecule.

A CAR may comprise a chimeric fusion protein comprising an extracellular antigen recognition element, a transmembrane element, and an intracellular signaling element, including a functional signaling element derived from a co-stimulatory molecule and a functional signaling element derived from a stimulatory molecule .

CAR can be a chimeric fusion protein, including an extracellular antigen recognition element, a transmembrane element, and an intracellular signaling element. The intracellular signaling element includes two functional signaling elements derived from one or more co-stimulatory molecules and a stimulatory molecule. Functional signal element.

A CAR may comprise a chimeric fusion protein comprising an extracellular antigen recognition element, a transmembrane element, and an intracellular signaling element comprising at least two functional signaling elements derived from one or more co-stimulatory molecules and one derived from a stimulatory molecule. The functional signal element.

The CAR may contain an optional leader sequence at the amino terminus (N-terminus) of the CAR fusion protein. The CAR may also include a leader sequence at the N-terminus of the extracellular antigen recognition element, wherein the leader sequence is optionally cleaved from the antigen recognition element (eg, single-chain antibody) during cellular processing and localization of the CAR to the cell membrane.

Choice of degradation system

The combination of binding elements and degradation initiators (eg, E3 ligase domains) used in the degradation system can be selected based on the target protein being degraded.

In some embodiments, a pair of DHD binding elements are used for targeted degradation of synthetic or exogenous transmembrane proteins. FIG. 4A is a schematic diagram 400 of an example of using 3+1 DHD pairs as binding elements for targeted degradation of transmembrane proteins. The degradation-targeted transmembrane protein ("TMP") is fused to one partner of the binding element pair (DHD-B) and the E3 ligase domain is fused to the second binding element partner of the binding element pair (DHD-A). The interaction of DHD-A and DHD-B binding partners brings the E3 ligase domain into proximity with the target protein, thereby promoting the ubiquitination of transmembrane proteins. Ubiquitination of transmembrane proteins facilitates subsequent receptor internalization, endosomal trafficking, and lysosomal degradation of transmembrane proteins.

In some embodiments, a pair of DHD binding elements is used to target degradation of synthetic or exogenous cytoplasmic proteins, such as transcription factors. Figure 4B is a schematic diagram 410 of an example of using 3+1 DHD pairs as binding elements for targeted degradation of cytoplasmic proteins. Cytoplasmic proteins targeted for degradation ("targets") are fused to one partner of the binding element pair (DHD-B), and the E3 ligase domain is fused to the second binding element partner of the binding element pair (DHD-A). The interaction of the DHD-A and DHD-B targeting domains of the fusion protein brings the E3 ligase domain into proximity with the target protein, thereby promoting the ubiquitination of the cytoplasmic protein target. Polyubiquitination ("Ub") of target proteins is induced upon interaction with DHD-E3 ligase and promotes downstream proteasomal degradation.

In some embodiments, small molecule regulated degradation systems are used for targeted degradation of transmembrane receptors. In one example, the transmembrane receptor is a chimeric receptor, such as a CAR. 5 is a schematic diagram 500 of an embodiment of a small molecule-mediated degradation system for degrading a chimeric receptor. In this example, a chimeric receptor targeting degradation is fused to the NS3a polypeptide and an E3 ligase domain is fused to the DNCR2 polypeptide. In the presence of the small molecule danoprevir, DNCR2 and NS3a polypeptides assembled with the small molecule danoprevir to form a dimeric complex. Formation of the dimeric complex brings the E3 ligase into proximity with the targeted chimeric receptor, thereby promoting ubiquitination (“Ub”) and subsequent degradation of the targeted receptor.

In some embodiments, a binding element can target a native motif on an endogenous protein of interest. In one example, the binding element can be a protein domain that interacts with a native motif on the intracellular region of the transmembrane protein of interest. In yet another example, a binding element can be a protein domain that interacts with a native motif on the transmembrane domain of a transmembrane protein of interest. The interaction of the binding element and native motifs on the target protein brings the E3 ligase into proximity with the target protein, thereby promoting the ubiquitination and subsequent degradation of the target protein.

Figure 6A is a schematic diagram 600 illustrating transmembrane receptor domains as binding elements for targeted degradation of chimeric transmembrane receptors. In this example, an E3 ligase domain is fused to a receptor-targeting transmembrane domain designed to bind a native transmembrane motif on a chimeric receptor. The interaction of the receptor-targeting transmembrane domain and the chimeric receptor brings the E3 ligase domain into proximity with the targeted receptor, thereby promoting ubiquitination of the chimeric receptor. Ubiquitination of chimeric receptors promotes subsequent lysosomal trafficking and receptor degradation.

In some embodiments, a binding element can be a protein domain that interacts with a specific post-translational modification on a protein of interest. For example, a binding element can interact with a post-translationally phosphorylated region of a target protein.

In one embodiment, the synthetic degradation system of the present disclosure uses a phosphotyrosine binding domain (PYBD) as a binding element to recruit an E3 ligase domain to a transmembrane receptor for degradation. Figure 6B is a schematic diagram 610 illustrating phosphotyrosine binding domains (PYBDs) as binders for targeting degradation of post-translational phosphorylated ("P") transmembrane receptors. Interaction of the PYBD domain with the phosphorylated domain of the transmembrane receptor brings the E3 ligase domain close to the target receptor, thereby promoting ubiquitination of the chimeric receptor. Ubiquitination of receptors facilitates subsequent lysosomal trafficking and receptor degradation.

In one specific example, binding elements can be used to target the endogenous T cell receptor (TCR) complex. For example, a construct encoding the CD3𝜁 transmembrane domain (TM ^CD3z ), which is used to recognize and disrupt the TCR complex, can be used to target an E3 ligase domain (such as ^RNF43 ) to the TCR complex The E3 ligase domain serves to facilitate receptor degradation, thus providing two mechanisms for modulating the activity of the TCR complex.

Carriers and Carrier Configurations

The polynucleotides of the disclosure can be provided as part of a vector. Examples of suitable vectors include expression vectors, viral vectors and plasmid vectors. Expression vectors may include plasmids, phagemids, viruses and derivatives thereof. In some aspects, polynucleotides of the disclosure can be provided as part of a homology directed repair vector.

In some aspects, a viral vector can include a polynucleotide encoding a gene editing polypeptide, such as a polypeptide useful in practicing gene editing techniques. Examples of such gene editing technologies include RNA/DNA-guided endonucleases (e.g., CRISPR (clustered regularly interspaced short palindromic repeats)), TALENs (transcriptional finger nuclease), recombinase, meganuclease or viral integration .

In some aspects, polynucleotides of the disclosure can be provided as part of a homology directed repair (HDR) vector. Homology-directed repair mechanisms can be used to integrate sets of polynucleotides into chromosomes. Examples of mechanisms that can be used to integrate sets of polynucleotides into chromosomes include sequence-specific nucleases such as transposases, CRISPR/Cas9, ZF nucleases, TALE nucleases, recombinases, and other homologs known in the art Recombinant targeting vector.

Vector components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Vectors for use in eukaryotic host cells may also encode signal sequences or other polypeptides with specific cleavage sites at the N-terminus of the mature protein or polypeptide of interest. The signal sequence selected is preferably one that is recognized and processed (ie, cleaved by a signal peptidase) by the host cell. In mammalian cell expression, mammalian signal sequences as well as viral secretory leaders can be used. Expression vectors for use in eukaryotic host cells also typically contain sequences necessary to terminate transcription and stabilize the mRNA. Such sequences are usually available from the 5' and occasionally 3' untranslated regions of eukaryotic or viral DNA or cDNA. One useful transcription termination element is the bovine growth hormone polyadenylation region.

Expression and cloning vectors can contain a selectable gene, also called a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies where relevant, or (c) Provides key nutrients not available from complex media.

The polynucleotides of the disclosure can in some cases be provided as part of a single vector. The polynucleotides of the present disclosure may be provided as part of a set of at least two vectors; a first vector comprising the first polynucleotide and a second vector comprising the second polynucleotide.

Examples of vectors suitable for use with polynucleotides of the present disclosure include adenoviral vectors, lentiviral vectors, baculoviral vectors, Epstein Barr viral vectors, polyvacuum viral vectors, vaccinia viral vectors, herpes simplex viral vectors, adeno-associated virus ( AAV) vectors and transposon vectors. The polynucleotides of the disclosure can be provided as part of a homology directed repair vector.

The disclosure provides a set of polynucleotides comprising, as part of one or more vectors: (i) a first polynucleotide encoding a first fusion protein comprising a first binding element; and (ii) a second polynucleotide encoding a second fusion protein comprising a corresponding second binding element; wherein: (iii) the first or second fusion protein also includes a degradation initiator; and (iv) the other of the first or second fusion protein also includes a target molecule; and (v) The interaction of the first and second elements effectively concentrates the degradation initiator to the molecule of interest for targeted degradation.

In some embodiments, the present disclosure provides a set of polynucleotides comprising, as part of one or more vectors: (i) a first polynucleotide encoding a first fusion protein comprising a first binding element, and (ii) a second polynucleotide encoding a second fusion protein comprising a corresponding second binding element, wherein: (iii) the first or second fusion protein further comprises an E3 ligase domain (or a functional variant thereof); and (iv) the other of the first or second fusion protein also includes a protein of interest; and (v) The interaction of the first and second binding elements efficiently focuses the E3 ligase to the protein of interest for targeted degradation.

In some embodiments, the present disclosure provides a set of polynucleotides comprising, as part of one or more vectors: (i) a first polynucleotide encoding a first fusion protein comprising a first binding element and an E3 ligase domain (or a functional variant thereof), and (ii) a second polynucleotide encoding a second fusion protein comprising a corresponding second binding element and a synthetic or exogenous protein of interest, wherein the interaction of the first and second binding elements Efficiently concentrates the E3 ligase domain to the target protein for targeted degradation. In some embodiments, the present disclosure provides sets of polynucleotides comprising, as part of one or more vectors: (i) a first polynucleotide encoding a first fusion protein comprising a DHD binding element and an E3 ligase domain (or a functional variant thereof); and (ii) a second polynucleotide encoding a second fusion protein comprising a corresponding second DHD binding element and a synthetic or exogenous protein of interest, wherein the interaction of the first and second binding elements Acts effectively to focus the E3 ligase domain to the target protein for targeted degradation.

In some embodiments, the present disclosure provides sets of polynucleotides comprising, as part of one or more vectors: (i) a first polynucleotide encoding a first fusion protein comprising a binding element comprising a reader protein and an E3 ligase domain (or a functional variant thereof); and (ii) a second polynucleotide encoding a second fusion protein comprising an NS3a binding element and a synthetic or exogenous protein of interest, wherein the interaction of the first and second binding elements is regulated by the presence of a small molecule, thereby linking the E3 Enzyme domains focus on proteins of interest for targeted degradation.

In some embodiments, the present disclosure provides sets of polynucleotides encoding fusion proteins comprising the following as part of a single vector: (i) degradation initiators; and (ii) A binding domain specific for a native motif on an endogenous target molecule, wherein binding of the fusion protein to the target molecule modulates the concentration of the degradation initiator to the target molecule for targeted degradation.

In some embodiments, the present disclosure provides sets of polynucleotides encoding fusion proteins comprising the following as part of a single vector: (i) E3 ligase domain; and (ii) A binding domain specific to a native motif on an endogenous target protein, wherein binding of the fusion protein to the target protein regulates the recruitment of the E3 ligase to the target protein for targeted degradation.

single carrier configuration

Figure 7A shows a schematic diagram of a single vector configuration 700 for encoding an inducible polynucleotide component and a constitutive polynucleotide component on a single vector. In this example, the vector is configured to express an inducible polynucleotide component 710 encoding a first fusion protein comprising an E3 ligase domain fused to a first binding element and encoding a second fusion protein The constitutive polynucleotide component 715 of the second fusion protein includes a protein of interest fused to a second binding element. For example, the first and second binding elements can be DHD polypeptides. The inducible promoter component consists of a minimal promoter with one or more 5' response element repeats that are recognized and bound by specific transcription factors in response to stimuli. An inducible promoter component may also include optional regulatory sequences, such as a polyA sequence. A constitutive polynucleotide component may also include optional regulatory sequences, such as polyA sequences.

In some embodiments, the vector further includes a transduction marker.

Two carrier configuration

In some embodiments, the set of polynucleotides comprising the first polynucleotide and the second polynucleotide are integrated on two vectors, wherein: (i) the first vector may comprise a first polynucleotide encoding a first fusion protein; and (ii) The second vector may include a second polynucleotide encoding a second fusion protein.

In some embodiments, the first vector is configured to express an inducible polynucleotide component encoding a first fusion protein comprising a degradation initiator fused to the first binding element, and the second vector is configured to A constitutive polynucleotide component encoding a second fusion protein comprising the molecule of interest and the second binding element is expressed.

In some embodiments, the first vector is configured to express an inducible polynucleotide component encoding a first fusion protein comprising an E3 ligase domain (or a functional variant thereof) fused to the first binding element, the second vector configured to express a constitutive polynucleotide component encoding a second fusion protein comprising a protein of interest and a second binding element.

Figure 7B shows a schematic diagram of a two-vector system 720 for encoding an inducible polynucleotide component and a constitutive polynucleotide component. In this example, a first vector 725 includes an inducible polynucleotide component 710 for expressing a first fusion protein comprising an E3 ligase domain fused to a first binding element, and a second vector 730 includes an inducible polynucleotide component 710 encoding a protein comprising the protein of interest and The constitutive polynucleotide component 715 of the second fusion protein of the second binding element.

Compositions comprising multiple carriers

The set of polynucleotides of the disclosure can be provided as part of a vector. In some embodiments, the first and second polynucleotide components of a set of polynucleotides can be provided as part of a single vector.

The present disclosure provides a composition comprising a single carrier comprising:

(i) a first polynucleotide component encoding a first binding element; and

(ii) a second polynucleotide component encoding a second binding element.

wherein the first or second polynucleotide component further encodes a degradation initiator, and the other of the first or second polynucleotide component further encodes a molecule of interest, and the interaction of the first and second binding elements Action-mediated degradation initiators focus on molecules of interest for targeted degradation.

In some embodiments, the present disclosure provides a composition comprising a single carrier comprising:

(i) a first polynucleotide component encoding a first binding element; and

(ii) a second polynucleotide component encoding a second binding element.

Wherein, any one of the first or second polynucleotide components further encodes an E3 ligase domain (or a functional variant thereof), and the other of the first or second polynucleotide components further encodes The protein of interest, and the interaction of the first and second binding elements regulates the recruitment of E3 ligases to the protein of interest for targeted degradation.

In some aspects, compositions are useful for treating a subject in need thereof. The disclosure provides a pharmaceutical composition comprising:

A single vector comprising a first polynucleotide component encoding a first binding element and a second polynucleotide component encoding a second binding element, wherein either the first or the second polynucleotide component is further encodes a degradation initiator, and the other of the first or second polynucleotide components further encodes a molecule of interest; and

A pharmaceutically acceptable carrier, excipient and/or stabilizer.

In some aspects, the present disclosure provides a pharmaceutical composition comprising:

(i) a single vector comprising a first polynucleotide component encoding a first binding element and a second polynucleotide component encoding a second binding element, wherein the first or second polynucleotide component Either further encodes an E3 ligase domain, and the other of the first or second polynucleotide components further encodes a protein of interest; and

(ii) Pharmaceutically acceptable carriers, excipients and/or stabilizers.

In some embodiments, the first and second polynucleotide components of the set of polynucleotides may be provided as part of a set of at least two vectors, wherein, for example, the first vector comprises the first polynucleotide component, the second polynucleotide component Secondary vectors include a second polynucleotide component.

The present disclosure provides a composition comprising:

(i) a first vector comprising a first polynucleotide component encoding a first binding element; and

(ii) a second vector comprising a second polynucleotide component encoding a second binding element.

Wherein, any one of the first or second polynucleotide components further encodes a degradation initiator, and the other of the first or second polynucleotide components further encodes a molecule of interest, and the first and second The interaction of the binding elements regulates the concentration of the degradation initiator to the molecule of interest for targeted degradation.

For some purposes, the compositions are useful for treating a subject in need thereof. The disclosure provides a pharmaceutical composition comprising:

(i) a first vector comprising a first polynucleotide component encoding a first binding element; and

(ii) a second vector comprising a second polynucleotide component encoding a second binding element. wherein either of the first or second polynucleotide components further encodes a degradation initiator, and the other of the first or second polynucleotide components further encodes a molecule of interest; and

(iii) Pharmaceutically acceptable carriers, excipients and/or stabilizers.

In some aspects, the present disclosure provides a pharmaceutical composition comprising:

(i) a first vector comprising a first polynucleotide component encoding a first binding element; and

(ii) a second vector comprising a second polynucleotide component encoding a second binding element, wherein either of the first or second polynucleotide components further encodes an E3 ligase (or a functional variant thereof), and the other of the first or second polynucleotide components further encodes a protein of interest; and

(iii) Pharmaceutically acceptable carriers, excipients and/or stabilizers.

The present disclosure provides a composition comprising a single vector comprising a polynucleotide encoding a fusion protein comprising a degradation trigger and a binding element as part of a single vector, wherein the binding element is specific to an endogenous The native motif on the molecule is specific.

In some aspects, the compositions are useful for treating a subject in need thereof. The disclosure provides a pharmaceutical composition comprising:

(i) a vector comprising a polynucleotide encoding a fusion protein comprising a degradation initiator and a binding element, wherein the binding element is specific for a native motif on an endogenous molecule of interest; and

(ii) Pharmaceutically acceptable carriers, excipients and/or stabilizers.

In some embodiments, the present disclosure provides a composition comprising a polynucleotide encoding a fusion protein comprising an E3 ligase domain and a binding element as part of a single vector, wherein the binding element pairs within The native motif on the derived protein of interest is specific.

In some aspects, the compositions are useful for treating a subject in need thereof. The disclosure provides a pharmaceutical composition comprising:

(i) a vector comprising a polynucleotide encoding a fusion protein comprising an E3 ligase domain and a binding element, wherein the binding element is specific for a native motif on the endogenous protein; and

(ii) Pharmaceutically acceptable carriers, excipients and/or stabilizers.

cell

The expression vectors of the present disclosure can be expressed in host cells. For example, a host cell can be a prokaryotic cell, such as a bacterial cell; or a eukaryotic cell, such as a yeast cell, a plant cell, or a mammalian cell. Examples of mammalian cells suitable for use in the present disclosure include human, mouse, rat, pig, rabbit, sheep, and goat cells. In some cases, the cells are synthetic cells.

For example, host cells may be selected from cardiomyocytes, lung cells, muscle cells, epithelial cells, pancreatic cells, skin cells, CNS cells, neurons, muscle cells, skeletal muscle cells, smooth muscle cells, liver cells, kidney cells, and glial cells .

In some embodiments, the host cell is an ex vivo human cell. In some embodiments, the host cell is a human cell in vivo.

In some embodiments, the host cell is a stem cell, such as a pluripotent stem cell or a hematopoietic stem cell.

In some embodiments, the host cell is a pluripotent cell or a mesenchymal cell or a mesenchymal stromal cell (MSC).

In some embodiments, the host cell is a stem cell and the polynucleotides of the disclosure are used to control the differentiation of cellular products produced by pluripotent cells, eg, pluripotent stem cells. For example, degradation systems that include inducible E3 ligase fusion proteins can be used to control the stability (e.g., half-life) of proteins that drive differentiation.

In some embodiments, the host cell is not pluripotent, and the polynucleotides of the disclosure are used to control reprogramming of the cell to regulate pluripotency. For example, degradation systems including inducible E3 ligase fusion proteins can be used to control the stability (e.g., half-life) of proteins driving reprogramming.

In some embodiments, a host cell is part of an organism. In addition to the therapeutic embodiments described elsewhere herein, cells can be part of a model organism. For example, degradation systems can be used to control protein stability, regulate cellular processes and generate signatures for scientific research, such as disease signatures or biological enhancement. Examples of suitable model organisms include yeast, Drosophila, nematodes, frogs, mice and fish (eg zebrafish). For example, a protein of interest may be a dysfunctional polypeptide, or a polypeptide that interacts with an organism's genes or regulates or interferes with metabolic processes. In one embodiment, the degradation system is inducible, and an inducing stimulus can be administered to modulate or titrate the expression of the E3 ligase fusion protein to produce a change in the characteristic of interest.

In some embodiments, the host cell is a cancer cell and/or a non-cancer cell from a human subject diagnosed with cancer.

In some embodiments, the host cell is selected from leukocytes, lymphocytes, T cells, regulatory T cells, effector T cells, CD4+ effector T cells, CD8+ effector T cells, memory T cells, autoreactive T cells, exhausted T cells group of immune cells, natural killer T cells, B cells, dendritic cells, and macrophages.

Host cells can be transformed with one or more polynucleotides or vectors of the present disclosure and cultured in a nutrient medium. Nutrient media can be formulated to induce promoters, select for transformants, or amplify a gene of interest. The cell is a mammalian cell or cell line. Non-limiting examples include: African green monkey kidney cells (VERO-76, ATCC CRL-1587); baby hamster kidney cells (BHK, ATCC CCL 10); BALB/c mouse myeloma line (NSO/I, ECACC No: 85110503); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); canine kidney cells (MDCK, ATCC CCL 34); Chinese hamster ovary (CHO) cells or cell line CHO-K1 cell line (see for example ATCC catalog number CCL -61™ and Lewis, N.E. et al. (2013), National Journal of Biology, 31:759-765); Chinese hamster ovary cells +/-DHFR (see e.g. Urlaub, G. and Chasin, L.A. (1980) Proc. Natl. Acad. Sci. 77:4216-4220); FS4 cells; HEK 293 cells; HT-1080 cells (ATCC® CCL-121™); human cervical cancer cells (HeLa, ATCC CCL-2); human embryonic kidney cell line ( 293 or subcloning of 293 cells for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); human liver cancer line (Hep G2); human hepatocytes (Hep G2, HB 8065); human Lung cells (W138, ATCC CCL 75); human retinoblastocytes (PER.C6, CruCell, Leiden, Netherlands); monkey kidney cells (CV1 ATCC CCL 70); SV40 transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); mouse mammary tumor (MMT 060562, ATCC CCL51); MRC 5 cells; TRI cells (Mather et al., Annals N. Y. Acad. Sci. 383:44-68 (1982)); and Engineered T cells and engineered natural killer cells.

Compositions comprising host cells

The set of polynucleotides of the disclosure can be provided in a host cell. Cells can be transiently or stably engineered to incorporate the disclosed set of polynucleotides.

The present disclosure provides a composition comprising a cell comprising a set of polynucleotides comprising a first polynucleotide component encoding a first binding element and a second polynucleotide encoding a second binding element An acid component, wherein any one of the first or second polynucleotide components further encodes a degradation initiator, and the other of the first or second polynucleotide components further encodes a molecule of interest, and the first and The interaction of the second binding element regulates the concentration of the degradation initiator to the molecule of interest for targeted degradation.

In some embodiments, the present disclosure provides a composition comprising a cell comprising a set of polynucleotides comprising a first polynucleotide component encoding a first binding element and a second binding element encoding The second polynucleotide component of the element, wherein any one of the first or second polynucleotide components further encodes an E3 ligase domain (or a functional variant thereof), and the first or second polynucleotide Another of the nucleotide components further encodes a protein of interest, and the interaction of the first and second binding elements regulates the concentration of the E3 ligase to the protein of interest for targeted degradation.

The present disclosure provides a composition comprising a cell comprising a polynucleotide encoding a fusion protein comprising a degradation initiator and a binding element, wherein the binding element binds to a naturally occurring gene on an endogenous molecule of interest. sequence is specific.

In some embodiments, the present disclosure provides compositions comprising a cell comprising a polynucleotide encoding a fusion protein comprising an E3 ligase (or a functional variant thereof) and a binding element, wherein the binding element Specific for a native motif on an endogenous protein of interest.

For some purposes, compositions comprising cells may be used to treat a subject in need thereof. The disclosure provides a pharmaceutical composition comprising:

(i) cells that have been modified to express the set of polynucleotides; and

(ii) A pharmaceutically acceptable carrier, excipient or stabilizer.

A cell may include a polynucleotide of the disclosure expressing a gene of interest that provides a therapeutic benefit. Expression of a gene of interest can confer on the cell the ability to attack tumor cells. The gene of interest may be a chimeric antigen receptor (CAR), such as a chimeric antigen receptor targeted to tumor cells. The gene of interest can express a single-chain antibody fragment attached to the hinge that connects to the transmembrane region. The transmembrane region can be linked to an intracellular signaling domain. The transmembrane region can be linked to a co-stimulatory domain.

The cells of the composition can be, for example, T cells. The cells of the composition can be, for example, CAR-T cells.

In some embodiments, the present disclosure provides a cellular composition comprising means for reducing, ameliorating or inhibiting exhaustion and/or dysfunction in a population of immune cells (eg, CAR-expressing immune cells). In some aspects, the method includes expressing a CAR as a gene of interest in a polynucleotide panel and modulating the degradation of the CAR, thereby inhibiting excessive CAR signaling that can lead to the undesirable exhaustion phenotype observed in many existing T cell therapies .

Methods of making small molecules

Small molecules of the present disclosure can be synthesized using known techniques. Danprevir ((2R, 6S, 12Z, 13aS, 14aR, 16aS)-14a-[(cyclopropylsulfonyl)carbamoyl]-6-({[(2-methyl-2-propane yl)oxy]carbonyl}amino)-5,16-dioxy-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a- Hexadecylhydrocyclopropane[e]pyrrole[1,2-a][1,4]diazocyclopentane-2-yl-4-fluoro-1,3-dihydro-2H-isoindole- 2-carboxylates) can be synthesized using known techniques. See, for example, Carreira, Erick Moran, Hisashi Yamamoto, and N.K. Yee. "Industrial Applications of Asymmetric Synthesis" In General Handbook 9, Amsterdam: Elsevier, 2012, section 9.19.6, danoprevir, the disclosure of which is incorporated herein by reference.

making polynucleotides

The disclosure provides methods of producing polynucleotides of the disclosure, such as DNA vectors of the disclosure and subcomponents thereof, as well as packaging vectors and plasmids of the disclosure. Standard molecular biology techniques can be used to assemble the polynucleotides of the disclosure. Polynucleotides can be chemically synthesized.

Making Encapsulated Viral Capsids

The disclosure includes methods of making capsids comprising polynucleotides of the disclosure. Typically, capsids of the present disclosure can be produced by supplying cells with packaging polynucleotides of the present disclosure. For example, packaging polynucleotides can be provided to packaging cells as plasmids. Packaging cells can be cultured to produce capsids containing polynucleotides of the disclosure. Preferably, the packaged viral capsids are replication incompetent.

A variety of commercially available kits are suitable for producing packaged viral capsids of the disclosure. Examples include: MISSION® Lentiviral Packaging Mix (available from Millipore Sigma); LV-Max Lentiviral Packaging Mix (available from ThermoFisher Scientific).

Capsids produced by packaging cells can be purified for use in downstream methods, such as delivery to cells for cell-based therapy, or delivery to a subject for gene therapy approaches. Purification can include treatments to remove contaminants from host cells or culture media. Purification steps may include steps based on the physical and/or chemical characteristics of the plasmid. Chemical properties may include, for example, hydrophilicity-hydrophobicity. Physical characteristics may include, for example, size. Examples of nuance-based purification strategies include density gradient ultracentrifugation, ultrafiltration, precipitation, two-phase extraction systems, and size exclusion chromatography. In some cases, precipitation can be used with centrifugation, for example, using polyethylene glycol, ammonium sulfate, or calcium phosphate. In some cases, aqueous two-phase separation systems with PEG, dextran, or polyvinyl alcohol can be used. In some cases, membrane-based tangential flow filtration techniques are used; examples include ultrafiltration, diafiltration, and microfiltration. In other embodiments, chromatographic methods can be used to purify viral capsids. In other embodiments, immunoaffinity methods can be used to capture capsids using monoclonal antibodies specific for the relevant capsids. See Morenweiser, R., "Downstream Processing of Viral Vectors and Vaccines", Gene Therapy, (2005) 12, S103-S110 (2005), the entire disclosure of which is incorporated herein by reference.

Examples of suitable capsids include, but are not limited to, adenovirus, retrovirus, lentivirus, Sendai virus, baculovirus, Epstein-Barr virus, papovavirus, vaccinia virus, herpes simplex virus, and adeno-associated virus (AAV).

making cells

The present disclosure provides methods for making cells modified to express a gene of interest.

In some embodiments, the present disclosure provides a method of making therapeutic cells expressing a set of polynucleotides for treating a subject in need of cell therapy. In one object, the present disclosure provides a method of producing or preparing a therapeutic cell expressing a gene of interest from a set of polynucleotides integrated into a single vector. In one object, the present disclosure provides a method of producing or preparing a therapeutic cell expressing a gene of interest from a set of polynucleotides integrated into two (or more) vectors.

In some embodiments, polynucleotides of the present disclosure are maintained in host cells as extrachromosomal polynucleotides. In some embodiments, a polynucleotide of the disclosure is present in a vector (eg, an expression vector) in a host cell. In some embodiments, polynucleotides of the disclosure, or a subset or subcomponent thereof, are integrated into the chromosome of the host cell.

Various methods can be used to introduce expression vectors of some embodiments of the present disclosure into cells to produce cells of the present disclosure. See, eg, Molecular Cloning: A Laboratory Manual by Green et al., Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press (2014).

Methods for introducing nucleic acid changes into a gene of interest are well known in the art. Examples include targeted homologous recombination (e.g. "Hit and run", "double replacement"), site-specific recombinases (e.g. Cre recombinase and Flp recombinase), PB transposases (e.g. Sleeping Beauty, piggyBac, To12 or Frog Prince), genome editing by engineered nucleases (e.g., meganucleases, zinc finger nucleases (ZFNs)), genome editing using recombinant adeno-associated virus (rAAV) platforms. Reagents for introducing nucleic acid changes into a gene of interest can be designed using publicly available resources or obtained commercially from Transposagen, Addgene, and Sangamo Biosciences. The vectors of the present disclosure can utilize these methods to integrate the polynucleotides of the present disclosure into the host genome. The polynucleotides and vectors of the disclosure may include polynucleotides encoding polypeptides required for carrying out the methods for integrating the polynucleotides of the disclosure into the host genome.

Various methods suitable for integrating polynucleotides into the host cell genome are known in the art, including random integration or site-specific integration (e.g., the "landing pad" approach); see, e.g., Zhao, M. el al. ( 2018), Microbial Biotechnology, 102:6105-6117; Li, J.S. et al., (2015) Journal of Science, 5:8572; and Gaidukov, L. et al., (2018) Journal of Nucleic Acids, 46:4072-4086. The vectors of the present disclosure can utilize these methods to integrate the polynucleotides of the present disclosure into the host genome. Vectors of the present disclosure may include polynucleotides encoding polypeptides required to perform the methods for integrating polynucleotides of the present disclosure into the genome of a host.

Examples of commercially available media suitable for culturing the host cells of the present disclosure include Ham's F10 (Sigma), Minimum Essential Medium ((MEM), (Sigma), RP MI-1640 (Sigma)) and Dulbecco's Modified Eagle Medium ((DMEM ), Sigma).

Media can be supplemented as needed with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the GENTAMYCIN™ drug), trace elements (defined as inorganic compounds usually present in final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature, pH, etc., will be apparent to those of ordinary skill.

Cell Therapy

The present disclosure provides methods of treating a subject in need of cell therapy. The method includes the following steps:

(i) administering to a subject an effective dose of a pharmaceutical composition comprising a therapeutic cell encoding a set of polynucleotides comprising degradation from an inducible promoter and expression of a therapeutic molecule of interest Initiator; and

(ii) administering to the subject a therapeutically effective dose of the inducing stimulus.

In some embodiments, the method includes the steps of:

(i) administering to the subject an effective amount of a pharmaceutical composition comprising a therapeutic cell encoding a set of polynucleotides comprising an E3 linkage expressed from an inducible promoter and a therapeutic molecule of interest an enzyme (or a functional variant thereof); and

(ii) administering to the subject a therapeutically effective dose of the inducing stimulus.

In one aspect, the present disclosure provides a method for treating cancer, eg, a tumor, in a subject in need thereof. Examples of cancers that may be treated using the pharmaceutical compositions of the present disclosure include, but are not limited to, melanoma, lymphoma, sarcoma, and cancer of the colon, kidney, stomach, bladder, brain (e.g., glioma, glioblastoma , astrocytoma, medulloblastoma), prostate, bladder, rectum, esophagus, pancreas, liver, lung, breast, uterus, cervix, ovary, blood (eg, acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, Burkitt lymphoma, EBV-induced B-cell lymphoma).

In one object, the present disclosure provides a method of controlling or modulating a T cell mediated immune response in a subject in need thereof.

In one object, the present disclosure provides a method of stimulating a T cell modulated immune response to a target cell population or tissue in a subject.

In one object, the present disclosure provides a method of providing anti-tumor immunity in a subject.

In one object, the present disclosure provides a method of preventing T cell exhaustion in a subject during CAR T cell cancer therapy.

gene therapy method

The present disclosure provides methods of delivering the set of polynucleotides of the present disclosure to a subject. A set of polynucleotides of the present disclosure can be delivered into cells of a subject. The method can comprise administering to the subject a pharmaceutically effective amount of the set of polynucleotides. Administration can be by administration of viral particles comprising one or more polynucleotides of the present disclosure. Administration can be by administration of a pharmaceutical composition comprising one or more polynucleotides of the present disclosure.

In some embodiments, the method includes the steps of:

(i) administering to the subject an effective dose of a pharmaceutical composition comprising a polynucleoside encoding a degradation initiator (e.g., an E3 ligase domain) expressed from a small molecule-regulated promoter and a therapeutic molecule of interest Acid group;

(ii) administering to the subject a therapeutically effective dose of the small molecule modulator;

(iii) monitoring the level of the therapeutic molecule in the subject; and

(iv) Optionally, adjusting the dose of the small molecule modulator to adjust the level of the therapeutic molecule in the subject to the desired level.

The subject can be a mammalian subject. A subject can be a human subject.

Examples of conditions that may be selected for gene therapy include, but are not limited to, cancer, cystic fibrosis, heart disease, diabetes, hemophilia, and AIDS.

Reagent test kit

The disclosure provides kits or articles of manufacture comprising the polynucleotides of the disclosure and formulations for delivering the polynucleotides to cells. A polynucleotide can be provided as part of a vector of the present disclosure. In some embodiments, the kit or article of manufacture further includes instructions for using the set of polynucleotides to transform cells to express a gene of interest to produce a polypeptide of interest and/or to modulate a gene of interest.

The present disclosure provides kits or articles of manufacture comprising pharmaceutical compositions comprising therapeutic cells encoding the set of polynucleotides of the present disclosure. The kit or article of manufacture may also include instructions for administering the pharmaceutical composition to a subject.

In some cases, a kit or article of manufacture may also include a small molecule modulator of the disclosure.

surface

Table A - Sequence name SEQ ID NO: sequence CD28 MLRLLLALNL FPSIQVTGNK ILVKQSPMLV AYDNAVNLSC KYSYNLFSRE FRASLHKGLD SAVEVCVVYG NYSQQLQVYS KTGFNCDGKL GNESVTFYLQ NLYVNQTDIY FCKIEVMYPP PYLDNEKSNG TIIHVKGKHL CPSPLFPGPS KPFWVLVVVG GVLACYSLLV TVAFIIFWVR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS (UniProtKB 登錄號 P10747-1) CD28 MLRLLLALNL FPSIQVTGKH LCPSPLFPGP SKPFWVLVVV GGVLACYSLL VTVAFIIFWV RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYRS (UniProtKB accession number P10747-2) CD28 MLRLLLALNL FPSIQVTGNK ILVKQSPMLV AYDNAVNLSY NEKSNGTIIH VKGKHLCPSP LFPGPSKPFW VLVVVGGVLA CYSLLVTVAF IIFWVRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRS (UniProtKB accession number P10747-3) CD28 MLRLLLALNL FPSIQVTGNK ILVKQSPMLV AYDNAVNLSW KHLCPSPLFP GPSKPFWVLV VVGGVLACYS LLVTVAFIIF WVRSKRSRLL HSDYMNMTPR RPGPTRKHYQ PYAPPRDFAA YRS (UniProtKB accession number P10747-4) CD28 MLRLLLALNL FPSIQVTGNK ILVKQSPMLV AYDNAVNLSY NEKSNGTIIH VKGEE (UniProtKB accession number P10747-5) CD28 MLRLLLALNL FPSIQVTGNK ILVKQSPMLV AYDNAVNLSY NEKSNGTIIH VKGKHLCPSP LFPGPSKPYA PPRDFAAYRS (UniProtKB accession number P10747-6) CD28 MPCGLSALIM CPKGMVAVVV AVDDGDSQAL AGNKILVKQS PMLVAYDNAV NLSCKYSYNL FSREFRASLH KGLDSAVEVC VVYGNYSQQL QVYSKTGFNC DGKLGNESVT FYLQNLYVNQ TDIYFCKIEV MYPPPYLDNE KSNGTIIHVK GKHLCPSPLF PGPSKPFWVL VVVGGVLACY SLLVTVAFII FWVRSKRSRL LHSDYMNMTP RRPGPTRKHY QPYAPPRDFA AYRS (UniProtKB 登錄號 P10747-7) 4-1BB MGNSCYNIVA TLLLVLNFER TRSLQDPCSN CPAGTFCDNN RNQICSPCPP NSFSSAGGQR TCDICRQCKG VFRTRKECSS TSNAECDCTP GFHCLGAGCS MCEQDCKQGQ ELTKKGCKDC CFGTFNDQKR GICRPWTNCS LDGKSVLVNG TKERDVVCGP SPADLSPGAS SVTPPAPARE PGHSPQIISF FLALTSTALL FLLFFLTLRF SVVKRGRKKL LYIFKQPFMR PVQTTQEEDG CSCRFPEEEE GGCEL (UniProtKB 登錄號Q07011-1) CD27 MARPHPWWLC VLGTLVGLSA TPAPKSCPER HYWAQGKLCC QMCEPGTFLV KDCDQHRKAA QCDPCIPGVS FSPDHHTRPH CESCRHCNSG LLVRNCTITA NAECACRNGW QCRDKECTEC DPLPNPSLTA RSSQALSPHP QPTHLPYVSE MLEARTAGHM QTLADFRQLP ARTLSTHWPP QRSLCSSDFI RILVIFSGMF LVFTLAGALF LHQRRKYRSN KGESPVEPAE PCHYSCPREE EGSTIPIQED YRKPEPACSP (UniProt 登錄號P26842-1) CD3 zeta MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF IYGVILTALF LRVKFSRSAD APAYQQGQNQ LYNELNLGRR EEYDVLDKRR GRDPEMGGKP QRRKNPQEGL YNELQKDKMA EAYSEIGMKG ERRRGKGHDG LYQGLSTATK P1DALHMQ CD3 zeta MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF IYGVILTALF LRVKFSRSAD APAYQQGQNQ LYNELNLGRR EEYDVLDKRR GRDPEMGGKP RRKNPQEGLY NELQKDKMAE AYSEIGMKGE RRRGKGHDGL PQGLSTATKD TYDALHMQAL PPR 6 ICOS MKSGLWYFFLFCLRIKVLTGEINGSANYEMFIFHNGGVQILCKYPDIVQQFKMQLLKGGQ ILCDLTKTKGSGNTVSIKSLKFCHSQLSNNSVSFFLYNLDHSHANYYFCNLSIFDPPPFK VTLTGGYLHIYESQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSR ICOS MKSGLWYFFLFCLRIKVLTGEINGSANYEMFIFHNGGVQILCKYPDIVQQFKMQLLKGGQ ILCDLTKTKGSGNTVSIKSLKFCHSQLSNNSVSFFLYNLDHSHANYYFCNLSIFDPPPFK VTLTGGYLHIYESQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKKM (UniProtKB accession number Q9Y6W8-2 OX40 MERVQPLEENVGNAARPRFERNKLLLVASVIQGLGLLLCFTYICLHFSALQVSHRYPRIQ SIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQ KDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGPLILIHQNPGEF3CVL OX40 MVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFS QEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGEL ILIHQNPGEFCVL (UniProtKB accession number P23510-2)

Form B disease or condition Target protein (or its antigen/epitope) autoimmunity ABCC8, ADIPOQ, ADRB3, AGPAT2, AKT2, ALMS1, ANGPTL8, APPL1, AQP2, AVP, AVPR2, BANK1, BCAR1, BLK, BSCL2, C4A, C4B, CAPN10, CAV1, CAVIN1, CCR5, CD38, CDKAL1, CEL, CELA2A, CISD2, CLEC16A, CLPS, CR2, CTLA4, DCAF17, DMXL2, DNAJC3, DNASE1, DNASE1L3, DYRK1B, EIF2AK3, ENPP1, FCGR2B, FOXP3, GCGR, GCK, GLIS3, GSK3A, GSK3B, GYS1, HNF1B, INF4A, IERH1IP1, IL2RA, INPPL1, INS, INSR, IRF5, IRS1, ITGAM, KCNJ11, KCNQ1, KLF11, LEP, LIPE, LTK, MAFA, MAPK8IP1, MBP, MCF2L2, MT-ND1, NEUROD1, PAX4, PDCD1, PDX1, PLAGL1, PPARG, PPP1R15B, PPP1R3A, PTF1A, PTPN22, PTPRN, PTPRN2, RASGRP1, RETN, RFX6, SH2B3, SLC16A11, SLC16A13, SLC19A2, SLC2A4, SLC30A8, SSB, STAT3, STAT4, SUMO4, TBC1D4, TCF4, TCF7L2N TRFS, TLR UCP1, UCP3, WFS1, XRCC5, XRCC6, ZFP57 blood ACSL4, ADA, AK1, ALDOA, AMMECR1, ANK1, ATP11C, BPGM, BRCA1, BRCA2, BRIP1, CD59, CDAN1, CDIN1, CPOX, CYBA, CYBB, CYBC1, EPB41, EPB42, ERCC4, F8, F9, FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL, G6PD, GCLC, GPI, GSR, GSS, HBA1, HBA2, HBB, KCNE5, KCNN4, KLF1, LPIN2, MAD2L2, NCF1, NCF2, NCF4, PALB2, PGK1, PIEZO1, PKLR, RAD51, RAD51C, RFWD3, RHAG, RPL11, RPL15, RPL18, RPL26, RPL27, RPL35, RPL35A, RPL5, RPS10, RPS15A, RPS17, RPS19, RPS24, RPS26, RPS27, RPS28, RPS29, RPS7, SEC23B SLC2A1, SLC4A1, SLX4, SPTA1, SPTB, TPI1, TSR2, UBE2T, XRCC2 skeleton ANO5, BMP1, CA2, CLCN7, COL1A1, COL1A2, CREB3L1, CRTAP, FKBP10, IFITM5, IKBKG, LRP5, MBTPS2, MESD, MITF, OSTM1, P3H1, P4HB, PLEKHM1, PLOD2, PPIB, SEC24D, SERPINF1, SERPINH1, SNX SP7, SPARC, SPG7, TCIRG1, TENT5A, TMEM38B, TNFRSF11A, TNFSF11, WNT1 Nervous system disease AARS1, AARS2, ABCA2, ABCA7, ABHD12, ACO2, ACOX1, ACTL6B, ADAM10, ADAM22, ADPRS, ADRA2B, ADSL, AFG3L2, AGTPBP1, AIFM1, ALDH18A1, ALDH7A1, ALG13, ALS2, AMPD2, ANG, ANO110, ANX1 AP2M1, AP3B2, AP4B1, AP4E1, AP4M1, AP4S1, AP5Z1, APLP1, APOA1, APOE, APP, APTX, AR, ARHGEF9, ARL6IP1, ARSA, ARSB, ARV1, ARX, ASAH1, ASCC1, ATG5, ATIC, ATL1, ATL3, ATM, ATN1, ATP13A2, ATP1A1, ATP2A2, ATP2B3, ATP6AP2, ATP6V1A, ATP7A, ATXN1, ATXN10, ATXN2, ATXN3, ATXN7, B4GALNT1, BCKDK, BEAN1, BICD2, BRAT1, BSCL2, C12orf65, C19orf9ACf19, C19orfNA17, C2 CACNA1B, CACNA1D, CACNA1E, CACNA1G, CACNA1H, CACNA2D2, CACNB4, CAD, CAPN1, CASR, CCDC88A, CCDC88C, CCT5, CDK19, CDK5, CDKL5, CERS1, CHCHD10, CHCHD2, CHD2, CHMP1CH, CHMP2B, RNA, CHP1, CHRNB2, CILK1, CLCN2, CLN3, CLN5, CLN6, CLN8, CLP1, CLPB, CNNM2, CNPY3, CNTN2, CNTNAP1, CNTNAP2, COA7, COASY, COQ2, COQ8A, COX6A1, CPA6, CPLX1, CPT1C, CRAT, CSF1B, CSNK2 CSTB, CTDP1, CTSD, CTSF, CUX2, CWF19L1, CYFIP2, CYP2U1, CYP7B1, DAB1, DAGLA, DALRD3, DBN1, DCAF8, DCTN1, DCX, DDHD1, DDHD2, DEAF1, DENND5A, DEPDC5, DGUOK, DHDDS, DHTKD1, DHX16, DIAPH1, DIAPH3, DLL1, DMXL2, DNAJB2, DNAJC13, DNAJC3, DNAJC6, DNM1, DNM2, DNMT1, DOCK7, DPP DST, DSTYK, DYNC1H1, ECHS1, EEF1A2, EEF2, EFHC1, EGR2, EIF2S3, EIF4G1, ELOVL4, ELOVL5, ELP1, ELP3, EMC1, EML1, ENTPD1, EPM2A, EPRS1, ERBB4, ERLIN1, ERLIN2, EXOSC3, EXOSC98, EXOSC98, EX EXT2, FA2H, FAR1, FARS2, FARSB, FAT2, FBLN5, FBXO38, FBXO7, FDXR, FGD4, FGF12, FGF14, FGGY, FIG4, FLVCR1, FOLR1, FRRS1L, FTL, FUS, GABBR2, GABRA1, GABRA2, GABRA5, GABRB1, GABRB2, GABRB3, GABRD, GABRG2, GAL, GAN, GARS1, GBA, GBA2, GBE1, GDAP1, GDAP2, GIGYF2, GJB1, GJC2, GLS, GNAO1, GNB4, GOSR2, GOT2, GRIA2, GRID2, GRIN2A, GRIN2B, GRIN2D, GRM1, GRM7, GRN, GUF1, HACE1, HARS1, HCN1, HEXA, HEXB, HINT1, HIP1, HK1, HNRNPA1, HNRNPU, HSD17B10, HSPB1, HSPB3, HSPB8, HSPD1, HTRA2, HTT, IARS2, IBA57, IER3IP1, IGHMBP2, INF2, IREB2, IRF2BPL, ITM2B, ITPA, ITPR1, KARS1, KCNA2, KCNB1, KCNC1, KCNC3, KCND2, KCND3, KCNH1, KCNJ10, KCNK4, KCNMA1, KCNQ2, KCNQ3, KCNT1, KCNT2, KCTD7, KIDINS220, KIF1A, KIF1B, KIF1C, KIF5A, KLC2, L1CAM, LAGE3, LGI1, LITAF, LMNA, LMNB2, LNPK, LRRK2, LRSAM1, MAG, MAPK10, MAPT, MARCHF6, MARS1, MARS2, MATR3, MCM3AP, MDH1, MDH2, MED25, MEF2C, MFN2, MFSD8, MME, MORC2, MPV17, MPV17, MPZ, MT-ATP6, MT-CO1, MT-CO3, MT-CYB, MTHFS, MTMR2, MT-ND1, MT-ND2, MT-ND4, MT-ND4L, MT-ND5, MT-ND6, MTOR, MTPAP, MYH14, NACC1, NAGA, NAGLU, NALCN, NAXD, NAXE, NDRG1, NECAP1, NEFH, NEFL, NEK1, NEUROD2, NGF, NHLRC1, NHLRC2, NIPA1, NKX6-2, NOP56, NOTCH2NLC, NPRL2, NPRL3, NRROS, NT5C2, NTRK2, NUP107, NUP133, OPA1, OPTN, OSGEP, OTOF, OTUD6B, OXR1, P4HTM, PACS2, PAK1, PANK2, PARK7, PARS2, PCDH12, PCDH19, PCLO, PCNA, PCYT2, PDK3, PDXK, PDYN, PFN1, PHACTR1, PHF21A, PHF6, PIGA, PIGB, PIGH, PIGK, PIGN, PIGP, PIGQ, PIGS, PIGT, PIGU, PIK3R5, PINK1, PLA2G6, PLCB1, PLD3, PLEKHG5, PLP1, PLPBP, PMP2, PMP22, PMPCA, PMPCB, PNKP, PNPLA6, PNPO, POLG, PPP2R2B, PPP3CA, PPP5C, PPT1, PRDM12, PRDM12, PRDM8, PRICKLE1, PRICKLE2, PRICKLE3, PRKCG, PRKN, PRPH, PR PS1, PRRT2, PRUNE1, PRX, PSAP, PSEN1, PSEN2, PTPN23, PUM1, QARS1, RAB10, RAB7A, RAPGEF2, RARS2, REEP1, REEP2, RELN, REPS1, RETREG1, RFC1, RHOBTB2, RNF13, ROGDI, RORA, RORB, RPIA, RRM2B, RTN2, RUBCN, SACS, SAMD12, SARS1, SBF1, SBF2, SBF2, SCARB2, SCN11A, SCN1A, SCN1B, SCN2A, SCN3A, SCN8A, SCN9A, SCYL1, SELENOI, SEMA6B, SEPSECS, SERPINI1, SETX,1A, SH3TC2, SIGMAR1, SIK1, SIRT2, SLC12A1, SLC12A5, SLC12A6, SLC13A5, SLC1A2, SLC1A4, SLC25A19, SLC25A22, SLC25A46, SLC2A1, SLC30A10, SLC33A1, SLC35A2, SLC35A3, SLC39A14, SLC44A1, SLC45A1, SLC5A6, SLC5A7, SLC6A1, SLC6A3, SLC9A1, SLC9A6, SMC1A, SMN1, SMN2, SMPD1, SNAP29, SNCA, SNCAIP, SNIP1, SNX14, SOD1, SORD, SORL1, SPART, SPAST, SPATA5, SPG11, SPG21, SPG7, SPTAN1, SPTBN2, SPTBN4, SPTLC1, SPTLC2, SQSTM1, SRPX2, ST3GAL3, ST3GAL5, STUB1, STX1B, STXBP1, SUMF1, SUOX, SURF1, SYN1, SYNE1, SYNJ1, SYT14, SZT2, TANGO2, TARDBP, TBC1D24, TBCD, TBCE, TBK1, TBP, TCF4, TDP1, TDP2 TECPR2, TFG, TGM6, THG1L, TIMM50, TMEM106B, TMEM175, TMEM230, TMEM240, TMX2, TNRC6A, TOE1, TP53RK, TPP1, TPRKB, TRAPPC2L, TRAPPC6B, TREM2, TREX1, TRIM2, TRIP4, TRPC3, TRPM7, TRPV4, TSC1, TSC2, TSEN2, TSEN34, TSEN54, TTBK2, TTR, TUBA4A, TUBNGKCP2, T TXN2, TYMP, UBA1, UBA5, UBAP1, UBQLN2, UBQLN4, UBTF, UCHL1, UGDH, UGP2, UNC5C, UNC80, VAC14, VAMP1, VAPB, VARS1, VCP, VPS13A, VPS13C, VPS13D, VPS35, VPS37A, VPS53, VRK1, VWA3B, WARS1, WASF1, WASHC5, WDR4, WDR45, WDR45B, WDR73, WNK1, WWOX, XRCC1, YARS1, YEATS2, YWHAG, ZEB2, ZFYVE26, ZFYVE27, ZNHIT3 Cardiovascular AARS2, ABCC9, ACADVL, ACTC1, ACTN2, AGK, ALPK3, BAG3, BRAF, CALR3, CAV3, CDH2, CRYAB, CSRP3, CTNNA3, DES, DMD, DNAJC19, DSC2, DSG2, DSP, DTNA, EMD, EYA4, FKRP, FKTN, FLNC, GATA4, GATAD1, GJA5, GRM7, GTPBP3, JPH2, JUP, KRAS, LAMA4, LDB3, LMNA, MAP2K1, MAP2K2, MIB1, MT-ATP6, MT-ATP8, MT-CYB, MTO1, MYBPC3, MYH6, MYH7, MYL2, MYL3, MYLK2, MYO6, MYOZ2, MYPN, NDUFB11, NEXN, NPPA, OPA1, PKP2, PLN, PPCS, PRDM16, PRKAG2, PSEN1, PSEN2, RAF1, RBM20, RYR2, SCN5A, SCO2, SDHA, SGCD, SLC25A4, TAZ, TBX20, TBX5, TCAP, TGFB3, TMEM43, TMPO, TNNC1, TNNI3, TNNI3K, TNNT2, TPM1, TSFM, TTN, VCL, ZFPM2 metabolism AGL, ALDOA, ARSB, BCKDHA, BCKDHB, DBT, ENO3, EPM2A, ETFA, ETFB, ETFDH, G6PC, GAA, GALNS, GBA, GBE1, GCDH, GCH1, GLB1, GNS, GUSB, GYG1, GYS2, HGSNAT, HYAL1, IDS, IDUA, LAMP2, LDHA, NAGLU, PAH, PFKM, PGAM2, PGM1, PHKA1, PHKA2, PHKB, PHKG2, PRKAG2, PSAP, PTS, PYGL, PYGM, QDPR, SGSH, SUGCT, SUMF1, VPS33A AAGAB, ANAPC1, AQP5, BMS1, BRAF, CAST, CD151, CDH1, CDH3, COL11A1, COL17A1, COL7A1, CST6, CTNND1, CTSC, CYP26C1, DDB2, DPH1, DSG1, DSP, DST, EDA, EDAR, EDARADD, ERCC6, ERCC2, ERCC3, ERCC4, ERCC5, EVC, EVC2, EXPH5, FGF10, FGFR2, FGFR3, GJA1, GJB2, GJB3, GJB4, GJB6, GRHL2, HOXC13, IFT122, IFT43, IKBKG, ITGA3, ITGA6, ITGB4, JUP, KANK2, KDF1, KDSR, KLHL24, KRAS, KREMEN1, KRT1, KRT14, KRT16, KRT17, KRT5, KRT6A, KRT6B, KRT6C, KRT74, KRT83, KRT85, KRT9, LAMA3, LAMB3, LAMC2, LORICRIN, MAP2K1, MAP2K2, MBTPS2, MSX1, NECTIN1, NECTIN4, NFKBIA, NLRP1, PKP1, PLEC, POLH, POMP, PRKD1, RHBDF2, RHOA, RIPK4, RSPO1, SASH1, SERPINB7, SLURP1, SMARCAD1, SNAP29, TAT, TP63, TRPM4, TRPV3, TSPEAR, TWIST2, WDR19, WDR35, WNT10A, XPA, XPC mitochondria AARS2, ACAD9, AGK, AIFM1, ATP5F1A, ATP5F1D, ATP5F1E, ATP5MD, ATPAF2, BCS1L, BOLA3, C12orf65, C1QBP, CARS2, COA3, COA5, COA6, COA8, COQ2, COQ4, COQ6, COQ7, X COQ0,8A, COQ1 COX14, COX15, COX20, COX4I1, COX5A, COX6A2, COX6B1, COX8A, CYC1, DGUOK, DNA2, EARS2, ELAC2, FARS2, FASTKD2, FBXL4, FDX2, FLAD1, FOXRED1, GATB, GATC, GFER, GFM1BP, G IBA57, ISCA2, LRPPRC, LYRM4, LYRM7, MARS2, MGME1, MICOS13, MIPEP, MPV17, MRM2, MRPL12, MRPL3, MRPL44, MRPS14, MRPS16, MRPS2, MRPS22, MRPS23, MRPS28, MRPS34, MRPS7, MT-ATP6, MT ATP8, MT-CO1, MT-CO2, MT-CO3, MT-CYB, MTFMT, MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-ND4L, MT-ND5, MT-ND6, MTO1, NARS2, NDUFA1, NDUFA10, NDUFA11, NDUFA12, NDUFA13, NDUFA2, NDUFA4, NDUFA6, NDUFA9, NDUFAF1, NDUFAF2, NDUFAF3, NDUFAF4, NDUFAF5, NDUFAF6, NDUFAF8, NDUFB11, NDUFB3, NDUFB8, NDUFB9, NDUFS1, NDUFS2, NDUFS3, NDUFS4, NDUFS6, NDUFS7, NDUFS8, NDUFV1, NDUFV2, NFU1, NSUN3, NUBPL, OPA1, PDHA1, PDSS1, PDSS2, PET100, PET117, PINK1, PNPLA4, PNPT1, POLG, POLG2, PRICKLE3, PUS1, Q TMEM TOP3A, TRIT1, TRMT10C, TRMT5, TSFM, TTC19, TUFM, TWNK, TXN2, TYMP, UQCC2, UQCC3, UQCRB, UQCRC2, UQCRFS1, UQCRQ, VARS2, WARS2, YARS2 muscle ANO5, B3GALNT2, B4GAT1, BVES, CAPN3, CAV3, CHKB, COL12A1, COL6A1, COL6A2, COL6A3, CRPPA, DAG1, DES, DNAJB6, DPM1, DPM2, DPM3, DYSF, EMD, FHL1, FKRP, FKTN, GIPC1, GMPPB, HNRNPDL, INPP5K, ITGA7, LAMA2, LARGE1, LIMS2, LMNA, MYOT, PLEC, POGLUT1, POMGNT1, POMGNT2, POMK, POMT1, POMT2, POPDC3, PYROXD1, RXYLT1, SGCA, SGCB, SGCD, SGCG, SYNE1, SYNE2, TCAP, TMEM43, TNPO3, TOR1AIP1, TRAPPC11, TRIM32, TRIP4, TTN liver ABCB11, ABCB4, AKR1D1, AMACR, ATP8B1, CYP7B1, HSD3B7, NR1H4, SLC25A13, TJP2 hearing ABCC1, ABHD12, ABHD5, ACOX1, ACSL4, ACTB, ACTG1, ADAMTS17, ADCY1, ADGRV1, AIFM1, ALMS1, AMMECR1, ANKH, AP000812.4, AP1B1, AP1S1, ARSG, ATP1A3, ATP6V1B1, ATP6V1B1, BCAPL, BCAPL BRAF, BSND, CABP2, CACNA1D, CCDC50, CD151, CD164, CDC14A, CDH11, CDH23, CEACAM16, CEP250, CEP78, CIB2, CISD2, CLCNKA, CLCNKB, CLDN14, CLIC5, CLPP, CLRN1, COCH, COL11A1, CO COL4A3, COL4A4, COL4A5, COL4A6, COL9A1, COL9A2, COQ6, CRYM, DCAF17, DCDC2, DCHS1, DIABLO, DIAPH1, DIAPH3, DLX5, DMXL2, DNAJC3, DNMT1, DSPP, EDN3, EDNRB, ELMOD3, EPS8, EPS8L2, ERCC2, ERCC3, ERCC5, ERCC6, ERCC8, ESPN, ESRP1, ESRRB, EXOSC2, EYA1, EYA4, FAT4, FDXR, FGF9, FGFR3, FITM2, FKBP14, FLNA, FOXC1, GAB1, GATA3, GFER, GIPC3, GJB2, GJB3, GJB6, GPC4, GPRASP2, GPSM2, GRAP, GRHL2, GRXCR1, GRXCR2, GSDME, HARS1, HARS2, HGF, HOMER2, HSD17B4, IARS2, IGF1, ILDR1, ITM2B, JAG1, KARS1, KCNE1, KCNE5, KCNH2, KCNJ10, KCNQ1, KCNQ4, KITLG, KRAS, LARS2, LHFPL5, LMX1A, LONP1, LOXHD1, LOXL3, MAF, MARS2, MARVELD2, MCM2, MET, MGP, MITF , MPZ, MPZL2, MSRB3, MT-CO1, MT-CYB, MYH14, MYH9, MYO15A, MYO1F, MYO3A, MYO6, MYO7A, NARS2, NDP, NF2, NLRP3, NOG, OPA1, OSBPL2, OTOA, OTOF, OTOG, OTOGL , P2RX2, PAX1, PAX3, PCDH15, PCNA, PDE1C, PDZD7, PEX1, PEX6, PEX7, PHYH, PIGL, PISD, PJVK, PLS1, PMP22, PNPT1, POU4F3, PPIP5K2, PPP2R3C, PRPS1, PTPN11, RAB40AL, RAF1, , REST, RIPOR2, ROR1, RPGR, RPS23, S1PR2, SALL1, SERAC1, SERPINB6, SIX1, SIX5, SLC17A8, SLC19A2, SLC26A4, SLC26A5, SLC33A1, SLC44A4, SLC4A11, SLC52A2, SLC52SN, SLC961, SMPXAI, SLITRK, X , SPATA5, SPNS2, SPTBN4, STRC, SYNE4, TBC1D24, TBL1X, TBL1Y, TBX22, TECTA, THRB, TIMM8A, TMC1, TMEM132E, TMEM67, TMIE, TMPRSS3, TNC, TNFRSF11A, TPRN, TRAPPC12, TRIOPEBP, TRMU, TSMU, TRARRAP , TUBB4B, TWNK, TXNL4A, TYR, USH1C, USH1G, USH2A, WBP2, WFS1, WHRN vision ARMS2, ABCA4, ABCA4, ABHD12, ABHD5, ADGRV1, AGBL1, AGBL5, AGK, AGPS, AHR, AIPL1, ARHGEF18, ARL6, ARSG, ASB10, BBS2, BEST1, BFSP1, BFSP2, C2, C3, C8orf37, C9, CA4, CACNA1F, CDH23, CEP290, CERKL, CFB, CFH, CFI, CHMP4B, CHST6, CIB2, CLCC1, CLPB, CLRN1, CNGA1, CNGA3, CNGB1, CNGB3, COL11A1, COL18A1, COL2A1, COL8A2, CRB1, CRB2, CRX, CRYAA CRYAA2, CRYAB, CRYBA1, CRYBA2, CRYBA4, CRYBB1, CRYBB2, CRYBB3, CRYGB, CRYGC, CRYGD, CRYGS, CST3, CTDP1, CWC27, CX3CR1, CYP1B1, CYP27A1, CYP4V2, DCN, DHDDNMB, DHX,38, EBP, ELOVL4, EPG5, EPHA2, ERCC1, ERCC2, ERCC6, ESPN, EXOSC2, EYS, FAM126A, FAM161A, FAR1, FBLN5, FLVCR1, FOXE3, FSCN2, FTL, FYCO1, GALK1, GALT, GCNT2, GDF6, GFER, GJA1, GJA3, GJA8, GLS, GNAT1, GNB3, GNPAT, GPATCH3, GRHL2, GRK1, GRM6, GSN, GUCA1B, GUCY2D, HARS1, HGSNAT, HK1, HMCN1, HMX1, HSF4, HTRA1, IARS2, IDH3B, IFT140, IFT172, IFT43, IMPDH1, IMPG2, INPP5K, IQCB1, KCNA4, KCNJ13, KIAA1549, KIF3B, KIZ, KLHL7, KRT12, KRT3, LCA5, LCAT, LEMD2, LIM2, LONP1, LOXL1, LRAT, LRIT3, LSS, LTBP2, MAF, MAK, MBNL1, MERTK, MFRP, MIP, MSMO1, MT-ATP6, MT-CO1, MT-CO3, MT-CYB, MT-ND1, MT-ND2, MT-ND4, MT-ND4L, MT-ND5, MT-ND6, MVK, MYH9, MYO7A, MYOC, NEK2, NHS, NMNAT1, NPHP1, NPHP4, NR2E3, NRL, NTF4, NUP188, NYX, OCRL, OFD1, OPA3, OPTN, OVOL2, PCARE, PCDH15, PDE6A, PDE6B, PDE6G, PDZD7, PEX7, PHYH, PIK3C2A, PIKFYVE, PISD, PITX3, PNPLA6, POMGNT1, PRCD, PRICKLE3, PROM1, PRPF3, PRPF31, PRPF4, PRPF6, PRPF8, PRPH2, PRPS1, RAB3GAP2, RAH2, RBPRD3, 1, RD3, RDH12, RECQL4, REEP6, RGR, RHO, ROM1, RP1, RP1L1, RP2, RP9, RPE65, RPGR, RPGRIP1, SAG, SCAPER, SDCCAG8, SEMA4A, SIPA1L3, SIX6, SLC16A12, SLC24A1, SLC2A1, SLC33A1, SLC4A14, SLC7A14, SNRNP200, SPATA7, SRD5A3, TACSTD2, TBK1, TCF4, TDRD7, TEK, TGFBI, TKFC, TLR4, TMCO1, TMEM67, TOPORS, TRAF3IP1, TRNT1, TRPM1, TTC8, TUBB4B, TULP1, UBIAD1, UNC45B, USH1C, USH2A, USP45, VCAN, VIM, VSX1, VSX2, WDR19, WDR36, WFS1, WHRN, ZEB1, ZNF408, ZNF513 cancer A2M, AARS2, ABCB1, ABCC1, ABCC2, ABCC3, ABCC5, ABCC6, ABCG2, ABI1, ABL1, ABL2, ACAP1, ACKR3, ACLY, ACO1, ACP3, ACSL3, ACVR1, ACVR1B, ACVR2A, ACVR2B, ADAM10, ADAM9, ADAMTS1, ADAMTS14, ADAMTS18, ADAMTS20, ADAMTS3, ADAMTS4, ADAMTS5, ADAMTS6, ADAMTS8, ADAMTS9, ADCY1, ADGRB1, ADM, ADNP, ADORA2A, ADRA1B, AFDN, AFF1, AFF3, AFF4, AFP, AGER, AHNAK2, AHR, AHSBA,G, AJU AK9, AKAP12, AKAP9, AKR1B1, AKT1, AKT2, AKT3, ALB, ALCAM, ALDOA, ALDOB, ALDOC, ALK, ALKBH6, ALPK2, ALPL, ALPP, AMER1, AMPH, ANAPC1, ANG, ANGPT1, ANGPT2, ANK3, ANKRD12, ANXA1, ANXA11, ANXA2, ANXA4, ANXA7, AOC3, AP2B1, APAF1, APC, APEX1, APOA1, APOA2, APOBEC3B, APOC1, APOC3, APOD, APOE, APOL2, APPBP2, AR, AREG, ARG2, ARHGAP26, ARHGAP33, ARHGEF12, ARHGEF6, ARID1A, ARID1B, ARID2, ARID5B, ARNT, ASPH, ASPM, ASPSCR1, ASXL1, ASXL2, ATF1, ATG13, ATIC, ATM, ATOH1, ATP1A1, ATP2B3, ATP7B, ATR, ATRX, AURKA, AURKA, KB, AXIN AXIN2, AZGP1, B2M, BAD, BAG1, BAP1, BARD1, BAX, BCL10, BCL11A, BCL11B, BCL2, BCL2A1, BCL2L1, BCL2L2, BCL2L2-PABPN1, BCL3, BCL6, BCL7A, BCL9, BCL9L, BCLAF1, BCOR, BCORL1, BCR, BDNF, BHMT2, BIRC2, BIRC3, BIRC5, BIRC6, BIVM-ERCC5, BLK, BLM, BLMH, BMI1, BMP2, BMP4, BMPR1A, BNIP3, BNIP3L, BRAF, BRCA1, BRCA2, BRD3, BRD4, BRIP1, BRMS1, BTG1, BTG2, BTK, BUB1B, C1QBP, C3orf70, C6, C7, CA8, CACNA1D, CAD, CALCA, CALR, CAMTA1, CANT1, CANX, CAP2, CAPN6, CARD11, CARM1, CARS1, CASC3, CASP1, CASP10, CASP2, CASP3, CASP4, CASP5, CASP6, CASP7, CASP8, CASP9, CAST, CAT, CAV1, CBFA2T3, CBFB, CBL, CBLB, CBLC, CCAR1, CCDC120, CCDC6, CCKBR, CCL11, CCL13, CCL14, CCL16, CCL18, CCL19, CCL2, CCL21, CCL23, CCL3, CCL4, CCL5, CCL7, CCL8, CCN2, CCN4, CCNA1, CCNA2, CCNB1, CCNB1IP1, CCNB2, CCND1, CCND2, CCND3, CCNE1, CCNE2, CCNG1, CCNG2, CCNH, CCR10, CCR7, CD14, CD1D, CD24, CD27, CD274, CD36, CD38, CD40, CD40LG, CD44, CD46, CD52, CD59, CD70, CD74, CD79A, CD79B, CD82, CD9, CDC16, CDC20, CDC25A, CDC25B, CDC25C, CDC27, CDC34, CDC37, CDC6, CDC73, CDH1, CDH11, CDH17, CDH5, CDK1, CDK12, CDK2, CDK4, CDK6, CDK7, CDKN1C, CDKN1B CDKN 2A, CDKN2C, CDX2, CEACAM5, CEACAM6, CEBPA, CENPF, CEP43, CEP76, CFH, CFHR1, CFLAR, CFTR, CGA, CHCHD7, CHD4, CHD7, CHD8, CHEK1, CHEK2, CHFR, CHGA, CHI3L1, CHP2, CIB2, CIC, CIITA, CKB, CKS1B, CKS2, CLDN3, CLDN4, CLDN7, CLEC3B, CLIC1, CLIP1, CLSTN1, CLTC, CLTCL1, CLU, CNBD1, CNBP, CNKSR1, CNN1, CNOT3, CNTF, CNTRL, COL11A1, COL17A1, COL18A1, COL1A1, COL1A2, COL2A1, COL4A2, COL4A3, COL4A4, COL4A5, COL5A1, COL5A3, COL6A1, COX17, CP, CRABP1, CRADD, CREB1, CREB3L1, CREB3L2, CREBBP, CRLF2, CRP, CRTC1, CRTC3CSL, DE, CRYAB, CSE, CSF1, CSF1R, CSF2, CSF2RA, CSF3, CSF3R, CSN1S1, CSNK1E, CSNK2A2, CSNK2B, CST3, CST6, CSTA, CSTB, CTAG1A, CTAG1B, CTAG2, CTCF, CTNNB1, CTNNBL1, CTNND1, CTSB, CTSD, SL, CTSH, CTTN, CUL1, CUL2, CUL4B, CUL5, CUX1, CXCL1, CXCL10, CXCL13, CXCL2, CXCL5, CXCL8, CXCL9, CXCR1, CXCR2, CXCR4, CYB5R3, CYLD, CYP19A1, CYP1A2, CYP2C19, CYP2E1, CYP3A5, C DAPK1, DAXX, DBI, DCC, DCN, DCTN1, DDB2, DDIT3, DDR2, DDX10, DDX3X, DDX5, DDX6, DEFA1, DEFA1B, DEFA3, DEK, DES, DHFR, DH X9, DIAPH1, DIAPH3, DICER1, DIS3, DLC1, DMD, DNAH12, DNAJB1, DNAJC2, DNER, DNM2, DNMT3A, DOCK2, DROSHA, DST, DUSP1, DUSP14, DUSP4, DVL3, DYNLL1, DYRK2, E2F1, E2F3, E2F5, EBAG9, EBF1, EDN1, EEF1A1, EEF2, EFNA1, EFNA2, EFNA5, EFNB1, EFNB2, EFNB3, EGF, EGFR, EGR1, EI24, EIF2S2, EIF3E, EIF3H, EIF4A2, EIF4E, EIF4EBP1, EIF4G1, EIF4H, EIF,5A, ELANE ELF3, ELF4, ELK3, ELK4, ELL, EML4, ENC1, ENG, ENO1, ENO2, ENPP2, EP300, EPAS1, EPCAM, EPHA1, EPHA2, EPHA3, EPHA4, EPHA7, EPHA8, EPHB2, EPHB3, EPHB4, EPHX1, EPO, EPOR, EPS15, ERBB2, ERBB3, ERBB4, ERC1, ERCC1, ERCC2, ERCC3, ERCC4, ERCC5, ERCC6, ERG, ESR1, ESR2, ETNK1, ETV1, ETV4, ETV5, ETV6, EWSR1, EXT1, EXT2, EZH1, EZH2, EZR, F13A1, F13B, F2, F3, FABP1, FABP2, FABP4, FABP5, FADD, FAF1, FAM166A, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FAP, FAS, FASLG, FASN, FAT1, FAT4, FBN2, FBXO11, FBXO6, FBXW7, FCER2, FCGR2B, FCRL4, FEN1, FES, FEV, FGA, FGB, FGF1, FGF17, FGF18, FGF19, FGF2, FGF23, FGF3, FGF4, FGF6, FGF7, FGF8, FGF9, FGFBP1, FGFR1, FGFR2, FGFR3, FGFR4, FGG, FH, FHIT, FIP1L1, FKBP5, FKBP8, FLCN, FLG, FLI1, FLT1, FLT3, FLT4, FMO5, FN1, FOLH1, FOS, FOSL1, FOXA1, FOXA2, FOXJ1, FOXL2, FOXM1, FOXO1, FOXO3, FOXO4, FOXP1, FOXQ1, FRMD7, FSCN1, FSHB, FST, FSTL3, FTH1, FTL, FUBP1, FUS, FZD1, FZD2, G6PD, GADD45A, GADD45G, GAS1, GAS7, GAST, GATA1, GATA2, GATA3, GCLM, GDF15, GDNF, GH1, GH2, GJA1, GJB5, GLO1, GMNN, GNA11, GNA13, GNAI1, GNAQ, GNAS, GNB1, GNPTAB, GOLGA5, GOPC, GOT1, GOT2, GPA33, GPC3, GPHN, GPI, GPS2, GPX1, GPX2, GRB10, GRB2, GRB7, GRIN2A, GSK3A, GSN, GSR, GSTM1, GSTM3, GSTP1, GTF2H1, GUSB, H2AC6, H3-3A, H3-3B, H3C2, H4C9, HDAC10, HDAC2, HDAC5, HERPUD1, HEY1, HGF, HGFAC, HIF1A, HIP1, HIP1R, HK1, HK2, HLA-A, HLA-B, HLA-G, HLF, HMGA1, HMGA2, HMGXB4, HMOX1, HNF1A, HNRNPA2B1, HOOK3, HOXA11, HOXA13, HOXA5, HOXA9, HOXC11, HOXC13, HOXD11, HOXD13, HP, HPGD, HPN, HRAS, HSF1, HSP90AA1, HSP90AB1, HSP90B1, HSPA1L, HSPA2, HSPA4, HSPA8, HSPB1, HSPD1, HSPE1, HSPH1, HUWE1, IBSP, ICAM1, ID1, ID2, ID3, IDH1, IDH2, IDO1, IFNA1, IFNA13, IFNAR1, I FNAR2, IFNB1, IFNG, IGF1R, IGF2, IGF2R, IGFBP2, IGFBP3, IKBKB, IKZF1, IL10, IL11, IL12A, IL13, IL13RA2, IL15, IL16, IL17A, IL17B, IL18, IL1A, IL1B, IL1R1, IL1R2, IL2, IL21R, IL24, IL2RA, IL2RB, IL2RG, IL4, IL4R, IL5, IL6, IL6R, IL6ST, IL7, IL7R, IL9, ILF3, ILK, ING1, INHBA, INHBB, INPPL1, INS, INTS12, IPO7, IRF1, IRF4, IRF6, IRS2, IRS4, ITGA1, ITGA2, ITGA2B, ITGA3, ITGA4, ITGA5, ITGA6, ITGAM, ITGAV, ITGB1, ITGB3, ITGB4, ITGB5, ITGB6, ITGB7, ITGB8, ITIH4, ITK, ITPKB, JAK1, JAK2, JAK3, JKAMP, JTB, JUN, JUND, JUP, KALRN, KAT2B, KAT6A, KAT6B, KCNJ5, KDM5A, KDM5C, KDM6A, KDR, KDSR, KEAP1, KEL, KIAA1109, KIF2A, KIF2C, KIF5B, KIFC3, KISS1, KIT, KITLG, KLF4, KLF5, KLF6, KLHL8, KLK10, KLK11, KLK13, KLK14, KLK15, KLK2, KLK3, KLK4, KLK5, KLK6, KLK7, KLK8, KLK9, KLRK1, KMT2A, KMT2B, KMT2C, KMT2D, KNL1, K KRT13, KRT14, KRT15, KRT17, KRT18, KRT19, KRT4, KRT8, KTN1, LALBA, LAMB1, LAMC1, LASP1, LATS1, LATS2, LCK, LCN1, LCP1, LCTL, LDHA, LEF1, LEP, LEPR, LGALS3, LGALS3BP, LGALS4, LGI1, LGMN, LHB, LHX1, LIF, LIFR, LIG4, LIMK1, LMNA, LMO1, LMO2, LPP, LRIG3, LRP1B, LRP6, LRRK2, LTA, LTA4H, LTB, LTBR, LTF, LUM, LYL1, LYN, LZTR1, MAD2L1, MAD2L2, MAF, MAFB, MAGEA3, MAGEA4, MAGEA6, MAGEB5, MAGEB6, MAGEC1, MAGEC2, MAGEC3, MAGED1, MAGED2, MAGI1, MALT1, MAML2, MAP2K1, MAP2K2, MAP2K4, MAP3K1, MAP3K13, MAP3K3, MAP3K4, MAP4K3, MAPK4, MAPK1, MAPK3, MAPK7, MAPK8, MAPK8IP1, MAPKAPK2, MAST2, MATK, MAX, MBD1, MBD2, MBD4, MCL1, MCM2, MCM3, MCM5, MCM7, MDC1, MDH1, MDK, MDM2, MDM4, MECOM, MECP2, MED1, MED12, MED13, MED17, MED23, MEF2A, MEN1, MET, METTL14, MFGE8, MGA, MGMT, MIA, MIF, MITF, MKI67, MLF1, MLH1, MLH3, MLLT1, MLLT10, MLLT11, MLLT3, MLLT6, MME, MMP1, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP2, MMP3, MMP7, MMP8, MMP9, MN1, MORC4, MPL, MPO, MRE11, MRTFA, MSH2, MSH6, MSI2, MSLN, MSMB, MSN, MSR1, MST1, MT1A, MT1G, MTA1, MTCP1, MTOR, MUC1, MUC17, MUTYH, MVP, MXI1, MXRA5, MYB, MYBL2, MYC, MYCL, MYCN, MYD88, MYH11, MYH9, MYO5A, MYOCD, MYOD1, MYOG, NAB2, NAGA, NAIP, NAMPT, NAT2, NAV3, NBN, NBPF1, NBPF10, NCAM1, NCOA1, NCOA2, NCOA3, NCOA4, NCOR1, NCOR2, NDRG1, NEB, NEDD4L, NEDD8, NEO1, NF1, NF2, NFATC2, NFE2L2, NFIB, NFKB1, NFKB2, NFKBIA, NFKBIE, NGF, NGFR, NIBAN1, NIN, NKX2-1, NKX3-1, NLRP3, NME1, NME2, NONO, NOS1, NOS2, NOS3, NOTCH1, NOTCH2, NOTCH3, NPM1, NQO1, NR0B1, NR1H2, NR4A2, NR4A3, NRAS, NRG1, NRG2, NRG3, NRP1, NRP2, NSD1, NSD2, NSD3, NT5C2, NTF3, NTF4, NTHL1, NTN1, NTN4, NTRK1, NTRK2, NTRK3, NUCB2, NUDT1, NUMA1, NUMB, NUP210L, NUP214, NUP93, NUP98, NUTM1, NUTM2B, NUTM2D, ODAM, OGG1, OLIG2, OMA1, OR4A16, OR51E2, OR52N1, ORM1, OSM, OTUD7A, P2RY8, PABPC1, PAFAH1B2, PAGE4, PALB2, PAPPA, PARP1, PARVB, PATZ1, PAX,3, PAX5, PAX8, PBRM1, PBX1, PCBP1, PCM1, PCNA, PDAP1, PDCD1LG2, PDCD2L, PDE4DIP, PDGFA, PDGFB, PDGFRA, PDGFRB, PDSS2, PDZD4, PECAM1, PER1, PF4, PGC, PGF, PGR, PHF20, PHF6, PHLDA1, PHOX2B, PICALM, PIGR, PIK3CA, PIK3CB, PIK3CG, PIK3R1, PIK3R2, PIK3R3, PIM1, PIM2, PIM3, PIN1, PIP4K2B, PIP5K1A, PKM, PLAG1, PLAT, PLAU, PLAUR, PLCG1, PLCG2, PLEC, P LG, PLK1, PLP1, PMEPA1, PML, PMP22, PMS1, PMS2, PNMT, POLD1, POLE, POLQ, POMC, PON1, POSTN, POT1, POTEF, POU2AF1, POU2F2, POU5F1, PPA2, PPARG, PPARGC1A, PPFIBP1, PPM1D, PPP1R15A, PPP2R1A, PPP6C, PPY, PRCC, PRDM1, PRDM13, PRDM16, PRDX2, PRDX4, PREX2, PRF1, PRKACA, PRKAR1A, PRKCA, PRKCB, PRKCD, PRKCE, PRKCH, PRKCI, PRKCQ, PRKDC, PRL, PROC, PRRX1, PRSS1, PSCA, PSEN1, PSIP1, PSMD4, PTCH1, PTCH2, PTEN, PTGS1, PTGS2, PTH, PTHLH, PTK2, PTK6, PTN, PTPN11, PTPN13, PTPRB, PTPRC, PTPRK, PTPRO, PTPRT, PTTG1, PURA, PZP, QKI, RAB11FIP3, RAB18, RAB25, RAB40A, RABEP1, RAC1, RAD21, RAD23A, RAD23B, RAD51, RAD51B, RAD51D, RAD52, RAD54B, RAF1, RANBP2, RANBP3, RAP1GDS1, RARA, RARB, RARG, RASA1, RB RBBP4, RBL1, RBL2, RBM10, RBM15, RBM6, RBMX, RBP4, RECQL4, REL, RELA, RELB, RET, RHEB, RHOA, RHOB, RHOC, RHOH, RIT1, RMC1, RMI2, RNF213, RNF43, ROS1, RPA2, RPGR, RPL10, RPL22, RPL27, RPL5, RPN1, RPS15, RPS2, RPS3, RPS6KA1, RPS6KA3, RSBN1L, RSPO2, RSPO3, RUNX1, RUNX1T1, RXRA, RXRB, RXRG, S100A1, S100A2, S 100A4, S100A6, S100A7, S100A8, S100A9, S100B, S1PR1, SACS, SALL4, SART1, SBDS, SCGB1A1, SCGB1D2, SCGB2A1, SCGB2A2, SDC1, SDC4, SDHA, SDHAF2, SDHB, SDHC, SDHD,SELL, SELE, SEMA3B, SEPTIN2, SERPINA1, SERPINA3, SERPINA5, SERPINB13, SERPINB2, SERPINB3, SERPINB4, SERPINE1, SERPINF1, SET, SETBP1, SETD2, SETDB1, SF3B1, SFN, SFPQ, SFRP4, SGK1, SH2B3, SH3B3, SIINSH2, SIINSH1, SIRT4, SIX1, SKP2, SLC19A1, SLC1A3, SLC26A3, SLC2A1, SLC34A2, SLC3A2, SLC44A3, SLC45A3, SLC4A5, SLPI, SMAD1, SMAD2, SMAD3, SMAD4, SMARCA4, SMARCB1, SMARCD1, SMARCE1, SMC3Y, SMC1A, S SND1, SNX25, SOCS1, SOD1, SOD2, SOS1, SOX1, SOX17, SOX2, SOX9, SP1, SPARC, SPARCL1, SPATA6, SPEN, SPINK1, SPINT1, SPINT2, SPOP, SPP1, SPRR1B, SPRR3, SPRY1, SRC, SRD5A1, SRD5A2, SRSF2, SRSF3, SS18, SS18L1, SST, SSX1, SSX2, SSX2B, SSX4, ST14, STAG2, STARD3, STAT3, STAT4, STAT5A, STAT5B, STAT6, STEAP1, STIL, STK11, STK19, STMN1, STRAP, STRN STT3A, STX2, SUFU, SULT1E1, SUZ12, SYK, SYNE1, TAF1, TAF15, TAGLN, TAL1, TAL2, TAP1, TBC1 D12, TBL1XR1, TBX3, TCEA1, TCF12, TCF3, TCF7, TCF7L2, TCL1A, TCP11L2, TDRD10, TDRD6, TEK, TENT5C, TERT, TET1, TET2, TF, TFAP2B, TFDP1, TFDP2, TFE3, TFEB, TFF1, TFF2, TFF3, TFG, TFRC, TG, TGFA, TGFB2, TGFB3, TGFBR2, TGFBR3, TGIF1, TGM4, TGM7, THBS1, THBS2, THBS4, THPO, THRA, THRB, TIE1, TIMM17A, TIMP1, TIMP2, TIMP3, TJP2, TK1, TLX1, TLX3, TMEM127, TMF1, TMPRSS2, TMPRSS3, TNC, TNF, TNFAIP2, TNFAIP3, TNFRSF10A, TNFRSF10B, TNFRSF10C, TNFRSF10D, TNFRSF11B, TNFRSF12A, TNFRSF14, TNFRSF17, TNFRSF1A, TNFRSF1B, TNFRSF4, TNFRSF8, TNFRSF9, TNFSF10, TNFSF11, TNFSF13, TNFSF13B, TNFSF4, TNFSF8, TNK2, TOM1, TOP1, TOP2A, TOP3A, TP53, TP53BP1, TP53BP2, TP63, TPD52, TPI1, TPM1, TPM2, TPM3, TPM4, TPR, TPX2, TRAF1, TRAF2, TRAF3, TRAF4, TRAF7, TRIM23, TRIM24, TRIM25, TRIM27, TRIM33, TRIM7, TRIO, TRIP11, TRIP4, TRO, TRRAP, TSC1, TSC2, TSG101, TSHR, TSPAN8, TSPO, TTLL9, TTR, TUBA1A, TUSC2, TWIST1, TXLNA, TXNDC8, TXNIP, TXNRD1, TYMP, TYMS, TYRO3, U2AF1, UBA1, UBE2C, UBE2I, UBE2N, UBR5, UGT1A1, UGT1A10, UGT1A3, UGT1A4, UGT1A9, UHRF1BP1L, USH1C, USP6, USP8, VAMP3, VCAM1, VEGFA, VEGFB, VEGFC, VEGFD, VHL, VIL1, VIP, VTN, VWF, WAS, WASF3, WDCP, WEE1, WFDC2, WIF1, WNT1, WNT2, WRN, WT1, WWTR1, XBP1, XIAP, XIRP2, XPA, XPC, XPO1, XRCC1, XRCC2, XRCC3, XRCC4, XRCC5, XRCC6, YBX1, YWHAB, YWHAE, YWHAH, ZBTB16, ZFHX3, ZFP36L1, ZFP36L2, ZMYM2, ZNF132, ZNF180, ZNF300, ZNF331, ZNF384, ZNF471, ZNF483, ZNF521, ZNF620, ZNF750, ZNF814, ZNF844, ZNF91, ZRANB3, ZRSR2 genetic disease AGPAT2, AGRN, AIPL1, ALG1, ALG11, ALG12, ALG13, ALG14, ALG2, ALG3, ALG6, ALG8, ALG9, ATP6AP1, ATP6AP2, B3GALNT2, B3GLCT, B4GALT1, B4GAT1, BPGM, BSCL2, CACNA1F, CAD, CAV1, CAVIN1, CCDC115, CDAN1, CDIN1, CEP290, CHAT, CHKB, CHRNA1, CHRNB1, CHRND, CHRNE, CNGA3, COG1, COG2, COG5, COG6, COG7, COG8, COL12A1, COL13A1, COL6A1, COL6A2, COL6A3, COLQ PA, CRB1, C CRX, CYP11B1, CYP17A1, CYP21A2, DAG1, DDOST, DOK7, DOLK, DPAGT1, DPM1, DPM2, DPM3, DUOX2, DUOXA2, EGLN1, EPAS1, EPO, EPOR, FCSK, FKRP, FKTN, FOXE1, FUT8, GALNT2, GDF6, GFPT1, GIPC1, GLIS3, GMPPB, GNAT1, GNB3, GRK1, GRM6, GUCY2D, HBB, HSD3B2, IGSF1, IMPDH1, INPP5K, IQCB1, IRS4, ITGA7, IYD, KCNJ13, KCNJ6, KLF1, LAMA2, LARGE1, LCA5, LMNA, LPIN2, LRAT, LRIT3, LRP4, MAGT1, MGAT2, MPDU1, MPI, MUSK, MYO7A, MYO9A, NKX2-1, NMNAT1, NPHP1, NPHP4, NUS1, NKX2-5, NYX, PAX8, PDE6B, PGM1, PMM2, POMGNT1, POMGNT2, POMK, POMT1, POMT2, POR, PREPL, RAPSN, RD3, RDH12, RFT1, RHO, RPE65, RPGRIP1, RXYLT1, SAG, SCN4A, SDCCAG8, SEC23B, SLC18A3, SLC24A1, SLC25A1, SLC35A1, SLC35A2, SLC35C1, SLC39A8, SLC5A5, SLC5A7, SNAP25, SPATA7, SRD5A3, SSR4, STAR, STT3A, STT3B, SYT2, TBL1X, TG, THRA, TMEM165, TMEM199, TPO, TRAF3IP1, TRHR, TRIP4, TS TUBB4B, TULP1, USP45, VAMP1, VHL, WDR19

Table 1. Amino acid sequences of dimerization domains (binding elements), including sequences of designed heterodimers (DHD). 6dlc_DHD37_1:234_A SEQ ID NO: 1 DSDEHLYKLKTFLENLRRHLDRLDKHIKQLRDILSENPEDERVKDAIDLSERSVRIVKTVIKIFEDSVRKKEKRPIDKRDDKELDKLLDTLEKILQTATKIIDDANKLLEYLRR 6dlc_DHD37_1:234_B SEQ ID NO: 2 DPKVVETYVELLKRHEKAVKELLEIAKTHAKKVE DHD13_1:234_A SEQ ID NO: 3 SEAMKRMLKLLEESLRLLKELLELSEESAQLLYEAR DHD13_1:234_B SEQ ID NO: 4 TTKRYLEEAERAHREQKEIIKKAQELHRRLEEIVRQSGSSEEAKKEAKKILEEIRELELSKRSLELLREILYLSQQVNDDVEKALERQRKIIERAQEIHRRQQEILEELERIIRKPGS DHD94_3:214_A SEQ ID NO: 5 SPERDENRKLLDKVRKLVEKSRRLVEELRKLVDQSTKN DHD94_3:214_B SEQ ID NO: 6 SDEKALRKQQEVLRKVEEVLEKQERVLRELEEISYRVITRGEDHKAEEDSRRVLERFVRVSREVLKVLEEFLRVSEELLREADDRDRDRRLEEYERQVDELREEIRRYKEEVDKFDKEVKYYKK DHD94_2:143_A SEQ ID NO: 7 SDRRLEEYERQVDELREEIRRYKEEVDKFDKEVKYYKK DHD94_2:143_B SEQ ID NO: 8 SPERDENRKLLDKVRKLVEKSRRLVEELRKLVDQSTKNGLIDEKALRKQQEVLRKVEEVLEKQERVLRELEEISYRVITRGEDHKAEEDSRRVLERFVRVSREVLKVLEEFLRVSEELLREADR ZCON_17A_8 SEQ ID NO: 9 DRKDLLKRNIKLLDRHLKILDTILKLLEKLSELLKKSSSEEVVKEYKKILDEIRKLLEESKEIHKESKEILERES ZCON_17B_8' SEQ ID NO: 10 DEEKLIERSKRLQEESEQLLEKFEQILRELTELLEKPDSEELARKIKKLHDELRKIIKRNQELIREHEEILRKRD DHD37-short-A (DHD-A) SEQ ID NO: 11 SPEFLENLRRHLDRLRKHIRKLEEILEEIDDQMVKEAIDLSRRSVEIVEEVIETFERAGDASPTKLDELEKILQRAEEIIDRADKLLEYRRR DHD37-short-A-KtoR (DHD-A-KtoR) SEQ ID NO: 12 SPEFLENLRRHLDRLRHIRRLEEILEEIDDQMVREAIDLSRRSVEIVEEVIETFERAGDASPTKLDELERILQRAEEIIDRADRLLEYRRR DHD37-short-B (DHD-B) SEQ ID NO: 13 DPKVVEEYVRLLEEHEKAVRELLEIAK DHD37-short-B-Y8A SEQ ID NO: 14 DPKVVEEAVRLLEEHEKAVRELLEIAK DHD37-short-B-Ntrunc (DHD-B-Ntrunc) SEQ ID NO: 15 KVVEEYVRLLEEHEKAVRELLEIAK DHD37-short-B-KtoR (DHD-B-KtoR) SEQ ID NO: 16 DPRVVEEYVRLLEEHERAVRELLEIAR DHD37-short-B-KtoR-var2 SEQ ID NO: 17 DPEVVAAYVRLLEEHERAVRELLEIAR DHDredesign1_YtoH_LtoV-A SEQ ID NO: 18 SPEFLENLRRHLDRMSKHARKLEEILEKGDDQAVKESIDLVRRSVSIVQAVIETFERAGDASPTKLDALAKILQRATEIIDRADKLLEALRR DHDredesign1_YtoH_LtoV-B SEQ ID NO: 19 DPKVVEEHVRLIEEHEKAIRELLEIAK DHDredesign2_YtoH_LtoV_shorter-A SEQ ID NO: 20 SPEFLENLARHLDRMSKHARKLEEILEKGSDQAVKESIDLVRRSVSIVQAVIKTGSGDPELAKILQRATEIIDRADKLLEALRR DHDredesign2_YtoH_LtoV_shorter-B SEQ ID NO: 21 DPKVVEEHVRLIEEHEKAIRELLK DHDredesign3.2_YtoH_shorter-A SEQ ID NO: 22 SENLARHLERLSKHARKLEEILERGSDQAVKESIELVRESVEIVQAVIKGSRGSPVLQRATEIIDRADQLLESLSR DHDredesign3_YtoH_shorter-A SEQ ID NO: 23 SPEFLENLARHLDRLSKHARKLEEILEKGSDQAVKESIDLVRESVSIVQAVIKTGSGSPELAKILQRATEIIDRADKLLEALRR DHDredesign3_YtoH_shorter-B SEQ ID NO: 24 DPRVVEEHVELIREHARAISELLR 40_-2_surface_redesign_DHD37-short-A SEQ ID NO: 25 SPEFASALNQHLNALRSHIEELQEIANTIDDPMVHTAINLSQRSVDIVETVIQTFSEAGDASPNAETTLTNILRRATSIIQQAQSLLTHRKR 40_-8_surface_redesign_DHD37-short-A SEQ ID NO: 26 SPEFARALHHHLTALQEHITALQQIANEGDDEMVHQAIKLSQRSVQIVNEVINTFNSSGDASPNAAKELQEILERATEIIEQATNLLTKRAR 40_-16_surface_redesign_DHD37-short-A SEQ ID NO: 27 SPEFAEELQEHLNALESHIEALQEIANTIDDEMVHQAIQLSQRSVNIVQTVEETFSSAGDASPNAATTLEQILHRATQIIEEATNLLTERAR 50_-3_surface_redesign_DHD37-short-A SEQ ID NO: 28 SPEFLEELRHHLKALKSHIEALQHIADKIDDEMVHQAIQLSQRSVNIVHQVIKTFQQAGDASPKKAETLTQILHRATEIIKEATKLLEKRAR 50_-9_surface_redesign_DHD37-short-A SEQ ID NO: 29 SPEFEEKLKHHLEELENHIKALENIANEIDDQMVHNAIKLSQRSVEIVHTVIETFSEAGDASPNAATKLTNILHRATKIINTAEELLTERAR 50_-19_surface_redesign_DHD37-short-A SEQ ID NO: 30 SPEFLEELNHHLSALESHITALQEIANEGDDEMVHQAIELSQRSVQIVKTVIETFEESGDASPEEASTLESILERATQIIQEATELLQERAR 60_-3_surface_redesign_DHD37-short-A SEQ ID NO: 31 SPEFEKNLKEHLDELRKHITELQNIANKIDDPMVHKAIKLSQRSVQIVEEVINTFSSAGDASPKAAKKLEQILQRASQIIRDAQELLEERAR 60_-10_surface_redesign_DHD37-short-A SEQ ID NO: 32 SPEFLENLEQHLEALEKHIEALQEIAKKGDDQMVEEAIKLSQRSVEIVQKVIQTFQKSGDASPPNKAKTLEEILQRAKEIIKEAEELLEHRAR 60_-23_surface_redesign_DHD37-short-A SEQ ID NO: 33 SPEFLEALEQHLSELERHIKALQEIADEIDDKMVHEAIELSQRSVNIVNTVIETFQEAGDASPNEEEEELEQILERATSIIEQAEELLEHRAR 70_-2_surface_redesign_DHD37-short-A SEQ ID NO: 34 SPEFLNELREHLRKLSEHITELQNIADEGDDEMVHRAIKLSQRSVEIVQKVEETFQRSGDASPNEATKLEEILKRADKIIEKARELLKKRAR 70_-12_surface_redesign_DHD37-short-A SEQ ID NO: 35 SPEFLENLRRHLDELEKHIQELQHIADEIDDEMVERAIELSQRSVEIVEEVIETFQQAGDASPNKAKELEEILQRAKEIINRADELLKKRAR 70_-24_surface_redesign_DHD37-short-A SEQ ID NO: 36 SPEFLEALSEHLEALSEHISELEEIANEGDDRMVEQAINLSQRSVEIVEEVKSTFEKSGDASPNKEETLEEILERAEEIIEHARELLEERER 80_-3_surface_redesign_DHD37-short-A SEQ ID NO: 37 SPEFLKELEEHLEKLEKHIQKLQEIADEGDDKMVEKAIELSQRSVEIVERVKKTFEESGDASPKKKDELSKILERATKIIKDANELLQKRKR 80_-13_surface_redesign_DHD37-short-A SEQ ID NO: 38 SPEFLKELEEHLRELEEHITELEQIARKGDDEMVEEAIELSQRSVQIVREVKKTFKDSGDASPNEAETLKEILKRAEKIIEDADKLLKERKR 80_-29_surface_redesign_DHD37-short-A SEQ ID NO: 39 SPEFARLLEQHLEELEEHIEALEEILDEIDDRMVEKAIELSQRSVEIVEEVKETFSEAGDASPEEKEELEEILERAEEIIEEAEKLLEERER 70_-26_Asp-surface_redesign_DHD37-short-A SEQ ID NO: 40 SKDFLDELDDHLLDLLEAHIAELEDIADKIDDDMVDAAIKLSERSEAIVDQVIKTFEAAGDASDDALRELEAILDRAEDIIDDADALLEARER 80_-29_Asp-surface_redesign_DHD37-short-A SEQ ID NO: 41 SEDFLDKLREHLDELDDHIKKLADIEDDIDDDMVADAEELSERSADIVKEVIDTFEDAGDASKKAAAADLDDILDRAEAIIDEADDLLEERER 70_-12_Asp-surface_redesign_DHD37-short-A SEQ ID NO: 42 SEKFLEKLEDHLAKLEDHIAKLEDIADNIDDDMVKEAIDLSDRSKAIVKAVIATFKAAGDASPNEADKLDKILDRADKIIEDAADLLKARER 80_-3_Asp-surface_redesign_DHD37-short-A SEQ ID NO: 43 SPDFEEELARHLKKLKKHEKKLRRAIAAKIDDKMVDDAIKLSKRSEAIVKDVEKTFKAAGDASPDEEAKLKKILDRADKIIDDADKLLEDRER 70_-12_Asp-surface_redesign_DHD37-short-A-KtoR SEQ ID NO: 44 SERFLERLEDHLARLEDHIARLEDIADNIDDDDMREAIDLSDRSRAIVRAVIATFRAAGDASPNEADRLDRILLDRADRIIEDDADLLRARER 70_-26_Asp-surface_redesign_DHD37-short-A-KtoR SEQ ID NO: 45 SRDFLDELDDHLLDLLEAHIAELEDIADRIDDDMVDAAIRLSERSEAIVDQVIRTFEAAGDASDDALRELEAILDRAEDIIDDADALLEARER 80_-29_Asp-surface_redesign_DHD37-short-A-KtoR SEQ ID NO: 46 SEDFLDRLREHLLDELDDHIRRLADIEDDIDDDMVADAEELSERSADIVREVIDTFEDAGDASRRAAADLDDILDRAEAIIDEADDLLEERER 2plus1_5400_dimer 1.1_short-B SEQ ID NO: 47 SELAERI KRSHERLEELLAEVKRLIER 2plus1_5400_dimer 1.1_short-A SEQ ID NO: 48 DPAKEVLEELYRASKRSLEEFERELREKGSDPESARELERIVKENAELAERVFREVEKIVR 2plus1_5400_dimer 1.2_short-A SEQ ID NO: 49 SELAERIKRSHERLEELLAEVKRLIERGSGSDPAKEVLEELYRASKRSLEEFERELREK 2plus1_5400_dimer 1.2_short-B SEQ ID NO: 50 DPESARELERIVKENAELAERVFREVEKIVR 2plus1_5400_shortest_dimer 1.1-B SEQ ID NO: 51 SELAERI KRSHERLEELLAR 2plus1_5400_shortest_dimer 1.1-A SEQ ID NO: 52 DPELYRASKRSLEEFERELREKGSDPESARELERIVKENAELAERVFRR 2plus1_5400_shortest_dimer 1.2-A SEQ ID NO: 53 SELAERIKRSHERLEELLARGSGDPELYRASKRSLEEFERELREK 2plus1_5400_shortest_dimer 1.2-B SEQ ID NO: 54 DPESARELERIVKENAELAERVFRR DHD94_2143_short-A SEQ ID NO: 55 SPLLDKVRKLVEKSRALVEELRKLVDQSTKGGLIDEKALRKQQEVLRKVEEVLEKQERVLRELEKGSGDPEVLERFVRVSREVLKVLEEFLRVSEELLRAADR DHD94_2143_short-B (noK) SEQ ID NO: 56 DPLEEYERQVDELREEIRRYAEEVDRFDREVR DHD94_2143_shorter2-A SEQ ID NO: 57 DPVRSLVEKSRALVEELRKLVDQSTKGGLIDEKALRKQQEVLRKVEEVLEKQERVLRGSEAFVEVSREVLKVLEEFLRVSEELLRAADR DHD94_2143_shorter2-A-KtoR_K9 SEQ ID NO: 58 DPVRSLVEKSRALVEELRRLVDQSTRGGLIDERALRRQQEVLRRVEEVLERQERVLRGSEAFVEVSREVLRVLEEFLRVSEELLRAADR DHD94_2143_shorter2-B (noK) SEQ ID NO: 59 DPLEEYERQVDELREEIRAYAEEVDRFDR DHD13_1234_shorter2-A SEQ ID NO: 60 SEAERAHREQKEIIKKAQELHRRGSDPEILEEIRELSKRSLELLREILKGSGSERQRKIIERAQEIHRRQAEILEALER DHD13_1234_shorter2-A-KtoR SEQ ID NO: 61 SEAERAHREQREIIRRAQELHRRGSDPEILEEIRELSRRSLELLREILRGSGSERQRRIIERAQEIHRRQAEILEALER DHD13_1234_shorter2-B (noK) SEQ ID NO: 62 SPELLEESLRLLRELLELSEESAR DHD37redesign3.2_YtoH_shorter-A SEQ ID NO: 63 SENLARHLERLSKHARKLEEILERGSDQAVKESIELVRESVEIVQAVIKGSRGSPVLQRATEIIDRADQLLESLSR DHD37redesign3_YtoH_shorter-A SEQ ID NO: 64 SPEFLENLARHLDRLSKHARKLEEILEKGSDQAVKESIDLVRESVSIVQAVIKTGSGSPELAKILQRATEIIDRADKLLEALRR DHD37redesign3_YtoH_shorter-B (noK) SEQ ID NO: 65 DPRVVEEHVELIREHARAISELLR Synzip1 SEQ ID NO: 66 NLVAQLENEVASLENENETLKKKNLHKKDLIAYLEKEIANLRKKIEE Synzip2 SEQ ID NO: 67 SARNAYLRKKIARLKKDNLQLERDEQNLEKIIANLRDEIARLENEVASHEQ Synzip3 SEQ ID NO: 68 NEVTTLENDAAFIENENAYLEKEIARLRKEKAALRNRLAHKK Synzip4 SEQ ID NO: 69 QKVAELKNRVAVKLNRNEQLKNKVEELKNRNAYLKNELATLENEVARLENDVAE Synzip5 SEQ ID NO: 70 NTVKELKNYIQELEERNAELKNLKEHLKFAKAELEFELAAHKFE Synzip6 SEQ ID NO: 71 QKVAQLKNRVAYKLKENAKLENIVARLENDNANLEKDIANLEKDIANLERDVAR Coil-coil-CZ-A SEQ ID NO: 72 ASAQLEKKLQALEKKLAQLEWKNQALEKKLAQ Coil-coil-NZ-B SEQ ID NO: 73 AQLKKELQANKKELAQLKWELQALKKELAQ

Table 2. Amino acid sequences of designed degradants: binding element-E3 ligase constructs. Glycine-serine flexible linker underlined. The sequences of DHD and other binding elements are shown in bold and can be replaced with any of the binding element sequences in Table 1. Shorthand names used in figures and text are in parentheses. RNF43 ^198-783 -DHD37-short-B (RNF43-v1) SEQ ID NO: 74 DVWILMTVVGTIFVIILASVLRIRCRPRHSRPDPLQQRTAWAISQLATRRYQASCRQARGEWPDSGSSCSSAPVCAICLEEFSEGQELRVISCLHEFHRNCVDPWLHQHRTCPLCMFNITEGDSFSQSLGPSRSYQEPGRRLHLIRQHPGHAHYHLPAAYLLGPSRSAVARPPRPGPFLPSQEPGMGPRHHRFPRAAHPRAPGEQQRLAGAQHPYAQGWGLSHLQSTSQHPAACPVPLRRARPPDSSGSGESYCTERSGYLADGPASDSSSGPCHGSSSDSVVNCTDISLQGVHGSSSTFCSSLSSDFDPLVYCSPKGDPQRVDMQPSVTSRPRSLDSVVPTGETQVSSHVHYHRHRHHHYKKRFQWHGRKPGPETGVPQSRPPIPRTQPQPEPPSPDQQVTRSNSAAPSGRLSNPQCPRALPEPAPGPVDASSICPSTSSLFNLQKSSLSARHPQRKRRGGPSEPTPGSRPQDATVHPACQIFPHYTPSVAYPWSPEAHPLICGPPGLDKRLLPETPGPCYSNSQPVWLCLTPRQPLEPHPPGEGPSEWSSDTAEGRPCPYPHCQVLSAQPGSEEELEELCEQAV GGGGSGGGGS GDPKVVEEYVRLLEEHEKAVRELLEIAK RNF43 ^198-364 -DHD37-short-B SEQ ID NO: 75 DVWILMTVVGTIFVIILASVLRIRCRPRHSRPDPLQQRTAWAISQLATRRYQASCRQARGEWPDSGSSSSSAPVCAICLEEFSEGQELRVISCLHEFHRNCVDPWLHQHRTCPLCMFNITEGDSFSQSLGPSRSYQEPGRRLHLIRQHPGHAHYHLPAAYLLGPSRS GGGGSGGGGS GDPKIAVVEEYVRLLEEHEKAVR RNF43 ^195-325 -DHD37-short-B (RNF43-v3) SEQ ID NO: 76 PDYDVWILMTVVGTIFVIILASVLRIRCRPRHSRPDPLQQRTAWAISQLATRRYQASCRQARGEWPDSGSSSAPVCACLEEFSEGQELRVISCLHEFHRNCVDPWLHQHRTCPLCMFNITEGDSFSQS GGGGSGGGGS GDPKVVEEYVRLLEEHEKAVRELLEIAK RNF43 ^198-317 -DHD37-short-B (RNF43-v2) SEQ ID NO: 77 DVWILMTVVGTIFVIILASVLRIRCRPRHSRPDPLQQRTAWAISQLATRRYQASCRQARGEWPDSGSSSSAPVCAICLEEFSEGQELRVISCLHEFHRNCVDPWLHQHRTCPLMFNIT GGGGSGGGGS GDPKVVEEYVRLLEEHEKAVRELLEIAK RNF128 ^208-428 -DHD37-short-B (RNF128-v1) SEQ ID NO: 78 IFFVSVSFFIITAATVGYFIFYSARRLRNARAQSRKQRQLKADAKKAIGRLQLRTLKQGDKEIGPDGDSCAVCIELYKPNDLVRILTCNHIFHKTCVDPWLLEHRTCPMCKCDILKALGIEVDVEDGSVSLQVPVSNEISNSASSHEEDNRSETASSGYASVQGTDEPPLEEHVQSTNESLQLVNHEANSVAVDVIPHVDNPTFEEDETPNQETAVREIKS GGGGSGGGGS GDPKVVEEYVRLLEEHEKAVRELLEIAK RNF128 ^208-333 -DHD37-short-B (RNF128-v2) SEQ ID NO: 79 IFFVSVSFFIITAATVGYFIFYSARRLRNARAQSRKQRQLKADAKKAIGRLQLRTLKQGDKEIGPDGDSCAVCIELYKPNDLVRILTCNHIFHKTCVDPWLLEHRTCPMCKCDILKALGIEVDVED GGGGSGGGGS GDPKVVEEYVRLLEEHEKAVRELLEIAK RNF128 ^208-325 -DHD37-short-B (RNF128-v3) SEQ ID NO: 80 IFFVSVSFFIITAATVGYFIFYSARRLRNARAQSRKQRQLKADAKKAIGRLQLRTLKQGDKEIGPDGDSCAVCIELYKPNDLVRILTCNHIFHKTCVDPWLLEHRTCPMCKCDILKAL GGGGSGGGGS GDPKVVEEYVRLLEEHEKAVRELLEIAK DHD37-short-B-3xUb SEQ ID NO: 81 GDPKVVEEYVRLLEEHEKAVRELLEIAK GGGGS MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRAARSMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRAARSMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGE DHD37-short-B-3xUb ^K48R SEQ ID NO: 82 GDPKVVEEYVRLLEEHEKAVRELLEIAK GGGGS MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGRQLEDGRTLSDYNIQKESTLHLVLRLRAARSMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGRQLEDGRTLSDYNIQKESTLHLVLRLRAARSMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGRQLEDGRTLSDYNIQKESTLHLVLRLRGE DHD37-short-B-3xUb ^K63R SEQ ID NO: 83 GDPKVVEEYVRLLEEHEKAVRELLEIAK GGGGS MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQRESTLHLVLRLRAARSMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQRESTLHLVLRLRAARSMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQRESTLHLVLRLRGE ZNRF3 ^219-936 -DHD37-short-B (ZNRF3-v1) SEQ ID NO: 84 TEYFDMGIFLAFFVVVSLVCLILLVKIKLKQRRSQNSMNRLAVQALEKMETRKFNSKSKGRREGSCGALDTLSSSSTSDCAICLEKYIDGEELRVIPCTHRFHRKCVDPWLLQHHTCPHCRHNIIEQKGNPSAVCVETSNLSRGRQQRVTLPVHYPGRVHRTNAIPAYPTRTSMDSHGNPVTLLTMDRHGEQSLYSPQTPAYIRSYPPLHLDHSLAAHRCGLEHRAYSPAHPFRRPKLSGRSFSKAACFSQYETMYQHYYFQGLSYPEQEGQSPPSLAPRGPARAFPPSGSGSLLFPTVVHVAPPSHLESGSTSSFSCYHGHRSVCSGYLADCPGSDSSSSSSSGQCHCSSSDSVVDCTEVSNQGVYGSCSTFRSSLSSDYDPFIYRSRSPCRASEAGGSGSSGRGPALCFEGSPPPEELPAVHSHGAGRGEPWPGPASPSGDQVSTCSLEMNYSSNSSLEHRGPNSSTSEVGLEASPGAAPDLRRTWKGGHELPSCACCCEPQPSPAGPSAGAAGSSTLFLGPHLYEGSGPAGGEPQSGSSQGLYGLHPDHLPRTDGVKYEGLPCCFYEEKQVARGGGGGSGCYTEDYSVSVQYTLTEEPPPGCYPGARDLSQRIPIIPEDVDCDLGLPSDCQGTHSLGSWGGTRGPDTPRPHRGLGATREEERALCCQARALLRPGCPPEEAGAVRANFPSALQDTQESSTTATEAAGPRSHSADSSSPGA GGGGSGGGGS GDPKVVEEYVRLLEEHEKAVRELLEIAK ZNRF3 ^219-528 -DHD37-short-B (ZNRF3-v2) SEQ ID NO: 85 TEYFDMGIFLAFFVVVSLVCLILLVKIKLKQRRSQNSMNRLAVQALEKMETRKFNSKSKGRREGSCGALDTLSSSSTSDCAICLEKYIDGEELRVIPCTHRFHRKCVDPWLLQHHTCPHCRHNIIEQKGNPSAVCVETSNLSRGRQQRVTLPVHYPGRVHRTNAIPAYPTRTSMDSHGNPVTLLTMDRHGEQSLYSPQTPAYIRSYPPLHLDHSLAAHRCGLEHRAYSPAHPFRRPKLSGRSFSKAACFSQYETMYQHYYFQGLSYPEQEGQSPPSLAPRGPARAFPPSGSGSLLFPTVVHVAPPSHLESGSTSS GGGGSGGGGS GDPKVVEEYVRLLEEHEKAVRELLEIAK ZNRF3 ^219-345 -DHD37-short-B (ZNRF3-v3) SEQ ID NO: 86 TEYFDMGIFLAFFVVVSLVCLILLVKIKLKQRRSQNSMNRLAVQALEKMETRKFNSKSKGRREGSCGALDTLSSSSSTSDCAICLEKYIDGEELRVIPCTHRFHRKCVDPWLLQHHTCPHCRHNIIEQKGNPS GGGGSGGGGS GDPKVVEEYVRLLEEHEKAVRELLEIAK DHD37-short-B-MARCH8 ^1-154 -KLRG1 ^37-60 (MARCH8-v1) SEQ ID NO: 87 GDPKVVEEYVRLLEEHEKAVRELLEIAK GGGGSGGGGS MSMPLHQISAIPSQDAISARVYRSKTKEKEREEQNEKTLGHFMSHSSNISKAGSPPSASAPAPVSSFSRTSITPSSQDICRICHCEGDDESPLITPCHCTGSLHFVHQACLQQWIKSSDTRCCELCKYEFIMETKLKPLRKWEKLQMTSSERALK GSGLVLLVAILL DHD37-short-B-MARCH8 ^1-228 (MARCH8-v2) SEQ ID NO: 88 GDPKVVEEYVRLLEEHEKAVRELLEIAK GGGGSGGGGS MSMPLHQISAIPSQDAISARVYRSKTKEKEREEQNEKTLGHFMSHSSNISKAGSPPSASAPAPVSSFSRTSITPSSQDICRICHCEGDDESPLITPCHCTGSLHFVHQACLQQWIKSSDTRCCELCKYEFIMETKLKPLRKWEKLQMTSSERRKIMCSVTFHVIAITCVVWSLYVLIDRTAEEIKQGQATGILEWPFWTKLVVVAIGFTGGLLFMYVQCKVYVQLWKR MARCH8 ^1-228 -DHD37-short-B (MARCH8-v3) SEQ ID NO: 89 MSMPLHQISAIPSQDAISARVYRSKTKEKEREEQNEKTLGHFMSHSSNISKAGSPPSASAPAPVSSFSRTSITPSSQDICRICHCEGDDESPLITPCHCTGSLHFVHQACLQQWIKSSDTRCCELCKYEFIMETKLKPLRKWEKLQMTSSERRKIMCSVTFHVIAITCVVWSLYVLIDRTAEEIKQGQATGILEWPFWTKLVVVAIGFTGGLLFMYVQCKVYVQLWKR GGGGSGGGGS GDPKVVEEYVRLLEEHEKAVRELLEIAK LNX1 ^1-171 -DHD37-short-B SEQ ID NO: 90 MNQPESANDPEPLCAVCGQAHSLEENHFYSYPEEVDDDLICHICLQALLDPLDTPCGHTYCTLCLTNFLVEKDFCPMDRKPLVLQHCKKSSILVNKLLNKLLVTCPFREHCTQVLQRCDLEHHFQTSCKGASHYGLTKDRKRRSQDGCPDGCASLTATAPSPEVSAAATIS GGGGSGGGGSVRHEEEEVRLL DHD37-short-B-VHL ^152-213 SEQ ID NO: 91 GDPKVVEEYVRLLEEHEKAVRELLEIAK GGGGSGGGGS TLPVYTLKERCLQVVRSLVKPENYRRLDIVRSLYEDLEDHPNVQKDLERLTQERIAHQRMGD DHD37-short-B- ^{NEDD4 921-1303} SEQ ID NO: 92 GDPKVVEEYVRLLEEHEKAVRELLEIAK GGGGSGGGGS EDPRLENVAITGPAVPYSRDYKRKYEFFRRKLKKQNDIPNKFEMKLRRATVLEDSYRRIMGVKRADFLKARLWIEFDGEKGLDYGGVAREWFFLISKEMFNPYYGLFEYSATDNYTLQINPNSGLCNEDHLSYFKFIGRVAGMAVYHGKLLDGFFIRPFYKMMLHKPITLHDMESVDSEYYNSLRWILENDPTELDLRFIIDEELFGQTHQHELKNGGSEIVVTNKNKKEYIYLVIQWRFVNRIQKQMAAFKEGFFELIPQDLIKIFDENELELLMCGLGDVDVNDWREHTKYKNGYSANHQVIQWFWKAVLMMDSEKRIRLLQFVTGTSRVPMNGFAELYGSNGPQSFTVEQWGTPEKLPRAHTCFNRLDLPPYESFEELWD DHD37-short-B-SPOP ^167-374 SEQ ID NO: 93 GDPKVVEEYVRLLEEHEKAVRELLEIAK GGGGSGGGGS VNISGQNTMNMVKVPECRLADELGGLWENSRFTDCCLCVAGQEFQAHKAILAARSPVFSAMFEHEMEESKKNRVEINDVEPEVFKEMMCFIYTGKAPNLDKMADDLLAAADKYALERLKVMCEDALCSNLSVENAAEILILADLHSADQLKTQAVDFINYHASDVLETSGWKSMVVSHPHLVAEAYRSLASAQCPFLGPPRKRLKQS DHD37-short-B-SOCS2 ^143-198 SEQ ID NO: 94 GDPKVVEEYVRLLEEHEKAVRELLEIAK GGGGSGGGGS PRNGTVHLYLTKPLYTSAPSLQHLCRLTINKCTGAIWGLPLPTRLKDYLEEYKFQV DHD37-short-B-CHIP ^128-303 SEQ ID NO: 95 GDPKVVEEYVRLLEEHEKAVRELLEIAK GGGGSGGGGS RLNFGDDIPSALRIAKKKRWNSIEERRIHQESELHSYLSRLIAAERERELEECQRNHEGDEDDSHVRAQQACIEAKHDKYMADMDELFSQVDEKRKKRDIPDYLCGKISFELMREPCITPSGITYDRKDIEEHLQRVGHFDPVTRSPLTNGKEQEQL FBXW7 ^1-293 -DHD37-short-B SEQ ID NO: 96 MNQELLSVGSKRRRTGGSLRGNPSSSQVDEEQMNRVVEEEQQQQLRQQEEEHTARNGEVVGVEPRPGGQNDSQQGQLEENNNRFISVDEDSSGNQEEQEEDEEHAGEQDEEDEEEEEMDQESDDFDQSDDSSREDEHTHTNSVTNSSSIVDLPVHQLSSPFYTKTTKMKRKLDHGSEVRSFSLGKKPCKVSEYTSTTGLVPCSATPTTFGDLRAANGQGQQRRRITSVQPPTGLQEWLKMFQSWSGPEKLLALDELIDSCEPTQVKHMMQVIEPQFQRDFISLLPKELALYVL GGGGSGGGGS GDPKVVEEYVRLLEEHEKAVRELLEIAK ELOC ^1-87 -DHD37-short-B SEQ ID NO: 97 MDGEEKTYGGCEGPDAMYVKLISSDGHEFIVKREHALTSGTIKAMLSGPGQFAENETNEVNFREIPSHVLSKVCMYFTYKVRYTNSS GGGGSGGGGSGDPKVVEEYVRLLEEHEKAVRELLEIAK FBW1A ^1-261 -DHD37-short-B SEQ ID NO: 98 MDPAEAVLQEKALKFMCSMPRSLWLGCSSLADSMPSLRCLYNPGTGALTAFQNSSEREDCNNGEPPRKIIPEKNSLRQTYNSCARLCLNQETVCLASTAMKTENCVAKTKLANGTSSMIVPKQRKLSASYEKEKELCVKYFEQWSESDQVEFVEHLISQMCHYQHGHINSYLKPMLQRDFITALPARGLDHIAENILSYLDAKSLCAAELVCKEWYRVTSDGMLWKKLIERMVRTDSLWRGLAERRGWGQYLFKNKPPDGN GGGGSGGGGS GDPKVVEEYVRLLEEHEKAVRELLEIAK DHD37-short-B-RNF4 ^71-190 SEQ ID NO: 99 GDPKVVEEYVRLLEEHEKAVRELLEIAK GGGGSGGGGS DERRRPRRNARRLPQDHADSCVVSSDDEELSRDRDVYVTTHTPRNARDEGATGLRPSGTVSCPICMDGYSEIVQNGRLIVSTECGHVFCSQCLRDSLKNANTCPTCRKKINHKRYHPIYI TRAF6 ^49-237 -DHD37-short-B SEQ ID NO: 100 MEEIQGYDVEFDPPLESKYECPICLMALREAVQTPCGHRFCKACIIKSIRDAGHKCPVDNEILLENQLFPDNFAKREILSLMVKCPNEGCLHKMELRHLEDHQAHCEFALMDCPQCQRPFQKFHINIHILKDCPRRQVSCDNCAASMAFEDKEIHDQNCPLANVICEYVRTILIREQMPNHYDLLEEEDCPT GGGLLGSKGG LNX1 ^1-171 -DHD37-short-A SEQ ID NO: 101 MNQPESANDPEPLCAVCGQAHSLEENHFYSYPEEVDDDLICHICLQALLDPLDTPCGHTYCTLCLTNFLVEKDFCPMDRKPLVLQHCKKSSILVNKLLNKLLVTCPFREHCTQVLQRCDLEHHFQTSCKGASHYGLTKDRKRRSQDGCPDGCASLTATAPSPEVSAAATIS GGGGSGGGGS PEFLENLRRHLDRLRKHIRKLEEILEEIDDQMVKEAIDLSRRSVEIVEEVIETFERAGDASPTKLDELEKILQRAEEIIDRADKLLEYRRR DHD37-short-A-VHL ^152-213 SEQ ID NO: 102 SPEFLENLRRHLDRLRKHIRKLEEILEEIDDQMVKEAIDLSRRSVEIVEEVIETFERAGDASPTKLDELEKILQRAEEIIDRADKLLEYRRR GGGGSGGGGS TLPVYTLKERCLQVVRSLVKPENYRRLDIVRSLYEDLEDHPNVQKDLERLTQERIAHQRMGD DHD37-short-A- ^{NEDD4 921-1303} SEQ ID NO: 103 SPEFLENLRRHLDRLRKHIRKLEEILEEIDDQMVKEAIDLSRRSVEIVEEVIETFERAGDASPTKLDELEKILQRAEEIIDRADKLLEYRRR GGGGSGGGGS EDPRLENVAITGPAVPYSRDYKRKYEFFRRKLKKQNDIPNKFEMKLRRATVLEDSYRRIMGVKRADFLKARLWIEFDGEKGLDYGGVAREWFFLISKEMFNPYYGLFEYSATDNYTLQINPNSGLCNEDHLSYFKFIGRVAGMAVYHGKLLDGFFIRPFYKMMLHKPITLHDMESVDSEYYNSLRWILENDPTELDLRFIIDEELFGQTHQHELKNGGSEIVVTNKNKKEYIYLVIQWRFVNRIQKQMAAFKEGFFELIPQDLIKIFDENELELLMCGLGDVDVNDWREHTKYKNGYSANHQVIQWFWKAVLMMDSEKRIRLLQFVTGTSRVPMNGFAELYGSNGPQSFTVEQWGTPEKLPRAHTCFNRLDLPPYESFEELWD DHD37-short-A-SPOP ^167-374 SEQ ID NO: 104 SPEFLENLRRHLDRLRKHIRKLEEILEEIDDQMVKEAIDLSRRSVEIVEEVIETFERAGDASPTKLDELEKILQRAEEIIDRADKLLEYRRR GGGGSGGGGS VNISGQNTMNMVKVPECRLADELGGLWENSRFTDCCLCVAGQEFQAHKAILAARSPVFSAMFEHEMEESKKNRVEINDVEPEVFKEMMCFIYTGKAPNLDKMADDLLAAADKYALERLKVMCEDALCSNLSVENAAEILILADLHSADQLKTQAVDFINYHASDVLETSGWKSMVVSHPHLVAEAYRSLASAQCPFLGPPRKRLKQS DHD37-short-A-SOCS2 ^143-198 SEQ ID NO: 105 SPEFLENLRRHLDRLRKHIRKLEEILEEIDDQMVKEAIDLSRRSVEIVEEVIETFERAGDASPTKLDELEKILQRAEEIIDRADKLLEYRRR GGGGSGGGGS PRNGTVHLYLTKPLYTSAPSLQHLCRLTINKCTGAIWGLPLPTRLKDYLEEYKFQV DHD37-short-A-CHIP ^128-303 SEQ ID NO: 106 SPEFLENLRRHLDRLRKHIRKLEEILEEIDDQMVKEAIDLSRRSVEIVEEVIETFERAGDASPTKLDELEKILQRAEEIIDRADKLLEYRRR GGGGSGGGGS RLNFGDDIPSALRIAKKKRWNSIEERRIHQESELHSYLSRLIAAERERELEECQRNHEGDEDDSHVRAQQACIEAKHDKYMADMDELFSQVDEKRKKRDIPDYLCGKISFELMREPCITPSGITYDRKDIEEHLQRVGHFDPVTRSPLTQEQLIPNLAMKEVIDAFISENGWVEDY FBXW7 ^1-293 -DHD37-short-A SEQ ID NO: 107 MNQELLSVGSKRRRTGGSLRGNPSSSQVDEEQMNRVVEEEQQQQLRQQEEEHTARNGEVVGVEPRPGGQNDSQQGQLEENNNRFISVDEDSSGNQEEQEEDEEHAGEQDEEDEEEEEMDQESDDFDQSDDSSREDEHTHTNSVTNSSSIVDLPVHQLSSPFYTKTTKMKRKLDHGSEVRSFSLGKKPCKVSEYTSTTGLVPCSATPTTFGDLRAANGQGQQRRRITSVQPPTGLQEWLKMFQSWSGPEKLLALDELIDSCEPTQVKHMMQVIEPQFQRDFISLLPKELALYVL GGGGSGGGGS PEFLENLRRHLDRLRKHIRKLEEILEEIDDQMVKEAIDLSRRSVEIVEEVIETFERAGDASPTKLDELEKILQRAEEIIDRADKLLEYRRR ELOC ^1-87 -DHD37-short-A SEQ ID NO: 108 MDGEEKTYGGCEGPDAMYVKLISSDGHEFIVKREHALTSGTIKAMLSGPGQFAENETNEVNFREIPSHVLSKVCMYFTYKVRYTNSS GGGGSGGGGS PEFLENLRRHLDRLRKHIRKLEEILEEIDDQMVKEAIDLSRRSVEIVEEVIETFERAGDASPTKLDELEKILQRAEEIIDRADKLLEYRRR FBW1A ^1-261 -DHD37-short-A SEQ ID NO: 109 MDPAEAVLQEKALKFMCSMPRSLWLGCSSLADSMPSLRCLYNPGTGALTAFQNSSEREDCNNGEPPRKIIPEKNSLRQTYNSCARLCLNQETVCLASTAMKTENCVAKTKLANGTSSMIVPKQRKLSASYEKEKELCVKYFEQWSESDQVEFVEHLISQMCHYQHGHINSYLKPMLQRDFITALPARGLDHIAENILSYLDAKSLCAAELVCKEWYRVTSDGMLWKKLIERMVRTDSLWRGLAERRGWGQYLFKNKPPDGN GGGGSGGGGS PEFLENLRRHLDRLRKHIRKLEEILEEIDDQMVKEAIDLSRRSVEIVEEVIETFERAGDASPTKLDELEKILQRAEEIIDRADKLLEYRRR DHD37-short-A-RNF4 ^71-190 SEQ ID NO: 110 SPEFLENLRRHLDRLRKHIRKLEEILEEIDDQMVKEAIDLSRRSVEIVEEVIETFERAGDASPTKLDELEKILQRAEEIIDRADKLLEYRRR GGGGSGGGGS DERRRPRRNARRLPQDHADSCVVSSDDEEELSRDRDVYVTTHTPRNARDEGATGLRPSGTVSCPICMDGYSEIVQNGRLIVSTECGHKVANRDYSLRKTCP Synzip2-RNF4 SEQ ID NO: 111 GGGGSARNAYLRKKIARLKKDNLQLERDEQNLEKIIANLRDEIARLENEVASHEQSGGGGSGGGGSDERRRPRRNARRLPQDHADSCVVSSDDEELSRDRDVYVTTHTPRNARDEGATGLRPSGTVSCPICMDGYSEIVQNGRLIVSTECGHVFCSQCLRDSLKNANTCPTCRKKINHKRYHPIYI Synzip4-RNF4 SEQ ID NO: 112 GGGGSQKVAELKNRVAVKLNRNEQLKNKVEELKNRNAYLKNELATLENEVARLENDVAESGGGGSGGGGSDERRRPRRNARRLPQDHADSCVVSSDDEELSRDRDVYVTTHTPRNARDEGATGLRPSGTVSCPICMDGYSEIVQNGRLIVSTECGHVFCSQCLRDSLKNANTCPTCRKKINHKRYHPIYI Synzip6-RNF4 SEQ ID NO: 113 GGGGSQKVAQLKNRVAYKLKENAKLENIVARLENDNNLEKDIANLEKDIANLERDVARSGGGGSGGGGSDERRRPRRNARRLPQDHADSCVVSSDDEELSRDRDVYVTTHTPRNARDEGATGLRPSGTVSCPICMDGYSEIVQNGRLIVSTECGHVFCSQCLRDSLKNANTCPTCRKKINHKRYHPIYI Coil-coil-NZ-RNF4 SEQ ID NO: 114 GGGGSAQLKKELQANKKELAQLKWELQALKKELAQSGGGGSGGGGSDERRRPRRNARRLPQDHADSCVVSSDDEELSRDRDVYVTTHTPRNARDEGATGLRPSGTVSCPICMDGYSEIVQNGRLIVSTECGHVFCSQCLRDSLKNANTCPTCRKKINHKRYHPIYI

Table 3. Amino acid sequences of target proteins used for degradation screening. Optional tags and glycine-serine flexible linkers are underlined. The sequences of DHD and other binding elements are shown in bold and can be replaced with any of the binding element sequences in Table 1. Abbreviated names used in figures and text are in parentheses. ROR1-CAR-DHD37-short-A (ROR1 CAR-DHD) or (CAR-DHD) SEQ ID NO: 115 [scFv-spacer]-MFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGS PEFLENLRRHLDRLRKHIRKLEEILEEIDDQMVKEAIDLSRRSVEIVEEVIETFERAGDASPTKLDELEKILQRAEEIIDRADKLLEYRRR ROR1 CAR-Synzip1 SEQ ID NO: 116 [scFv-spacer]-MFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGS NLVAQLENEVASLENENETLKKKNLHKKDLIAYLEKEIANLRKKIEE ROR1 CAR-Synzip3 SEQ ID NO: 117 [scFv-spacer]-MFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGS NEVTTLENDAAFIENENAYLEKEIARLRKEKAALRNRLAHKK ROR1 CAR-Synzip5 SEQ ID NO: 118 [scFv-spacer]-MFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGS NTVKELKNYIQELEERNAELKNLKEHLKFAKAELEFELAAHKFE ROR1 CAR-Coilcoil-CZ SEQ ID NO: 119 [scFv-spacer]-MFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGS ASAQLEKKLQALEKKLAQLEWKNQALEKKLAQ DHD37-short-B-BACH2 (DHD-BACH2) with optional N-terminal 3X-FLAG tag SEQ ID NO: 120 DYKDHDGDYKDHDIDYKDDDDKGGSGSGDPKVVEEYVRLLEEHEKAVRELLEIAKGGGGSGGGGS _ _

Table 4. Amino Acid Sequences of Optimized RNF43-DHD-B Constructs Exhibiting Enhanced Target Degradation. The sequence of the endocytic motif is underlined and that of the DHD is in bold. Some figures and short names used in the text are in parentheses. RNF43 ^198-317 -DHD37-short-B-KtoR (RNF43-DHD-KtoR) SEQ ID NO: 121 DVWILMTVVGTIFVIILASVLRIRCRPRHSRPDPLQQRTAWAISQLATRRYQASCRQARGEWPDSGSSSSAPVCAICLEEFSEGQELRVISCLHEFHRNCVDPWLHQHRTCPLMFNITGGGGSGGGGS GDPRVVEEYVRLLEEHERAVRELLEIAR RNF43 ^198-317 -DHD37-short-B-endo-var1 (RNF43-DHD-endo) SEQ ID NO: 122 DVWILMTVVGTIFVIILASVLRIRCRPRHSRPDPLQQRTAWAISQLATRRYQASCRQARGEWPDSGSSSSAPVCNACLEEFSEGQELRVISCLHEFHRNCVDPWLHQHRTCPLCMFNITGGGGSGGGGS GDPKVVEEYVRLLEEHEKAVRELLEIAK GGSGSGGSGGA EAAALL AV RNF43 ^198-317 -DHD37-short-B-endo-var2 SEQ ID NO: 123 DVWILMTVVGTIFVIILASVLRIRCRPRHSRPDPLQQRTAWAISQLATRRYQASCRQARGEWPDSGSSSCAPVCAICLEEFSEGQELRVISCLHEFHRNCVDPWLHQHRTCPLCMFNITGGGGSGGGGS GDPKVVEEYVRLLEEHEKAVRELLEIAK GGSGSGGSGGA EYVRLL AV RNF43 ^198-317 -DHD37-short-B-endo-var3 SEQ ID NO: 124 DVWILMTVVGTIFVIILASVLRIRCRPRHSRPDPLQQRTAWAISQLATRRYQASCRQARGEWPDSGSSSCAPVCNACLEEFSEGQELRVISCLHEFHRNCVDPWLHQHRTCPLCMFNITGGGGSGGGGS GDPKVVEEYVRLLEEHEKAVRELLEIAK GGSGSGGSGGAA YAAL AAV RNF43 ^198-317 -KtoR-DHD37-short-B-endo-var1 (RNF43-DHD-KtoR-endo) SEQ ID NO: 125 DVWILMTVVGTIFVIILASVLRIRCRPRHSRPDPLQQRTAWAISQLATRRYQASCRQARGEWPDSGSSSAPVCNACLEEFSEGQELRVISCLHEFHRNCVDPWLHQHRTCPLCMFNITGGGGSGGGGS GDPRVVEEYVRLLEEHERAVRELLEIAR GGSGSGGSGGA EAAALL AV

Table 5. Amino acid sequences of DNCR-E3 and CAR-NS3a constructs. The endocytic motif and the glycine-serine flexible linker are underlined, and the sequences of DNCR and NS3a are in bold. ROR1 CAR-NS3a SEQ ID NO: 126 [scFv-間隔物]- MFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR GSASGSASGSAS MKKKGSVVIVGRINLSGDTAYSQQTRGLEGCQETSQTGRDKNQVEGEVQVVSTATQSFLATSINGVLWTVYHGAGTRTIASPKGPVTQMYTNVDKDLVGWQAPQGSRSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVSTRGVAKAVDFIPVESLETTMRSP RNF43 ^198-317- DNCR SEQ ID NO: 127 PDYDVWILMTVVGTIFVIILASVLRIRCRPRHSRPDPLQQRTAWAISQLATRRYQASCRQARGEWPDSGSSCSSAPVCAICLEEFSEGQELRVISCLHEFHRNCVDPWLHQHRTCPLCMFNIT GGGGSGGGGS SSDEEEARELIERAKEAAERAQEAAERTGDPRVRELARELKRLAQEAAEEVKRDPSSSDVNEALKLIVEAIEAAVDALEAAERTGDPEVRELARELVRLAVEAAEEVQRNPSSSDVNEALHSIVYAIEAAIFALEAAERTGDPEVRELARELVRLAVEAAEEVQRNPSSRNVEHALMRIVLAIYLAEENLREAEESGDPEKREKARERVREAVERAEEVQRDPSGWLNH DNCR-RNF4 ^71-190 SEQ ID NO: 128 SSDEEEARELIERAKEAAERAQEAAERTGDPRVRELARELKRLAQEAAEEVKRDPSSSDVNEALKLIVEAIEAAVDALEAAERTGDPEVRELARELVRLAVEAAEEVQRNPSSSDVNEALHSIVYAIEAAIFALEAAERTGDPEVRELARELVRLAVEAAEEVQRNPSSRNVEHALMRIVLAIYLAEENLREAEESGDPEKREKARERVREAVERAEEVQRDPSGWLNH GGGGSGGGGS DERRRPRRNARRLPQDHADSCVVSSDDEELSRDRDVYVTTHTPRNARDEGATGLRPSGTVSCPICMDGYSEIVQNGRLIVSTECGHVFCSQCLRDSLKNANTCPTCRKKINHKRYHPIYI LNX1 ^1-171- DNCR SEQ ID NO: 129 MNQPESANDPEPLCAVCGQAHSLEENHFYSYPEEVDDDLICHICLQALLDPLDTPCGHTYCTLCLTNFLVEKDFCPMDRKPLVLQHCKKSSILVNKLLNKLLVTCPFREHCTQVLQRCDLEHHFQTSCKGASHYGLTKDRKRRSQDGCPDGCASLTATAPSPEVSAAATIS GGGGSGGGGS SSDEEEARELIERAKEAAERAQEAAERTGDPRVRELARELKRLAQEAAEEVKRDPSSSDVNEALKLIVEAIEAAVDALEAAERTGDPEVRELARELVRLAVEAAEEVQRNPSSSDVNEALHSIVYAIEAAIFALEAAERTGDPEVRELARELVRLAVEAAEEVQRNPSSRNVEHALMRIVLAIYLAEENLREAEESGDPEKREKARERVREAVERAEEVQRDPSGWLNH RNF43 ^198-317 -DNCR-endo SEQ ID NO: 130 PDYDVWILMTVVGTIFVIILASVLRIRCRPRHSRPDPLQQRTAWAISQLATRRYQASCRQARGEWPDSGSSCSSAPVCAICLEEFSEGQELRVISCLHEFHRNCVDPWLHQHRTCPLCMFNIT GGGGSGGGGS SSDEEEARELIERAKEAAERAQEAAERTGDPRVRELARELKRLAQEAAEEVKRDPSSSDVNEALKLIVEAIEAAVDALEAAERTGDPEVRELARELVRLAVEAAEEVQRNPSSSDVNEALHSIVYAIEAAIFALEAAERTGDPEVRELARELVRLAVEAAEEVQRNPSSRNVEHALMRIVLAIYLAEENLREAEESGDPEKREKARERVREAVERAEEVQRDPSGWLNH GGSGSGGSGGAEAAALLAV RNF43 ^198-317 -DNCR-KtoR SEQ ID NO: 131 PDYDVWILMTVVGTIFVIILASVLRIRCRPRHSRPDPLQQRTAWAISQLATRRYQASCRQARGEWPDSGSSCSSAPVCAICLEEFSEGQELRVISCLHEFHRNCVDPWLHQHRTCPLCMFNIT GGGGSGGGGS SSDEEEARELIERAREAAERAQEAAERTGDPRVRELARELRRLAQEAAEEVRRDPSSSDVNEALRLIVEAIEAAVDALEAAERTGDPEVRELARELVRLAVEAAEEVQRNPSSSDVNEALHSIVYAIEAAIFALEAAERTGDPEVRELARELVRLAVEAAEEVQRNPSSRNVEHALMRIVLAIYLAEENLREAEESGDPERRERARERVREAVERAEEVQRDPSGWLNH RNF43 ^198-317 -DNCR-KtoR-endo SEQ ID NO: 132 PDYDVWILMTVVGTIFVIILASVLRIRCRPRHSRPDPLQQRTAWAISQLATRRYQASCRQARGEWPDSGSSCSSAPVCAICLEEFSEGQELRVISCLHEFHRNCVDPWLHQHRTCPLCMFNIT GGGGSGGGGS SSDEEEARELIERAREAAERAQEAAERTGDPRVRELARELRRLAQEAAEEVRRDPSSSDVNEALRLIVEAIEAAVDALEAAERTGDPEVRELARELVRLAVEAAEEVQRNPSSSDVNEALHSIVYAIEAAIFALEAAERTGDPEVRELARELVRLAVEAAEEVQRNPSSRNVEHALMRIVLAIYLAEENLREAEESGDPERRERARERVREAVERAEEVQRDPSGWLNH GGSGSGGSGGAEAAALLAV DNCR1 SEQ ID NO: 178 SSDEEEARELIERAKEAAERAQEAAAERTGDPRVRELARELKRLAQEAAEEVKRDPSSSDVNEALKLIVEAIEAAVDALEAAERTGDPEVRELARELVRLAVEAAEEVQRNPSSSDVNEALLSIVIAIEAAVHALEAAERTGDPEVRELARELVRLAVEAAEEVQRNPSSREVEHALMKIVLAIYEAEESLREAEESGDPEKRE KARERVREEAGRAWEEV DNCR2 SEQ ID NO: 179 SSDEEEARELIERAREAAERAQEAAERTGDPRVRELARELRRRLAQEAAEEVRRDPSSSDVNEALRLIVEAIEAAVDALEAAERTGDPEVRELARELVRLAVEAAEEVQRNPSSSDVNEALHSIVYAIEAAIFALEAAERTGDPEVRELARELVRLAVEAAEEVQRNPSSRNVEHALMRIVLAIYLAEENLREAEESGDPERRERAREVRREAVERALEEVH GNCR1 SEQ ID NO: 180 DIEKLCKKAEEEEAKEAQEKADELRQRHPDSQAAEDAEDLANLAVAAVLTACLLAQEHPNADIAKLCIKAASEAAEAASKAAELAQRHPDSQAARDAIKLASQAAARAVILAIMLAAENPNADIAKLCIKAAASEAAEAASKAAELAQRHPDSQAARDAIKLASQAAEAVERAIWLAAENPNADIAKKCIKAASEAAEEKADEASKAAEEAQR NS3a SEQ ID NO: 181 MKKKGSVVIVGRINLSGDTAYSQQTRGLEGCQETSQTGRDKNQVEGEVQVVSTATQSFLATSINGVLWTVYHGAGTRTIASPKGPVTQMYTNVDKDLVGWQAPQGSRSLTPCTCGSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVSTRGVAKAVDFIPVESLETT

Table 6. Amino acid sequences of TM ^CD3z -, SH2 ^ZAP70 -, SH3 ^{NCK1 -E3} fusion constructs. The glycine-serine flexible linker is underlined and the sequences of the transmembrane domain, SH2 domain and SH3 domain are in bold. TM ^CD3z -RNF43 ^226-317 (v1) SEQ ID NO: 133 QSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRS HSRPDPLQQRTAWAISQLATRRYQASCRQARGEWPDSGSSSSAPVCAICLEEFSEGQELRVISCLHEFHRNCVDPWLHQHRTCPLCMFNIT TM ^CD3z-K54R- RNF43 ^226-317 (v2) SEQ ID NO: 134 QSFGLLDPKLCYLLDGILFIYGVILTALFLRVRFSRS HSRPDPLQQRTAWAISQLATRRYQASCRQARGEWPDSGSSSSAPVCAICLEEFSEGQELRVISCLHEFHRNCVDPWLHQHRTCPLCMFNIT TM ^CD3z -RNF43 ^236-317 (v3) SEQ ID NO: 135 QSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRS GGGGSGGGGS TAWAISQLATRRYQASCRQARGEWPDSGSSSSAPVCAICLEEFSEGQELRVISCLHEFHRNCVDPWLHQHRTCPLMFNIT TM ^CD3z-K54R- RNF43 ^236-317 (v4) SEQ ID NO: 136 QSFGLLDPKLCYLLDGILFIYGVILTALFLRVRFSRS GGGGSGGGGS TAWAISQLATRRYQASCRQARGEWPDSGSSSSAPVCAICLEEFSEGQELRVISCLHEFHRNCVDPWLHQHRTCPLMFNIT ZAP70 ^nSH2-cSH2- RNF4 ^71-190 SEQ ID NO: 137 MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHDVRFHHFPIERQLNGTYAIAGGKAHCGPAELCEFYSRDPDGLPCNLRKPCNRPSGLEPQPGVFDCLRDAMVRDYVRQTWKLEGEALEQAIISQAPQVEKLIATTAHERMPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYHYLISQDKAGKYCIPEGTKFDTLWQLVEYLKLKADGLIYCLKEACPNSSAS GGGGSGGGGS DERRRPRRNARRLPQDHADSCVVSSDDEELSRDRDVYVTTHTPRNARDEGATGLRPSGTVSCPICMDGYSEIVQNGRLIVSTECGHVFCSQCLRDSLKNANTCPTCRKKINHKRYHPIYI ZAP70 ^cSH2 -RNF4 ^71-190 SEQ ID NO: 138 MPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYHYLISQDKAGKYCIPEGTKFDTLWQLVEYLKLKADGLIYCLKEACPNSSAS GGGGSGGGGS DERRRPRRNARRLPQDHADSCVVSSDDEELSRDRDVYVTTHTPRNARDEGATGLRPSGTVSCPICMDGYSEIVQNGRLIVSTECGHVFCSQCLRDSLKNANTCPTCRKKINHKRYHPIYI LNX1 ^1-171 -ZAP70 ^nSH2-cSH2 (LNX1-nSH2-cSH2) SEQ ID NO: 139 MNQPESANDPEPLCAVCGQAHSLEENHFYSYPEEVDDDLICHICLQALLDPLDTPCGHTYCTLCLTNFLVEKDFCPMDRKPLVLQHCKKSSILVNKLLNKLLVTCPFREHCTQVLQRCDLEHHFQTSCKGASHYGLTKDRKRRSQDGCPDGCASLTATAPSPEVSAAATIS GGGGS MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHDVRFHHFPIERQLNGTYAIAGGKAHCGPAELCEFYSRDPDGLPCNLRKPCNRPSGLEPQPGVFDCLRDAMVRDYVRQTWKLEGEALEQAIISQAPQVEKLIATTAHERMPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYHYLISQDKAGKYCIPEGTKFDTLWQLVEYLKLKADGLIYCLKEACPNSSAS LNX1 ^1-171- ZAP70 ^cSH2 SEQ ID NO: 140 MNQPESANDPEPLCAVCGQAHSLEENHFYSYPEEVDDDLICHICLQALLDPLDTPCGHTYCTLCLTNFLVEKDFCPMDRKPLVLQHCKKSSILVNKLLNKLLVTCPFREHCTQVLQRCDLEHHFQTSCKGASHYGLTKDRKRRSQDGCPDGCASLTATAPSPEVSAAATIS GGGGS MPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYHYLISQDKAGKYCIPEGTKFDTLWQLVEYLKLKADGLIYCLKEACPNSSAS NCK1 ^SH3.1- RNF4 ^71-19 SEQ ID NO: 141 MAEEVVVVAKFDYVAQQEQELDIKKNERLWLLDDSKSWWRVRNSMNKTGFVPSNYVERKNS GGGGSGGGGS DERRRPRRNARRLPQDHADSCVVSSDDEELSRDRDVYVTTHTPRNARDEGATGLRPSGTVSCPICMDGYSEIVQNGRLIVSTECGHVFCSQCLRDSLKNANTCPTCRKKINHKRYHPIYI LNX1 ^1-171- NCK1 ^SH3.1 SEQ ID NO: 142 MNQPESANDPEPLCAVCGQAHSLEENHFYSYPEEVDDDLICHICLQALLDPLDTPCGHTYCTLCLTNFLVEKDFCPMDRKPLVLQHCKKSSILVNKLLNKLLVTCPFREHCTQVLQRCDLEHHFQTSCKGASHYGLTKDRKRRSQDGCPDGCASLTATAPSPEVSAAATIS GGGGS MAEEVVVVAKFDYVAQQEQELDIKKNERLWLLDDSKSWWRVRNSMNKTGFVPSNYVERKNS

Table 7. DNA sequence of NFAT-AP1 promoter. The minimal CMV promoter is underlined, and the sequence of the 4-repeat NFAT-AP1 binding site is shown in bold. NFAT-AP1 promoter SEQ ID NO: 143 ggaggaaaaactgtttcatacagaaggcgtggaggaaaaactgtttcatacagaaggcgtggaggaaaaactgtttcatacagaaggcgtggaggaaaaactgtttcatacagaaggcgt gaattccacgtg gaggtaggcgtgtacggtgggcgcctataaaagcagagctcgtccttagtaggaaccg minimal CMV promoter SEQ ID NO: 176 gaggtaggcgtgtacggtgggcgcctataaaagcagagctcgtttagtgaaccgtcagatcgcctgga NFAT-AP1 binding site SEQ ID NO: 177 ggaggaaaaactgtttcatacagaaggcgt

Table 9. DNA sequences of constitutive promoters. MND SEQ ID NO: 144 TGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGGATCAAGGTTAGGAACAGAGAGACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGTTGGAACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCCGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTCACTCGGC hPGK SEQ ID NO: 145 GGGGTTGGGGTTGCGCCTTTTCCAAGGCAGCCCTGGGTTTGCGCAGGGACGCGGCTGCTCTGGGCGTGGTTCCGGGAAACGCAGCGGCGCCGACCCTGGGTCTCGCACATTCTTCACGTCCGTTCGCAGCGTCACCCGGATCTTCGCCGCTACCCTTGTGGGCCCCCCGGCGACGCTTCCTGCTCCGCCCCTAAGTCGGGAAGGTTCCTTGCGGTTCGCGGCGTGCCGGACGTGACAAACGGAAGCCGCACGTCTCACTAGTACCCTCGCAGACGGACAGCGCCAGGGAGCAATGGCAGCGCGCCGACCGCGATGGGCTGTGGCCAATAGCGGCTGCTCAGCAGGGCGCGCCGAGAGCAGCGGCCGGGAAGGGGCGGTGCGGGAGGCGGGGTGTGGGGCGGTAGTGTGGGCCCTGTTCCTGCCCGCGCGGTGTTCCGCATTCTGCAAGCCTCCGGAGCGCACGTCGGCAGTCGGCTCCCTCGTTGACCGAATCACCGACCTCTCTCCCCAG CMV SEQ ID NO: 146 CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGCTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGAT CAG SEQ ID NO: 147 ATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGATCGATT SFFV SEQ ID NO: 148 GTAACGCCATTTTGCAAGGCATGGAAAAATACCAAACCAAGAATAGAGAAGTTCAGATCAAGGGCGGGTACATGAAAATAGCTAACGTTGGGCCAAACAGGATATCTGCGGTGAGCAGTTTCGGCCCCGGCCCGGGGCCAAGAACAGATGGTCACCGCAGTTTCGGCCCCGGCCCGAGGCCAAGAACAGATGGTCCCCAGATATGGCCCAACCCTCAGCAGTTTCTTAAGACCCATCAGATGTTTCCAGGCTCCCCCAAGGACCTGAAATGACCCTGCGCCTTATTTGAATTAACCAATCAGCCTGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTTCCCGAGCTCTATAAAAGAGCTCACAACCCCTCACTCGGCGCGCCAGTCCTCCGACAGACTGAGTCGCCCGGG EF1alpha SEQ ID NO: 149 GGGCAGAGCGCACATCGCCCACAGTCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGAGAACCGTATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGAGCCGCCAGAAC UBC SEQ ID NO: 150 GGCCTCCGCGCCGGGTTTTGGCGCCTCCCGCGGGCGCCCCCCTCCTCACGGCGAGCGCTGCCACGTCAGACGAAGGGCGCAGCGAGCGTCCTGATCCTTCCGCCCGGACGCTCAGGACAGCGGCCCGCTGCTCATAAGACTCGGCCTTAGAACCCCAGTATCAGCAGAAGGACATTTTAGGACGGGACTTGGGTGACTCTAGGGCACTGGTTTTCTTTCCAGAGAGCGGAACAGGCGAGGAAAAGTAGTCCCTTCTCGGCGATTCTGCGGAGGGATCTCCGTGGGGCGGTGAACGCCGATGATTATATAAGGACGCGCCGGGTGTGGCACAGCTAGTTCCGTCGCAGCCGGGATTTGGGTCGCGGTTCTTGTTTGTGGATCGCTGTGATCGTCACTTGGTGAGTAGCGGGCTGCTGGGCTGGCCGGGGCTTTCGTGGCCGCCGGGCCGCTCGGTGGGACGGAAGCGTGTGGAGAGACCGCCAAGGGCTGTAGTCTGGGTCCGCGAGCAAGGTTGCCCTGAACTGGGGGTTGGGGGGAGCGCAGCAAAATGGCGGCTGTTCCCGAGTCTTGAATGGAAGACGCTTGTGAGGCGGGCTGTGAGGTCGTTGAAACAAGGTGGGGGGCATGGTGGGCGGCAAGAACCCAAGGTCTTGAGGCCTTCGCTAATGCGGGAAAGCTCTTATTCGGGTGAGATGGGCTGGGGCACCATCTGGGGACCCTGACGTGAAGTTTGTCACTGACTGGAGAACTCGGTTTGTCGTCTGTTGCGGGGGCGGCAGTTATGGCGGTGCCGTTGGGCAGTGCACCCGTACCTTTGGGAGCGCGCGCCCTCGTCGTGTCGTGACGTCACCCGTTCTGTTGGCTTATAATGCAGGGTGGGGCCACCTGCCGGTAGGTGTGCGGTAGGCTTTTCTCCGTCGCAGGACGCAGGGTTCGGGCCTAGGGTAGGCTCTCCTGAATCGACAGGCGCCGGACCTCTGGTGAGGGGAGGGATAAGTGA GGCGTCAGTTTCTTGGTCGGTTTTATGTACCTATCTTTCTTAAGTAGCTGAAGCTCCGGTTTTGAACTATGCGCTCGGGGTTGGCGAGTGTGTTTTGTGAAGTTTTTTAGGCACCTTTTGAAATGTAATCATTTGGGTCAATATGTAATTTTCAGTGTTAGACTAGTAAATTGTCTAAATTCTGGCCGTTTTTGGCTTTTTTGTTAGACATCGATT CD43 SEQ ID NO: 151 AGTTCTCAGGCTCACATTCCCACCACCCACCTCTGAGCCCAGCCCTCCCTAGCATCACCACTTCCATCCCATTCCTCAGCCAAGAGCCAGGAATCCTGATTCCAGATCCCACGCTTCCCTGCCTCCCTCAGGTGAGCCCCAGACCCCCAGGCACCCCGCTGGCCCCTGAAGGAGCAGGTGATGGTGCTGTCTTCGCCCAGCAGCTGTGGGAGCAGGCGGGTGGGGCAGGATGGAGGGGTGGGTGGGGTGGGTGGAGCCAGGGCCCACTTCCTTTCCCCTTGGGGCCCTGTCCTTCCCAGTCTTGCCCCAGCCTCGGGAGGTGGTGGAGTGACCTGGCCCCAGTGCTGCGTCCTTATCAGCCGAGCCGGTAAGAGGGTGAGACTTGGTGGGGTAGGGGCCTCAGTGGGCCTGGGAATGTGCCTGTGGCTTGAAAAGACTCTGACAGGTTATGATGGGAAGAGATTGGGAGCCATTGGGCTGCACAGGGTCAGGGAAGGCCAGGAGGGGCTGGTCACTGCTGGAATCTAAGCTGCTGAGGCTGGAGGGAGCCTCAGGATGGGGCTGATGGGGGAGCTGCCAGCATCTGTTCCTCTGTCATTTCTGATAACAGTAAAAGCCAGCATGGAAAAAACCGTTAAACCGCAGGTTGGGCCTGGCCGTTGGCAGGGAAGTGGGCAGAGGGGAGGCCCGGCCAGGTCCTCCGGCAACTCCCGCGTGTTCTGCTTCTCCGGCTGCCCACCTGCAGGTCCCAGCTCTTGCTCCTGCCTGTTTGCCTGgaa  

Table 10. Amino Acid Sequences for Combination Screening Components. DAP10-V5 SEQ ID NO: 157 MIHLGHILFLLLLPVAAAGKPIPNPLLGLDSTQTTPGERSSLPAFYPGTSGSSSGSGSLSLP CD8 TMD SEQ ID NO: 158 IYIWAPLAGTCGVLLLSLVITLYCSL RNF43 SEQ ID NO: 159 DVWILMTVVGTIFVIILASVLRIRCRPRHSRPDPLQQRTAWAISQLATRRYQASCRQARGEWPDSGSSSSAPVCAICLEEFSEGQELRVISCLHEFHRNCVDPWLHQHRTCPLMFNIT LNX1 SEQ ID NO: 160 MNQPESANDPEPLCAVCGQAHSLEENHFYSYPEEVDDDLICHICLQALLDPLDTPCGHTYCTLCLTNFLVEKDFCPMDRKPLVLQHCKKSSILVNKLLNKLLVTCPFREHCTQVLQRCDLEHHFQTSCKGASHYGLTKDRKRRSQDGCPDGCASLTATAPSPEVSAAATISGGG LNX1 KtoR SEQ ID NO: 161 MNQPESANDPEPLCAVCGQAHSLEENHFYSYPEEVDDDLICHICLQALLDPLDTPCGHTYCTLCLTNFLVERDFCPMDRRPLVLQHCRRSSILVNRLLNRLLVTCPFREHCTQVLQRCDLEHHFQTSCRGASHYGLTRDRRRRSQDGCPDGCASLTATAPSPEVSAAATISGGG RNF4 SEQ ID NO: 162 DERRRPRRNARRLPQDHADSCVVSSDDEELSRDRDVYVTTHTPRNARDEGATGLRPSGTVSCPICMDGYSEIVQNGRLIVSTECGHVFCSQCLRDSLRNANTCPTCRRRRINHRRYHPIYI RNF4 KtoR SEQ ID NO: 163 DERRRPRRNARRLPQDHADSCVVSSDDEELSRDRDVYVTTHTPRNARDEGATGLRPSGTVSCPICMDGYSEIVQNGRLIVSTECGHVFCSQCLRDSLKNANTCPTCRKKINHKRYHPIYI RING finger protein 128 (RNF128) SEQ ID NO: MGPPPGAGVSCRGGCGFSRLLAWCFLLALSPQAPGSRGAEAVWTAYLNVSWRVPHTGVNRTVWELSEEGVYGQDSPLEPVAGVLVPPDGPGALNACNPHTNFTVPTVWGSTVQVSWLALIQRGGGCTFADKIHLAYERGASGAVIFNFPGTRNEVIPMSHPGAVDIVAIMIGNLKGTKILQSIQRGIQVTMVIEVGKKHGPWVNHYSIFFVSVSFFIITAATVGYFIFYSARRLRNARAQSRKQRQLKADAKKAIGRLQLRTLKQGDKEIGPDGDSCAVCIELYKPNDLVRILTCNHIFHKTCVDPWLLEHRTCPMCKCDILKALGIEVDVEDGSVSLQVPVSNEISNSASSHEEDNRSETASSGYASVQGTDEPPLEEHVQSTNESLQLVNHEANSVAVDVIPHVDNPTFEEDETPNQETAVREIKS Zinc/ring finger protein 3 (ZNRF3) SEQ ID NO: MRPRSGGRPGATGRRRRRLRRRPRGLRCSRLPPPPPLPLLLGLLLAAAGPGAARAKETAF VEVVLFESSPSGDYTTYTTGLTGRFSRAGATLSAEGEIVQMHPLGLCNNNDEEDLYEYGW VGVVKLEQPELDPKPCLTVLGKAKRAVQRGATAVIFDVSENPEAIDQLNQGSEDPLKRPV VYVKGADAIKLMNIVNKQKVARARIQHRPPRQPTEYFDMGIFLAFFVVVSLVCLILLVKI KLKQRRSQNSMNRLAVQALEKMETRKFNSKSKGRREGSCGALDTLSSSSTSDCAICLEKY IDGEELRVIPCTHRFHRKCVDPWLLQHHTCPHCRHNIIEQKGNPSAVCVETSNLSRGRQQ RVTLPVHYPGRVHRTNAIPAYPTRTSMDSHGNPVTLLTMDRHGEQSLYSPQTPAYIRSYP PLHLDHSLAAHRCGLEHRAYSPAHPFRRPKLSGRSFSKAACFSQYETMYQHYYFQGLSYP EQEGQSPPSLAPRGPARAFPPSGSGSLLFPTVVHVAPPSHLESGSTSSFSCYHGHRSVCS GYLADCPGSDSSSSSSSGQCHCSSSDSVVDCTEVSNQGVYGSCSTFRSSLSSDYDPFIYR SRSPCRASEAGGSGSSGRGPALCFEGSPPPEELPAVHSHGAGRGEPWPGPASPSGDQVST CSLEMNYSSNSSLEHRGPNSSTSEVGLEASPGAAPDLRRTWKGGHELPSCACCCEPQPSP AGPSAGAAGSSTLFLGPHLYEGSGPAGGEPQSGSSQGLYGLHPDHLPRTDGVKYEGLPCC FYEEKQVARGGGGGSGCYTEDYSVSVQYTLTEEPPPGCYPGARDLSQRIPIIPEDVDCDL GLPSDCQGTHSLGSWGGTRGPDTPRPHRGLGATREEERALCCQARALLRPGCPPEEAGAV RANFPSALQDTQESSTTATEAAGPRSHSADSSSPGA Membrane-associated RING-CH protein VIII (MARCH8) SEQ ID NO: MSMPLHQISAIPSQDAISARVYRSKTKEKEREEQNEKTLGHFMSHSSNISKAGSPPSASA PAPVSSFSRTSITPSSQDICRICHCEGDDESPLITPCHCTGSLHFVHQACLQQWIKSSDT RCCELCKYEFIMETKLKPLRKWEKLQMTSSERRKIMCSVTFHVIAITCVVWSLYVLIDRT AEEIKQGQATGILEWPFWTKLVVVAIGFTGGLLFMYVQCKVYVQLWKRLKAYNRVIYVQN CPETSKKNIFEKSPLTEPNFENKHGYGICHSDTNSSCCTEPEDTGAEIIHV Leucine-rich alpha-2-glycoprotein (LRG1) SEQ ID NO: MSSWSRQRPKSPGGIQPHVSRTLFLLLLLAASAWGVTLSPKDCQVFRSDHGSSISCQPPA EIPGYLPADTVHLAVEFFNLTHLPANLLQGASKLQELHLSSNGLESLSPEFLRPVPQLRV LDLTRNALTGLPPGLFQASATLDTLVLKENQLEVLEVSWLHGLKALGHLDLSGNRLRKLP PGLLANFTLLRTLDLGENQLETLPPDLLRGPLQLERLHLEGNKLQVLGKDLLLPQPDLRY LFLNGNKLARVAAGAFQGLRQLDMLDLSNNSLASVPEGLWASLGQPNWDMRDGFDISGNP WICDQNLSDLYRWLQAQKDKMFSQNDTRCAGPEAVKGQTLLAVAKSQ Neural progenitor cells express developmental downregulated protein 4 (NEDD4) SEQ ID NO: MAQSLRLHFAARRSNTYPLSETSGDDLDSHVHMCFKRPTRISTSNVVQMKLTPRQTALAP LIKENVQSQERSSVPSSENVNKKSSCLQISLQPTRYSGYLQSSNVLADSDDASFTCILKD GIYSSAVVDNELNAVNDGHLVSSPAICSGSLSNFSTSDNGSYSSNGSDFGSCASITSGGS YTNSVISDSSSYTFPPSDDTFLGGNLPSDSTSNRSVPNRNTTPCEIFSRSTSTDPFVQDD LEHGLEIMKLPVSRNTKIPLKRYSSLVIFPRSPSTTRPTSPTSLCTLLSKGSYQTSHQFI ISPSEIAHNEDGTSAKGFLSTAVNGLRLSKTICTPGEVRDIRPLHRKGSLQKKIVLSNNT PRQTVCEKSSEGYSCVSVHFTQRKAATLDCETTNGDCKPEMSEIKLNSDSEYIKLMHRTS ACLPSSQNVDCQININGELERPHSQMNKNHGILRRSISLGGAYPNISCLSSLKHNCSKGG PSQLLIKFASGNEGKVDNLSRDSNRDCTNELSNSCKTRDDFLGQVDVPLYPLPTENPRLE RPYTFKDFVLHPRSHKSRVKGYLRLKMTYLPKTSGSEDDNAEQAEELEPGWVVLDQPDAA CHLQQQQEPSPLPPGWEERQDILGRTYYVNHESRRTQWKRPTPQDNLTDAENGNIQLQAQ RAFTTRRQISEETESVDNRESSENWEIIREDEATMYSNQAFPSPPPSSNLDVPTHLAEEL NARLTIFGNSAVSQPASSSNHSSRRGSLQAYTFEEQPTLPVLLPTSSGLPPGWEEKQDER GRSYYVDHNSRTTTWTKPTVQATVETSQLTSSQSSAGPQSQASTSDSGQQVTQPSEIEQG FLPKGWEVRHAPNGRPFFIDHNTKTTTWEDPRLKIPAHLRGKTSLDTSNDLGPLPPGWEE RTHTDGRIFYINHNIKRTQWEDPRLENVAITGPAVPYSRDYKRKYEFFRRKLKKQNDIPN KFEMKLRRATVLEDSYRRIMGVKR ADFLKARLWIEFDGEKGLDYGGVAREWFFLISKEMF NPYYGLFEYSATDNYTLQINPNSGLCNEDHLSYFKFIGRVAGMAVYHGKLLDGFFIRPFY KMMLHKPITLHDMESVDSEYYNSLRWILENDPTELDLRFIIDEELFGQTHQHELKNGGSE IVVTNKNKKEYIYLVIQWRFVNRIQKQMAAFKEGFFELIPQDLIKIFDENELELLMCGLG DVDVNDWREHTKYKNGYSANHQVIQWFWKAVLMMDSEKRIRLLQFVTGTSRVPMNGFAEL YGSNGPQSFTVEQWGTPEKLPRAHTCFNRLDLPPYESFEELWDKLQMAIENTQGFDGVD Carboxy-terminal (CHIP) of Hsp70 Interacting Protein SEQ ID NO: MKGKEEKEGGARLGAGGGSPEKSPSAQELKEQGNRLFVGRKYPEAAACYGRAITRNPLVA VYYTNRALCYLKMQQHEQALADCRRALELDGQSVKAHFFLGQCQLEMESYDEAIANLQRA YSLAKEQRLNFGDDIPSALRIAKKKRWNSIEERRIHQESELHSYLSRLIAAERERELEEC QRNHEGDEDDSHVRAQQACIEAKHDKYMADMDELFSQVDEKRKKRDIPDYLCGKISFELM REPCITPSGITYDRKDIEEHLQRVGHFDPVTRSPLTQEQLIPNLAMKEVIDAFISENGWV EDY Speckled POZ protein (SPOP) SEQ ID NO: MSRVPSPPPPAEMSSGPVAESWCYTQIKVVKFSYMWTINNFSFCREEMGEVIKSSTFSSG ANDKLKWCLRVNPKGLDEESKDYLSLYLLLVSCPKSEVRAKFKFSILNAKGEETKAMESQ RAYRFVQGKDWGFKKFIRRDFLLDEANGLLPDDKLTLFCEVSVVQDSVNISGQNTMNMVK VPECRLADELGGLWENSRFTDCCLCVAGQEFQAHKAILAARSPVFSAMFEHEMEESKKNR VEINDVEPEVFKEMMCFIYTGKAPNLDKMADDLLAAADKYALERLKVMCEDALCSNLSVE NAAEILILADLHSADQLKTQAVDFINYHASDVLETSGWKSMVVSHPHLVAEAYRSLASAQ CPFLGPPRKRLKQS F-box/WD repeat protein 7 (FBXW7) SEQ ID NO: MNQELLSVGSKRRRTGGSLRGNPSSSQVDEEQMNRVVEEEQQQQLRQQEEEHTARNGEVV GVEPRPGGQNDSQQGQLEENNNRFISVDEDSSGNQEEQEEDEEHAGEQDEEDEEEEEMDQ ESDDFDQSDDSSREDEHTHTNSVTNSSSIVDLPVHQLSSPFYTKTTKMKRKLDHGSEVRS FSLGKKPCKVSEYTSTTGLVPCSATPTTFGDLRAANGQGQQRRRITSVQPPTGLQEWLKM FQSWSGPEKLLALDELIDSCEPTQVKHMMQVIEPQFQRDFISLLPKELALYVLSFLEPKD LLQAAQTCRYWRILAEDNLLWREKCKEEGIDEPLHIKRRKVIKPGFIHSPWKSAYIRQHR IDTNWRRGELKSPKVLKGHDDHVITCLQFCGNRIVSGSDDNTLKVWSAVTGKCLRTLVGH TGGVWSSQMRDNIIISGSTDRTLKVWNAETGECIHTLYGHTSTVRCMHLHEKRVVSGSRD ATLRVWDIETGQCLHVLMGHVAAVRCVQYDGRRVVSGAYDFMVKVWDPETETCLHTLQGH TNRVYSLQFDGIHVVSGSLDTSIRVWDVETGNCIHTLTGHQSLTSGMELKDNILVSGNAD STVKIWDIKTGQCLQTLQGPNKHQSAVTCLQFNKNFVITSSDDGTVKLWDLKTGEFIRNL VTLESGGSGGVVWRIRASNTKLVCAVGSRNGTEETKLLVLDFDVDMK Elongin-C (ELOC) SEQ ID NO: MDGEEKTYGGCEGPDAMYVKLISSDGHEFIVKREHALTSGTIKAMLSGPGQFAENETNEVNFREIPSHVLSKVCMYFTYKVRYTNSSTEIPEFPIAPEIALELLMAANFLDC F-box/WD repeat protein 1A (FBW1A) SEQ ID NO: MDPAEAVLQEKALKFMCSMPRSLWLGCSSLADSMPSLRCLYNPGTGALTAFQNSSEREDC NNGEPPRKIIPEKNSLRQTYNSCARLCLNQETVCLASTAMKTENCVAKTKLANGTSSMIV PKQRKLSASYEKEKELCVKYFEQWSESDQVEFVEHLISQMCHYQHGHINSYLKPMLQRDF ITALPARGLDHIAENILSYLDAKSLCAAELVCKEWYRVTSDGMLWKKLIERMVRTDSLWR GLAERRGWGQYLFKNKPPDGNAPPNSFYRALYPKIIQDIETIESNWRCGRHSLQRIHCRS ETSKGVYCLQYDDQKIVSGLRDNTIKIWDKNTLECKRILTGHTGSVLCLQYDERVIITGS SDSTVRVWDVNTGEMLNTLIHHCEAVLHLRFNNGMMVTCSKDRSIAVWDMASPTDITLRR VLVGHRAAVNVVDFDDKYIVSASGDRTIKVWNTSTCEFVRTLNGHKRGIACLQYRDRLVV SGSSDNTIRLWDIECGACLRVLEGHEELVRCIRFDNKRIVSGAYDGKIKVWDLVAALDPR APAGTLCLRTLVEHSGRVFRLQFDEFQIVSSSHDDTILIWDFLNDPAAQAEPPRSPSRTY TYISR TNF receptor-associated factor 6 (TRAF6) SEQ ID NO: MSLLNCENSCGSSQSESDCCVAMASSCSAVTKDDSVGGTASTGNLSSSFMEEIQGYDVEF DPPLESKYECPICLMALREAVQTPCGHRFCKACIIKSIRDAGHKCPVDNEILLENQLFPD NFAKREILSLMVKCPNEGCLHKMELRHLEDHQAHCEFALMDCPQCQRPFQKFHINIHILK DCPRRQVSCDNCAASMAFEDKEIHDQNCPLANVICEYCNTILIREQMPNHYDLDCPTAPI PCTFSTFGCHEKMQRNHLARHLQENTQSHMRMLAQAVHSLSVIPDSGYISEVRNFQETIH QLEGRLVRQDHQIRELTAKMETQSMYVSELKRTIRTLEDKVAEIEAQQCNGIYIWKIGNF GMHLKCQEEEKPVVIHSPGFYTGKPGYKLCMRLHLQLPTAQRCANYISLFVHTMQGEYDS HLPWPFQGTIRLTILDQSEAPVRQNHEEIMDAKPELLAFQRPTIPRNPKGFGYVTFMHLE ALRQRTFIKDDTLLVRCEVSTRFDMGSLRREGFQPRSTDAGV von Hippel-Lindau tumor suppressor (VHL) SEQ ID NO: MPRRAENWDEAEVGAEEAGVEEYGPEEDGGEESGAEESGPEESGPEELGAEEEMEAGRPR PVLRSVNSREPSQVIFCNRSPRVVLPVWLNFDGEPQPYPTLPPGTGRRIHSYRGHLWLFR DAGTHDGLLVNQTELFVPSLNVDGQPIFANITPVYTLKERCLQVVRSLVKPENYRRLDIRMVRDSLYKHLT Suppressor of cytokine signaling 2 (SOCS2) SEQ ID NO: MTLRCLEPSGNGGEGTRSQWGTAGSAEEPSPQAARLAKALRELGQTGWYWGSMTVNEAKE KLKEAPEGTFLIRDSSSHSDYLLTISVKTSAGPTNLRIEYQDGKFRLDSIICVKSKLKQFD SVVHLIDYYVQMCKDKRTGPEAPRNGTVHLYLTKPLYTSAPSLVHLCRLTINKCTGAIWGYLELPYKD 1X GS Linker SEQ ID NO: 164 GGGGS 2X GS linker SEQ ID NO: 165 GGGGSGGGGS 3X GS linker SEQ ID NO: 166 GGGGSGGGGSGGGGS 4X GS linker SEQ ID NO: 167 GGGGSGGGGSGGGGSGGGGS Combined sequence DHD-B SEQ ID NO: 169 GDPRVVEEYVRLLEEHERAVRELLEIAR

Table 11, CAR-DHD construct, the amino acid sequence of DHD DHD37_A(-8, RtoK) SEQ ID NO: 170 SPEFAKALHHHLTALQEHITALQQIANEGDDEMVHQAIKLSQKSVQIVNEVINTFNSSGDASPNAAKELQEILEKATEIIEQATNLLTKKAK DHD37_A(-8) SEQ ID NO: 171 SPEFARALHHHLTALQEHITALQQIANEGDDEMVHQAIKLSQRSVQIVNEVINTFNSSGDASPNAAKELQEILERATEIIEQATNLLTKRAR DHD37_A(fl-V97S-L104A-L114S, RtoK) SEQ ID NO: 172 PEFLENLKKHLDKLKKHIKKLEEILEEGDDQMVKEAIDLSKKSVEIVEEEVIETFEKAGDASPTKLDELEKILQKAEEIIDKADKLLEYKKKGGSGSGDPKVSEEYVKLAEEHEKAVKESLEIAK DHD37A(fl-V97S-L104A-L114S) SEQ ID NO: 173 PEFLENLRRHLDRLRKHIRKLEEILEEGDDQMVKEAIDLSRRSVEIVEEVIETFERAGDASPTKLDELEKILQRAEEIIDRADKLLEYRRRGGSGSGDPKVSEEYVRLAEEHEKAVRESLEIAK

Table 12, DHD-e3 sequence DAP10 V5 RNF43 G4S DHD37-short-B-KtoR SEQ ID NO: 174 MIHLGHILFLLLLPVAAAGKPIPNPLLGLDSTQTTPGERSSLPAFYPGTSGSSSGSGSLSLPGSDVWILMTVVGTIFVIILASVLRIRCRPRHSRPDPLQQRTAWAISQLATRRYQASCRQARGEWPDSGSSSSAPVCAICLEEFSEGQELRVISCLHEFHRNCVDPWLHQHRTCPLMCMFNITSGGGGGSSSGDPRVVERAEYLELLEEGS DHD37A(-8, RtoK) SEQ ID NO: SPEFAKALHHHLTALQEHITALQQIANEGDDEMVHQAIKLSQKSVQIVNEVINTFNSSGDASPNAAKELQEILEKATEIIEQATNLLTKKAK DHD37A(fl-V97S-L104A-L114S, RtoK) SEQ ID NO: PEFLENLKKHLDKLKKHIKKLEEILEEGDDQMVKEAIDLSKKSVEIVEEEVIETFEKAGDASPTKLDELEKILQKAEEIIDKADKLLEYKKKGGSGSGDPKVSEEYVKLAEEHEKAVKESLEIAK DHD37A(fl-V97S-L104A-V111S, RtoK) SEQ ID NO: PEFLENLKKHLDKLKKHIKKLEEILEEGDDQMVKEAIDLSKKSVEIVEEEVIETFEKAGDASPTKLDELEKILQKAEEIIDKADKLLEYKKKGGSGSGDPKVSEEYVKLAEEHEKASKELLEIAK DHD37A(fl-V97S-L104A-V111S) SEQ ID NO: PEFLENLRRHLDRLRKHIRKLEEILEEGDDQMVKEAIDLSRRSVEIVEEVIETFERAGDASPTKLDELEKILQRAEEIIDRADKLLEYRRRGGSGSGDPKVSEEYVRLAEEHEKASRELLEIAK DHD37A(fl-V97S-L104A-L114S) SEQ ID NO: PEFLENLRRHLDRLRKHIRKLEEILEEGDDQMVKEAIDLSRRSVEIVEEVIETFERAGDASPTKLDELEKILQRAEEIIDRADKLLEYRRRGGSGSGDPKVSEEYVRLAEEHEKAVRESLEIAK DHD37A(fl-V97S-L114S, RtoK) SEQ ID NO: PEFLENLKKHLDKLKKHIKKLEEILEEGDDQMVKEAIDLSKKSVEIVEEEVIETFEKAGDASPTKLDELEKILQKAEEIIDKADKLLEYKKKGGSGSGDPKVSEEYVKLLEEHEKAVKESLEIAK DHD37A(fl-V97S-L104A, RtoK) SEQ ID NO: PEFLENLKKHLDKLKKHIKKLEEILEEGDDQMVKEAIDLSKKSVEIVEEEVIETFEKAGDASPTKLDELEKILQKAEEIIDKADKLLEYKKKGGSGSGDPKVSEEYVKLAEEHEKAVKELLEIAK

Table 13. Target molecules target name sequence Nuclear receptor subfamily 4 group A member 1 (NR4A1), isoform 1, UniProtKB accession number P22736-1 SEQ ID NO: 182 MPCIQAQYGTPAPSPGPRDHLASDPLTPEFIKPTMDLASPEAAPAAPTALPSFSTFMDGYTGEFDTFLYQLPGTVQPCSSASSSASSTSSSSATSPASASFKFEDFQVYGCYPGPLSGPVDEALSSSGSDYYGSPCSAPSPSTPSFQPPQLSPWDGSFGHFSPSQTYEGLRAWTEQLPKASGPPQPPAFFSFSPPTGPSPSLAQSPLKLFPSQATHQLGEGESYSMPTAFPGLAPTSPHLEGSGILDTPVTSTKARSGAPGGSEGRCAVCGDNASCQHYGVRTCEGCKGFFKRTVQKNAKYICLANKDCPVDKRRRNRCQFCRFQKCLAVGMVKEVVRTDSLKGRRGRLPSKPKQPPDASPANLLTSLVRAHLDSGPSTAKLDYSKFQELVLPHFGKEDAGDVQQFYDLLSGSLEVIRKWAEKIPGFAELSPADQDLLLESAFLELFILRLAYRSKPGEGKLIFCSGLVLHRLQCARGFGDWIDSILAFSRSLHSLLVDVPAFACLSALVLITDRHGLQEPRRVEELQNRIASCLKEHVAAVAGEPQPASCLSRLLGKLPELRTLCTQGLQRIFYLKLEDLVPPPPIIDKIFMDTLPF Nuclear receptor subfamily 4 group A member 1 (NR4A1), isoform 2, UniProtKB accession number P22736-2 SEQ ID NO: 183 MWLAKACWSIQSEMPCIQAQYGTPAPSPGPRDHLASDPLTPEFIKPTMDLASPEAAPAAPTALPSFSTFMDGYTGEFDTFLYQLPGTVQPCSSASSSASSTSSSSATSPASASFKFEDFQVYGCYPGPLSGPVDEALSSSGSDYYGSPCSAPSPSTPSFQPPQLSPWDGSFGHFSPSQTYEGLRAWTEQLPKASGPPQPPAFFSFSPPTGPSPSLAQSPLKLFPSQATHQLGEGESYSMPTAFPGLAPTSPHLEGSGILDTPVTSTKARSGAPGGSEGRCAVCGDNASCQHYGVRTCEGCKGFFKRTVQKNAKYICLANKDCPVDKRRRNRCQFCRFQKCLAVGMVKEVVRTDSLKGRRGRLPSKPKQPPDASPANLLTSLVRAHLDSGPSTAKLDYSKFQELVLPHFGKEDAGDVQQFYDLLSGSLEVIRKWAEKIPGFAELSPADQDLLLESAFLELFILRLAYRSKPGEGKLIFCSGLVLHRLQCARGFGDWIDSILAFSRSLHSLLVDVPAFACLSALVLITDRHGLQEPRRVEELQNRIASCLKEHVAAVAGEPQPASCLSRLLGKLPELRTLCTQGLQRIFYLKLEDLVPPPPIIDKIFMDTLPF Nuclear receptor subfamily 4 group A member 1 (NR4A1), isoform 3, UniProtKB accession number P22736-3 SEQ ID NO: 184 MPCIQAQYGTPAPSPGPRDHLASDPLTPEFIKPTMDLASPEAAPAAPTALPSFSTFMDGYTGEFDTFLYQLPGTVQPCSSASSSASSTSSSSATSPASASFKFEDFQVYGCYPGPLSGPVDEALSSSGSDYYGSPCSAPSPSTPSFQPPQLSPWDGSFGHFSPSQTYEGLRAWTEQLPKASGPPQPPAFFSFSPPTGPSPSLAQSPLKLFPSQATHQLGEGESYSMPTAFPGLAPTSPHLEGSGILDTPVTSTKARSGAPGGSEGRCAVCGDNASCQHYGVRTCEGCKGFFKVPRSPRWGLLLEMERGWPHPIGTCGLPLGSPPS Nuclear receptor subfamily 4 group A member 2 (NR4A2), isoform 1, UniProtKB accession number P43354-1 SEQ ID NO: 185 MPCVQAQYGSSPQGASPASQSYSYHSSGEYSSDFLTPEFVKFSMDLTNTEITATTSLPSFSTFMDNYSTGYDVKPPCLYQMPLSGQQSSIKVEDIQMHNYQQHSHLPPQSEEMMPHSGSVYYKPSSPPTPTTPGFQVQHSPMWDDPGSLHNFHQNYVATTHMIEQRKTPVSRLSLFSFKQSPPGTPVSSCQMRFDGPLHVPMNPEPAGSHHVVDGQTFAVPNPIRKPASMGFPGLQIGHASQLLDTQVPS PPSRGSPSNEGLCAVCGDNAACQHYGVRTCEGCKGFFKRTVQKNAKYVCLANKNCPVDKRRRNRCQYCRFQKCLAVGMVKEVVRTDSLKGRRGRLPSKPKSPQEPSPPSPPVSLISALVRAHVDSNPAMTSLDYSRFQANPDYQMSGDDTQHIQQFYDLLTGSMEIIRGWAEKIPGFADLPKADQDLLFESAFLELFVLRLAYRSNPVEGKLIFCNGVVLHRLQCVRGFGEWIDSIVEFSSNLQNMNIDISAFSCIAALAMVTERHGLKEPKRVEELQNKIVNCLKDHVTFNNGGLNRPNYLSKLLGKLPELRTLCTQGLQRIFYLKLEDLVPPPAIIDKLFLDTLPF Nuclear receptor subfamily 4 group A member 2 (NR4A2), isoform 2, UniProtKB accession number P43354-2 SEQ ID NO: 186 MDNYSTGYDVKPPCLYQMPLSGQQSSIKVEDIQMHNYQQHSHLPPQSEEMMPHSGSVYYKPSSPPTPTTPGFQVQHSPMWDDPGSLHNFHQNYVATTHMIEQRKTPVSRLSLFSFKQSPPGTPVSSCQMRFDGPLHVPMNPEPAGSHHVVDGQTFAVPNPIRKPASMGFPGLQIGHASQLLDTQVPSPPSRGSPSNEGLCAVCGDNAACQHYGVRTCEGCKGFFKRTVQKNAKYVCLANKNCPVDKRRRNRCQYCRFQKCLAVGMVKEVVRTDSLKGRRGRLPSKPKSPQEPSPPSPPVSLISALVRAHVDSNPAMTSLDYSRFQANPDYQMSGDDTQHIQQFYDLLTGSMEIIRGWAEKIPGFADLPKADQDLLFESAFLELFVLRLAYRSNPVEGKLIFCNGVVLHRLQCVRGFGEWIDSIVEFSSNLQNMNIDISAFSCIAALAMVTERHGLKEPKRVEELQNKIVNCLKDHVTFNNGGLNRPNYLSKLLGKLPELR Nuclear receptor subfamily 4 group A member 3 (NR4A3), α-isomer, UniProtKB accession number Q92570-1 SEQ ID NO: 187 MPCVQAQYSPSPPGSSYAAQTYSSEYTTEIMNPDYTKLTMDLGSTEITATATTSLPSISTFVEGYSSNYELKPSCVYQMQRPLIKVEEGRAPSYHHHHHHHHHHHHHHQQQHQQPSIPPASSPEDEVLPSTSMYFKQSPPSTPTTPAFPPQAGALWDEALPSAPGCIAPGPLLDPPMKAVPTVAGARFPLFHFKPSPPHPPAPSPAGGHHLGYDPTAAAALSLPLGAAAAAGSQAAALESHPYGLPLAKRAAPLAFPPLGLTPSPTASSLLGESPSLPSPPSRSSSSGEGTCAVCGDNAACQHYGVRTCEGCKGFFKRTVQKNAKYVCLANKNCPVDKRRRNRCQYCRFQKCLSVGMVKEVVRTDSLKGRRGRLPSKPKSPLQQEPSQPSPPSPPICMMNALVRALTDSTPRDLDYSRYCPTDQAAAGTDAEHVQQFYNLLTASIDVSRSWAEKIPGFTDLPKEDQTLLIESAFLELFVLRLSIRSNTAEDKFVFCNGLVLHRLQCLRGFGEWLDSIKDFSLNLQSLNLDIQALACLSALSMITERHGLKEPKRVEELCNKITSSLKDHQSKGQALEPTESKVLGALVELRKICTLGLQRIFYLKLEDLVSPPSIIDKLFLDTLPF Nuclear receptor subfamily 4 group A member 3 (NR4A3), β isomer, UniProtKB accession number Q92570-2 SEQ ID NO: 188 MPCVQAQYSPSPPGSSYAAQTYSSEYTTEIMNPDYTKLTMDLGSTEITATATTSLPSISTFVEGYSSNYELKPSCVYQMQRPLIKVEEGRAPSYHHHHHHHHHHHHHHQQQHQQPSIPPASSPEDEVLPSTSMYFKQSPPSTPTTPAFPPQAGALWDEALPSAPGCIAPGPLLDPPMKAVPTVAGARFPLFHFKPSPPHPPAPSPAGGHHLGYDPTAAAALSLPLGAAAAAGSQAAALESHPYGLPLAKRAAPLAFPPLGLTPSPTASSLLGESPSLPSPPSRSSSSGEGTCAVCGDNAACQHYGVRTCEGCKGFFKRTVQKNAKYVCLANKNCPVDKRRRNRCQYCRFQKCLSVGMVKEVVRTDSLKGRRGRLPSKPKSPLQQEPSQPSPPSPPICMMNALVRALTDSTPRDLDYSRVSFMISCFQMNDQGLYLWLLVIRVD Nuclear receptor subfamily 4 group A member 3 (NR4A3), isoform 3, UniProtKB accession number Q92570-3 SEQ ID NO: 189 MHDSIRFGNVDMPCVQAQYSPSPPGSSYAAQTYSSEYTTEIMNPDYTKLTMDLGSTEITATATTSLPSISTFVEGYSSNYELKPSCVYQMQRPLIKVEEGRAPSYHHHHHHHHHHHHHHQQQHQQPSIPPASSPEDEVLPSTSMYFKQSP PSTPTTPAFPPQAGALWDEALPSAPGCIAPGPLLDPPMKAVPTVAGARFPLFHFKPSPPHPPAPSPAGGHHLGYDPTAAAALSLPLGAAAAAGSQAAALESHPYGLPLAKRAAPLAFPPLGLTPSPTASSLLGESPSLPSPPSRSSSSGEGTCAVCGDNAACQHYGVRTCEGCKGFFKRTVQKNAKYVCLANKNCPVDKRRRNRCQYCRFQKCLSVGMVKEVVRTDSLKGRRGRLPSKPKSPLQQEPSQPSPPSPPICMMNALVRALTDSTPRDLDYSRYCPTDQAAAGTDAEHVQQFYNLLTASIDVSRSWAEKIPGFTDLPKEDQTLLIESAFLELFVLRLSIRSNTAEDKFVFCNGLVLHRLQCLRGFGEWLDSIKDFSLNLQSLNLDIQALACLSALSMITERHGLKEPKRVEELCNKITSSLKDHQSKGQALEPTESKVLGALVELRKICTLGLQRIFYLKLEDLVSPPSIIDKLFLDTLPF Thymocyte selection-associated high-mobility group box protein TOX (TOX), isoform 1, UniProtKB accession number O94900-1 SEQ ID NO: 190 MDVRFYPPPAQPAAAPDAPCLGPSPCLDPYYCNKFDGENMYMSMTEPSQDYVPASQSYPGPSLESEDFNIPPITPPSLPDHSLVHLNEVESGYHSLCHPMNHNGLLPFHPQNMDLPEITVSNMLGQDGTLLSNSISVMPDIRNPEGTQYSSHPQMAAMRPRGQPADIRQQPGMMPHGQLTTINQSQLSAQLGLNMGGSNVPHNSPSPPGSKSATPSPSSSVHEDEGDDTSKINGGEKRPASDMGKKPKTP KKKKKKDPNEPQKPVSAYALFFRDTQAAIKGQNPNATFGEVSKIVASMWDGLGEEQKQVYKKKTEAAKKEYLKQLAAYRASLVSKSYSEPVDVKTSQPPQLINSKPSVFHGPSQAHSALYLSSHYHQQPGMNPHLTAMHPSLPRNIAPKPNNQMPVTVSIANMAVSPPPPLQISPPLHQHLNMQQHQPLTMQQPLGNQLPMQVQSALHSPTMQQGFTLQPDYQTIINPTSTAAQVVTQAMEYVRSGCRNPPPQPVDWNNDYCSSGGMQRDKALYLT TOX High Mobility Box Family Member 2 (TOX2), Isomer 1, UniProtKB Accession No. Q96NM4-1 SEQ ID NO: 191 MQQTRTEAVAGAFSRCLGFCGMRLGLLLLARHWCIAGVFPQKFDGDSAYVGMSDGNPELLSTSQTYNGQSENNEDYEIPPITPPNLPEPSLLHLGDHEASYHSLCHGLTPNGLLPAYSYQAMDLPAIMVSNMLAQDSHLLSGQLPTIQEMVHSEVAAYDSGRPGPLLGRPAMLASHMSALSQSQLISQMGIRSSIAHSSPSPPGSKSATPSPSSSTQEEESEVHFKISGEKRPSADPGKKAKNPKKKKKKDPNEPQKPVSAYALFFRDTQAAIKGQNPSATFGDVSKIVASMWDSLGEEQKQSSPDQGETKSTQANPPAKMLPPKQPMYAMPGLASFLTPSDLQAFRSGASPASLARTLGSKSLLPGLSASPPPPPSFPLSPTLHQQLSLPPHAQGALLSPPVSMSPAPQPPVLPTPMALQVQLAMSPSPPGPQDFPHISEFPSSSGSCSPGPSNPTSSGDWDSSYPSGECGISTCSLLPRDKSLYLT TOX high mobility group box family member 2 (TOX2), isoform 2, UniProtKB accession number Q96NM4-2 SEQ ID NO: 192 MQQTRTEAVAGAFSRCLGFCGMRLGLLLLARHWCIAGVFPQKFDGDSAYVGMSDGNPELLSTSQTYNGQSENNEDYEIPPITPPNLPEPSLLHLGDHEASYHSLCHGLTPNGLLPAYSYQAMDLPAIMVSNMLAQDSHLLSGQLPTIQEMVHSEVAAYDSGRPGPLLGRPAMLASHMSALSQSQLISQMGIRSSIAHSSPSPPGSKSATPSPSSSTQEEESEVHFKISGEKRPSADPGKKAKNPKKKKKKDPNEPQKPVSAYALFFRDTQAAIKGQNPSATFGDVSKIVASMWDSLGEEQKQAYKRKTEAAKKEYLKALAAYRASLVSKSSPDQGETKSTQANPPAKMLPPKQPMYAMPGLASFLTPSDLQAFRSGASPASLARTLGSKSLLPGLSASPPPPPSFPLSPTLHQQLSLPPHAQGALLSPPVSMSPAPQPPVLPTPMALQVQLAMSPSPPGPQDFPHISEFPSSSGSCSPGPSNPTSSGDWDSSYPSGECGISTCSLLPRDKSLYLT TOX high mobility cassette family member 2 (TOX2), isoform 3, UniProtKB accession number Q96NM4-3 SEQ ID NO: 193 MSDGNPELLSTSQTYNGQSENNEDYEIPPITPPNLPEPSLLHLGDHEASYHSLCHGLTPNGLLPAYSYQAMDLPAIMVSNMLAQDSHLLSGQLPTIQEMVHSEVAAYDSGRPGPLLGRPAMLASHMSALSQSQLISQMGIRSSIAHSSPSPPGSKSATPSPSSSTQEEESEVHFKISGEKRPSADPGKKAKNPKKKKKKDPNEPQKPVSAYALFFRDTQAAIKGQNPSATFGDVSKIVASMWDSLGEEQKQAYKRKTEAAKKEYLKALAAYRASLVSKSSPDQGETKSTQANPPAKMLPPKQPMYAMPGLASFLTPSDLQAFRSGASPASLARTLGSKSLLPGLSASPPPPPSFPLSPTLHQQLSLPPHAQGALLSPPVSMSPAPQPPVLPTPMALQVQLAMSPSPPGPQDFPHISEFPSSSGSCSPGPSNPTSSGDWDSSYPSGECGISTCSLLPRDKSLYLT TOX high mobility cassette family member 2 (TOX2), isoform 4, UniProtKB accession number Q96NM4-4 SEQ ID NO: 194 MDVRLYPSAPAVGARPGAEPAGLAHLDYYHGGKFDGDSAYVGMSDGNPELLSTSQTYNGQSENNEDYEIPPITPPNLPEPSLLHLGDHEASYHSLCHGLTPNGLLPAYSYQAMDLPAIMVSNMLAQDSHLLSGQLPTIQEMVHSEVAAYDSGRPGPLLGRPAMLASHMSALSQSQLISQMGIRSSIAHSSPSPPGSKSATPSPSSSTQEEESEVHFKISGEKRPSADPGKKAKNPKKKKKKDPNEPQKPVSAYALFFRDTQAAIKGQNPSATFGDVSKIVASMWDSLGEEQKQAYKRKTEAAKKEYLKALAAYRASLVSKSSPDQGETKSTQANPPAKMLPPKQPMYAMPGLASFLTPSDLQAFRSGASPASLARTLGSKSLLPGLSASPPPPPSFPLSPTLHQQLSLPPHAQGALLSPPVSMSPAPQPPVLPTPMALQVQLAMSPSPPGPQDFPHISEFPSSSGSCSPGPSNPTSSGDWDSSYPSGECGISTCSLLPRDKSLYLT Interferon regulatory factor 4 (IRF4), isoform 1, UniProtKB accession number Q15306-1 SEQ ID NO: 195 MNLEGGGRGGEFGMSAVSCGNGKLRQWLIDQIDSGKYPGLVWENEEKSIFRIPWKHAGKQDYNREEDAALFKAWALFKGKFREGIDKPDPPTWKTRLRCALNKSNDFEELVERSQLDISDPYKVYRIVPEGAKKGAKQLTLEDPQMSMSHPYTMTTPYPSLPAQQVHNYMMPPLDRSWRDYVPDQPHPEIPYQCPMTFGPRGHHWQGPACENGCQVTGTFYACAPPESQAPGVPTEPSIRSAEALAFSDCRLHICLYYREILVKELTTSSPEGCRISHGHTYDASNLDQVLFPYPEDNGQRKNIEKLLSHLERGVVLWMAPDGLYAKRLCQSRIYWDGPLALCNDRPNKLERDQTCKLFDTQQFLSELQAFAHHGRSLPRFQVTLCFGEEFPDPQRQRKLITAHVEPLLARQLYYFAQQNSGHFLRGYDLPEHISNPEDYHRSIRHSSIQE Interferon regulatory factor 4 (IRF4), isoform 2, UniProtKB accession number Q15306-2 SEQ ID NO: 196 MNLEGGGRGGEFGMSAVSCGNGKLRQWLIDQIDSGKYPGLVWENEEKSIFRIPWKHAGKQDYNREEDAALFKAWALFKGKFREGIDKPDPPTWKTRLRCALNKSNDFEELVERSQLDISDPYKVYRIVPEGAKKGAKQLTLEDPQMSMSHPYTMTTPYPSLPAQVHNYMMPPLDRSWRDYVPDQPHPEIPYQCPMTFGPRGHHWQGPACENGCQVTGTFYACAPPESQAPGVPTEPSIRSAEALAFSDCRLHICLYYREILVKELTTSSPEGCRISHGHTYDASNLDQVLFPYPEDNGQRKNIEKLLSHLERGVVLWMAPDGLYAKRLCQSRIYWDGPLALCNDRPNKLERDQTCKLFDTQQFLSELQAFAHHGRSLPRFQVTLCFGEEFPDPQRQRKLITAHVEPLLARQLYYFAQQNSGHFLRGYDLPEHISNPEDYHRSIRHSSIQE Basic leucine zipper transcription factor ATF class (BATF) 1, UniProtKB accession number Q16520-1 SEQ ID NO: 197 MPHSSSSSSSFSRSPPPGKQDSSDDDVRRVQRREKNRIAAQKSRQRQTQKADTLHLESEDLEKQNAALRKEIKQLTEELKYFTSVLNSHEPLCSVLAASTPSPPEVVYSAHAFHQPHVSSPRFQP Basic leucine zipper transcription factor ATF-2 (BATF2), isoform 1, UniProtKB accession number Q8N1L9-1 SEQ ID NO: 198 MHLCGGNGLLTQTDPKEQQRQLKKQKNRAAAQRSRQKHTDKADALHQQHESLEKDNLALRKEIQSLQAELAWWSRTLHVHERLCPMDCASCSAPGLLGCWDQAEGLLGPGPQGQHGCREQLELFQTPGSCYPAQPLSPGPQPHDSPSLLQCPLPSLSLGPAVVAEPPVQLSPSPLLFASHTGSSLQGSSSKLSALQPSLTAQTAPPQPLELEHPTRGKLGSSPDNPSSALGLARLQSREHKPALSAATWQGLVVDPSPHPLLAFPLLSSAQVHF Basic leucine zipper transcription factor ATF class-2 (BATF2), isoform 2, UniProtKB accession number Q8N1L9-2 SEQ ID NO: 199 MDCASCSAPGLLGCWDQAEGLLGPGPQGQHGCREQLELFQTPGSCYPAQPLSPGPQPHDSPSLLQCPLPSLSLGPAVVAEPPPVQLSSPPLLFASHTGSSLQGSSSKLSALQPSLTAQTAPPQPLELEHPTRGKLGSPDNPSSALGLARLQSREHKPALSAATWQGLVVDPSPPHPLLAFPLLSSAQVHF Basic leucine zipper transcription factor ATF class-3 (BATF3), isoform 1, UniProtKB accession number Q9NR55-1 SEQ ID NO: 200 MSQGLPAAGSVLQRSVAAPGNQPQPQPQQQSPEDDDRKVRRREKNRVAAQRSRKKQTQKADKLHEEYESLEQENTMLRREIGKLTEELKHLTEALKEHEKMCPLLLCPMNFVPVPPRPDPVAGCLPR X-box binding protein 1 (XBP1), isoform 1, UniProtKB accession number P17861-1 SEQ ID NO: 201 MVVVAAAPNPADGTPKVLLLSGQPASAAGAPAGQALPLMVPAQRGASPEAASGGLPQARKRQRLTHLSPEEKALRRKLKNRVAAQTARDRKKARMSELEQQVVDLEEENQKLLLENQLLREKTHGLVVENQELRQRLGMDALVAEEEAEAKGNEVRPVAGSAESAALRLRAPLQQVQAQLSPLQNISPWILAVLTLQIQSLISCWAFWTTWTQSCSSNALPQSLPAWRSSQRSTQKDPVPYQPPFLCQWGRHQPSWKPLMN X-box binding protein 1 (XBP1), isoform 2, UniProtKB accession number P17861-2 SEQ ID NO: 202 MVVVAAAPNPADGTPKVLLLSGQPASAAGAPAGQALPLMVPAQRGASPEAASGGLPQARKRQRLTHLSPEEKALRRKLKNRVAAQTARDRKKARMSELEQQVVDLEEENQKLLLENQLLREKTHGLVVENQELRQRLGMDALVAEEEAEAKGNEVRPVAGSAESAAGAGPVVTPPEHLPMDSGGIDSSDSESDILLGILDNLDPVMFFKCPSPEPASLEELPEVYPEGPSSLPASLSLSVGTSSAKLEAINELIRFDHIYTKPLVLEIPSETESQANVVVKIEEAPLSPSENDHPEFIVSVKEEPVEDDLVPELGISNLLSSSHCPKPSSCLLDAYSDCGYGGSLSPFSDMSSLLGVNHSWEDTFANELFPQLISV Transcription factor AP-1 (c-Jun), isoform 1, UniProtKB accession number P05412-1 SEQ ID NO: 203 MTAKMETTFYDDALNASFLPSESGPYGYSNPKILKQSMTLNLADPVGSLKPHLRAKNSDLLTSPDVGLLKLASPELERLIIQSSNGHITTTPTPTQFLCPKNVTDEQEGFAEGFVRALAELHSQNTLPSVTSAAQPVNGAGMVAPAVASVAGGSGSGGFSASLHSEPPVYANLSNFNPGALSSGGGAPSYGAAGLAFPAQPQQQQQPPHHLPQQMPVQHPRLQALKEEPQTVPEMPGETPPLSPIDMESQ ERIKAERKRMRNRIAASKCRKRKLERIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNHVNSGCQLMLTQQLQTF Proto-oncogene c-Fos (Fos), isoform 1, UniProtKB accession number P01100-1 SEQ ID NO: 204 MMFSGFNADYEASSSRCSSASPAGDSLSYYHSPADSFSSMGSPVNAQDFCTDLAVSSANFIPTVTAISTSPDLQWLVQPALVSSVAPSQTRAPHPFGVPAPSAGAYSRAGVVKTMTGGRAQSIGRRGKVEQLSPEEEEKRRIRRERNKMAAAKCRNRRRELTDTLQAETDQLEDEKSALQTEIANLLKEKEKLEFILAAHRPACKIPDDLGFPEEMSVASLDLTGGLPEVATPESEEAFTLPLLNDPEPKPSVEPVKSISSMELKTEPFDDFLFPASSRPSGSETARSVPDMDLSGSFYAADWEPLHSGSLGMGPMATELEPLCTPVVTCTPSCTAYTSSFVFTYPEADSFPSCAAAHRKGSSSNEPSSDSLSSPTLLAL Proto-oncogene c-Fos (Fos), isoform 2, UniProtKB accession number P01100-2 SEQ ID NO: 205 MTGGRAQSIGRRGKVEQLSPEEEEKRRIRRERNKMAAAKCRNRRRELTDTLQAETDQLEDEKSALQTEIANLLKEKEKLEFILAAHRPACKIPDDLGFPEEMSVASLDLTGGLPEVATPESEEAFTLPLLNDPEPKPSVEPVKSISSMELKTEPFDDFLFPASSRPSGSETARSVPDMDLSGSFYAADWEPLHSGSLGMGPMATELEPLCTPVVTCTPSCTAYTSSFVFTYPEADSFPSCAAAHRKGSSSNEPSSDSLSSPTLLAL Proto-oncogene c-Fos (Fos), isoform 3, UniProtKB accession number P01100-3 SEQ ID NO: 206 MMFSGFNADYEASSSRCSSASPAGDSLSYYHSPADSFSSMGSPVNAQDFCTDLAVSSANFIPTVTAISTSPDLQWLVQPALVSSVAPSQTRAPHPFGVPAPSAGAYSRAGVVKTMTGGRAQSIGRRGKVEQETDQLEDEKSALQTEIANLLKEKEKLEFILAAHRPACKIPDDLGFPEEMSVASLDLTGGLPEVATPESEEAFTLPLLNDPEPKPSVEPVKSISSMELKTEPFDDFLFPASSRPSGSETARSVPDMDLSGSFYAADWEPLHSGSLGMGPMATELEPLCTPVVTCTPSCTAYTSSFVFTYPEADSFPSCAAAHRKGSSSNEPSSDSLSSPTLLAL AP-1 complex subunit sigma 2 (AP1S2), isoform 1, UniProtKB accession number P56377-1 SEQ ID NO: 207 MQFMLLFSRQGKLRLQKWYVPLSKEKKKITRELVQTVLARKPKMCSFLEWRDLKIVYKRYASLYFCCAIEDQDNELITLEIIHRYVELLDKYFGSVCELDIIFNFEKAYFILDEFLLGGEVQETSKKNVLKAIEQADLLQEEAETPRSVLEEIGLT AP-1 complex subunit sigma 2 (AP1S2), isoform 2, UniProtKB accession number P56377-2 SEQ ID NO: 208 MPAGCPPHSTTASLPQHGDRGFPFAAAAAAGQAPPRPAAAMQFMLLFSRQGKLRLQKWYVPLSKEKKKITRELVQTVLARKPKMCSFLEWRDLKIVYKRYASLYFCCAIEDQDNELITLEIIHRYVELLDKYFGSVCELDIIFNFEKAYFILDEFLLGGEVQETSKKNVLKAIEQADLLKSKTEGFKTM AP-1 complex subunit sigma-1A (AP1S1), isoform 1, UniProtKB accession number P61966-1 SEQ ID NO: 209 MMRFMLLFSRQGKLRLQKWYLATSKERKKMVRELMQVVLARKPKMCSFLEWRDLKVVYKRYASLYFCCAIEGQDNELITLELIHRYVELLDKYFGSVCELDIIFNFEKAYFILDEFLMGGDVQDTSKKSVLKAIEQADLLQEEDESPRSVLEEMGLA AP-1 complex subunit sigma-1A (AP1S1), isoform 2, UniProtKB accession number P61966-2 SEQ ID NO: 210 MMRFMLLFSRQGKLRLQKWYLATSSDKERKKMVRELMQVVLARKPKMCSFLEWRDLKVVYKRYASLYFCCAIEGQDNELITLELIHRYVELLDKYFGSVCELDIIFNFEKAYFILDEFLMGGDVQDTTFPFSH AP-1 complex subunit sigma 3 (AP1S3), isoform 1, UniProtKB accession number Q96PC3-1 SEQ ID NO: 211 MIHFILLFSRQGKLRLQKWYITLPDKERKKITREIVQIILSRGHRTSSFVDWKELKLVYKRYASLYFCCAIENQDNELLTLEIVHRYVELLDKYFGNVCELDIIFNFEKAYFILDEFIIGGEIQETSKKIAVKAIEDSDMLQEVSTVSQTMGER AP-1 complex subunit sigma 3 (AP1S3), isoform 2, UniProtKB accession number Q96PC3-2 SEQ ID NO: 212 MIHFILLFSRQGKLRLQKWYITLPDKERKKITREIVQIILSRGHRTSSFVDWKELKLVYKRYASLYFCCAIENQDNELLTLEIVHRYVELLDKYFGNVCELDIIFNFEKAYFILDEFIIGGEIQETSKKIAVKAIEDSDMLQENRLSPRGRDCSEPRSCHCTLA AP-1 complex subunit sigma 3 (AP1S3), isoform 3, UniProtKB accession number Q96PC3-3 SEQ ID NO: 213 MIHFILLFSRQGKLRLQKWYITLPDKERKKITREIVQIILSRGHRTSSFVDWKELKLVYKRYASLYFCCAIENQDNELLTLEIVHRYVELLDKYFGNTWPFARA AP-1 complex subunit sigma 3 (AP1S3), isoform 4, UniProtKB accession number Q96PC3-4 SEQ ID NO: 214 MIHFILLFSRQGKLRLQKWYITLPDKERKKITREIVQIILSRGHRTSSFVDWKELKLVYKRYASLYFCCAIENQDNELLTLEIVHRYVELLDKYFGNVCELDIIFNFEKAYFILDEFIIGGEIQETSKKIAVKAIEDSDMLQETMEEYMNKPTF AP-1 complex subunit gamma-1 (AP1G1), isoform 1, UniProtKB accession number O43747-1 SEQ ID NO: 215 MPAPIRLRELIRTIRTARTQAEEREMIQKECAAIRSSFREEDNTYRCRNVAKLLYMHMLGYPAHFGQLECLKLIASQKFTDKRIGYLGAMLLLDERQDVHLLMTNCIKNDLNHSTQFVQGLALCTLGCMGSSEMCRDLAGEVEKLLKTSNSYLRKKAALCAVHVIRKVPELMEMFLPATKNLLNEKNHGVLHTSVVLLTEMCERSPDMLAHFRKLVPQLVRILKNLIMSGYSPEHDVSGISDPFLQVRIL RLLRILGRNDDDSSEAMNDILAQVATNTETSKNVGNAILYETVLTIMDIKSESGLRVLAINILGRFLLNNDKNIRYVALTSLLKTVQTDHNAVQRHRSTIVDCLKDLDVSIKRRAMELSFALVNGNNIRGMMKELLYFLDSCEPEFKADCASGIFLAAEKYAPSKRWHIDTIMRVLTTAGSYVRDDAVPNLIQLITNSVEMHAYTVQRLYKAILGDYSQQPLVQVAAWCIGEYGDLLVSGQCEEEEPIQVTEDEVLDILESVLISNMSTSVTRGYALTAIMKLSTRFTCTVNRIKKVVSIYGSSIDVELQQRAVEYNALFKKYDHMRSALLERMPVMEKVTTNGPTEIVQTNGETEPAPLETKPPPSGPQPTSQANDLLDLLGGNDITPVIPTAPTSKPSSAGGELLDLLGDINLTGAPAAAPAPASVPQISQPPFLLDGLSSQPLFNDIAAGIPSITAYSKNGLKIEFTFERSNTNPSVTVITIQASNSTELDMTDFVFQAAVPKTFQLQLLSPSSSIVPAFNTGTITQVIKVLNPQKQQLRMRIKLTYNHKGSAMQDLAEVNNFPPQSWQ AP-1 complex subunit gamma-1 (AP1G1), isoform 2, UniProtKB accession number O43747-2 SEQ ID NO: 216 MPAPIRLRELIRTIRTARTQAEEREMIQKECAAIRSSFREEDNTYRCRNVAKLLYMHMLGYPAHFGQLECLKLIASQKFTDKRIGYLGAMLLLDERQDVHLLMTNCIKNDLNHSTQFVQGLALCTLGCMGSSEMCRDLAGEVEKLLKTSNSYLRKKAALCAVHVIRKVPELMEMFLPATKNLLNEKNHGVLHTSVVLLTEMCERSPDMLAHFRKNEKLVPQLVRILKNLIMSGYSPEHDVSGISDPFLQVRILRLLRILGRNDDDSSEAMNDILAQVATNTETSKNVGNAILYETVLTIMDIKSESGLRVLAINILGRFLLNNDKNIRYVALTSLLKTVQTDHNAVQRHRSTIVDCLKDLDVSIKRRAMELSFALVNGNNIRGMMKELLYFLDSCEPEFKADCASGIFLAAEKYAPSKRWHIDTIMRVLTTAGSYVRDDAVPNLIQLITNSVEMHAYTVQRLYKAILGDYSQQPLVQVAAWCIGEYGDLLVSGQCEEEEPIQVTEDEVLDILESVLISNMSTSVTRGYALTAIMKLSTRFTCTVNRIKKVVSIYGSSIDVELQQRAVEYNALFKKYDHMRSALLERMPVMEKVTTNGPTEIVQTNGETEPAPLETKPPPSGPQPTSQANDLLDLLGGNDITPVIPTAPTSKPSSAGGELLDLLGDINLTGAPAAAPAPASVPQISQPPFLLDGLSSQPLFNDIAAGIPSITAYSKNGLKIEFTFERSNTNPSVTVITIQASNSTELDMTDFVFQAAVPKTFQLQLLSPSSSIVPAFNTGTITQVIKVLNPQKQQLRMRIKLTYNHKGSAMQDLAEVNNFPPQSWQ AP-1 complex subunit mu-1 (AP1M1), isoform 1, UniProtKB accession number Q9BXS5-1 SEQ ID NO: 217 MSASAVYVLDLKGKVLICRNYRGDVDMSEVEHFMPILMEKEEEGMLSPILAHGGVRFMWIKHNNLYLVATSKKNACVSLVFSFLYKVVQVFSEYFKELEEESIRDNFVIIYELLDELMDFGYPQTTDSKILQEYITQEGHKLETGAPRPPATVTNAVSWRSEGIKYRKNEVFLDVIESVNLLVSANGNVLRSEIVGSIKMRVFLSGMPELRLGLNDKVLFDNTGRGKSKSVELEDVKFHQCVRLSRFENDRTISFIPPDGEFELMSYRLNTHVKPLIWIESVIEKHSHSRIEYMIKAKSQFKRRSTANNVEIHIPVPNDADSPKFKTTVGSVKWVPENSEIVWSIKSFPGGKEYLMRAHFGLPSVEAEDKEGKPPISVKFEIPYFTTSGIQVRYLKIIEK SGYQALPWVRYITQNGDYQLRTQ AP-1 complex subunit mu-1 (AP1M1), isoform 2, UniProtKB accession number Q9BXS5-2 SEQ ID NO: 218 MSASAVYVLDLKGKVLICRNYRGDVDMSEVEHFMPILMEKEEEGMLSPILAHGGVRFMWIKHNNLYLVATSKKNACVSLVFSFLYKVVQVFSEYFKELEEESIRDNFVIIYELLDELMDFGYPQTTDSKILQEYITQEGHKLETGAPRPPATVTNAVSWRSEGIKYRKNEVFLDVIESVNLLGKYPGVGWLGHTVSANGNVLRSEIVGSIKMRVFLSGMPELRLGLNDKVLFDNTGRGKSKSVELEDVKF HQCVRLSRFENDRTISFIPPDGEFELMSYRLNTHVKPLIWIESVIEKHSHSRIEYMIKAKSQFKRRSTANNVEIHIPVPNDADSPKFKTTVGSVKWVPENSEIVWSIKSFPGGKEYLMRAHFGLPSVEAEDKEGKPPISVKFEIPYFTTSGIQVRYLKIIEKSGYQALPWVRYITQNGDYQLRTQ AP-1 complex subunit beta-1 (AP1B1), isoform A, UniProtKB accession number Q10567-1 SEQ ID NO: 219 MTDSKYFTTTKKGEIFELKAELNSDKKEKKKEAVKKVIASMTVGKDVSALFPDVVNCMQTDNLELKKLVYLYLMNYAKSQPDMAIMAVNTFVKDCEDPNPLIRALAVRTMGCIRVDKITEYLCEPLRKCLKDEDPYVRKTAAVCVAKLHDINAQLVEDQGFLDTLKDLISDSNPMVVANAVAALSEIAESHPSSNLLDLNPQSINKLLTALNECTEWGQIFILDCLANYMPKDDREAQSICERVTPRLSHANSAVVLSAVKVLMKFMEMLSKDLDYYGTLLKKLAPPLVTLLSAEPELQYVALRNINLIVQKRPEILKHEMKVFFVKYNDPIYVKLEKLDIMIRLASQANIAQVLAELKEYATEVDVDFVRKAVRAIGRCAIKVEQSAERCVSTLLDLIQTKVNYVVQEAIVVIKDIFRKYPNKYESVIATLCENLDSLDEPEARAAMIWIVGEYAERIDNADELLESFLEGFHDESTQVQLQLLTAIVKLFLKKPTETQELVQQVLSLATQDSDNPDLRDRGYIYWRLLSTDPVAAKEVVLAEKPLISEETDLIEPTLLDELICYIGTLASVYHKPPSAFVEGGRGVVHKSLPPRTASSESAESPETAPTGAPPGEQPDVIPAQGDLLGDLLNLDLGPPVSGPPLATSSVQMGAVDLLGGGLDSLMGDEPEGIGGTNFVAPPTAAVPANLGAPIGSGLSDLFDLTSGVGTLSGSYVAPKAVWLPAMKAKGLEISGTFTRQVGSISMDLQLTNKALQVMTDFAIQFNRNSFGLAPATPLQVHAPLSPNQTVEISLPLSTVGSVMKMEPLNNLQVAVKNNIDVFYFSTLYPLHILFVEDGKMDRQMFLATWKDIPNENEAQFQIRDCPLNAEAASSKLQSSNIFTVAKRNVEGQDMLYQSLKLTNGIWVLAELRIQPGNPSCTDLELSLKCRAPEVSQHVYQAYETILKN AP-1 complex subunit beta-1 (AP1B1), isoform B, UniProtKB accession number Q10567-2 SEQ ID NO: 220 MTDSKYFTTTKKGEIFELKAELNSDKKEKKKEAVKKVIASMTVGKDVSALFPDVVNCMQTDNLELKKLVYLYLMNYAKSQPDMAIMAVNTFVKDCEDPNPLIRALAVRTMGCIRVDKITEYLCEPLRKCLKDEDPYVRKTAAVCVAKLHDINAQLVEDQGFLDTLKDLISDSNPMVVANAVAALSEIAESHPSSNLLDLNPQSINKLLTALNECTEWGQIFILDCLANYMPKDDREAQSICERVTPRLSHANSAVVLSAVKVLMKFMEMLSKDLDYYGTLLKKLAPPLVTLLSAEPELQYVALRNINLIVQKRPEILKHEMKVFFVKYNDPIYVKLEKLDIMIRLASQANIAQVLAELKEYATEVDVDFVRKAVRAIGRCAIKVEQSAERCVSTLLDLIQTKVNYVVQEAIVVIKDIFRKYPNKYESVIATLCENLDSLDEPEARAAMIWIVGEYAERIDNADELLESFLEGFHDESTQVQLQLLTAIVKLFLKKPTETQELVQQVLSLATQDSDNPDLRDRGYIYWRLLSTDPVAAKEVVLAEKPLISEETDLIEPTLLDELICYIGTLASVYHKPPSAFVEGGRGVVHKSLPPRTASSESAESPETAPTGAPPGEQPDVIPAQGDLLGDLLNLDLGPPVSGPPLATSSVQMGAVDLLGGGLDSLIGGTNFVAPPTAAVPANLGAPIGSGLSDLFDLTSGVGTLSGSYVAPKAVWLPAMKAKGLEISGTFTRQVGSISMDLQLTNKALQVMTDFAIQFNRNSFGLAPATPLQVHAPLSPNQTVEISLPLSTVGSVMKMEPLNNLQVAVKNNIDVFYFSTLYPLHILFVEDGKMDRQMFLATWKDIPNENEAQFQIRDCPLNAEAASSKLQSSNIFTVAKRNVEGQDMLYQSLKLTNGIWVLAELRIQPGNPSCTDLELSLKCRAPEVSQHVYQAYETILKN AP-1 complex subunit beta-1 (AP1B1), isoform C, UniProtKB accession number Q10567-3 SEQ ID NO: 221 MTDSKYFTTTKKGEIFELKAELNSDKKEKKKEAVKKVIASMTVGKDVSALFPDVVNCMQTDNLELKKLVYLYLMNYAKSQPDMAIMAVNTFVKDCEDPNPLIRALAVRTMGCIRVDKITEYLCEPLRKCLKDEDPYVRKTAAVCVAKLHDINAQLVEDQGFLDTLKDLISDSNPMVVANAVAALSEIAESHPSSNLLDLNPQSINKLLTALNECTEWGQIFILDCLANYMPKDDREAQSICERVTPRLSHANSAVVLSAVKVLMKFMEMLSKDLDYYGTLLKKLAPPLVTLLSAEPELQYVALRNINLIVQKRPEILKHEMKVFFVKYNDPIYVKLEKLDIMIRLASQANIAQVLAELKEYATEVDVDFVRKAVRAIGRCAIKVEQSAERCVSTLLDLIQTKVNYVVQEAIVVIKDIFRKYPNKYESVIATLCENLDSLDEPEARAAMIWIVGEYAERIDNADELLESFLEGFHDESTQVQLQLLTAIVKLFLKKPTETQELVQQVLSLATQDSDNPDLRDRGYIYWRLLSTDPVAAKEVVLAEKPLISEETDLIEPTLLDELICYIGTLASVYHKPPSAFVEGGRGVVHKSLPPRTASSESAESPETAPTGAPPGEQPDVIPAQGDLLGDLLNLDLGPPVSGPPLATSSVQMGAVDLLGGGLDSLIGGTNFVAPPTAAVPANLGAPIGSGLSDLFDLTSGVGTLSGSYVAPKAVWLPAMKAKGLEISGTFTRQVGSISMDLQLTNKALQVMTDFAIQFNRNSFGLAPATPLQVHAPLSPNQTVEISLPLSTVGSVMKMEPLNNLQVAVKNNIDVFYFSTLYPLHILFVEDGKMDRQMFLATWKDIPNENEAQFQIRDCPLNAEAASSKLQSSNIFTVAKRNVEGQDMLYQSLKLTNGIWVLAELRIQPGNPSCTLSLKCRAPEVSQHVYQAYETILKN AP-1 complex subunit beta-1 (AP1B1), isoform 4, UniProtKB accession number Q10567-4 SEQ ID NO: 222 MTDSKYFTTTKKGEIFELKAELNSDKKEKKKEAVKKVIASMTVGKDVSALFPDVVNCMQTDNLELKKLVYLYLMNYAKSQPDMAIMAVNTFVKDCEDPNPLIRALAVRTMGCIRVDKITEYLCEPLRKCLKDEDPYVRKTAAVCVAKLHDINAQLVEDQGFLDTLKDLISDSNPMVVANAVAALSEIAESHPSSNLLDLNPQSINKLLTALNECTEWGQIFILDCLANYMPKDDREAQSICERVTPRLSHANSAVVLSAVKVLMKFMEMLSKDLDYYGTLLKKLAPPLVTLLSAEPELQYVALRNINLIVQKRPEILKHEMKVFFVKYNDPIYVKLEKLDIMIRLASQANIAQVLAELKEYATEVDVDFVRKAVRAIGRCAIKVEQSAERCVSTLLDLIQTKVNYVVQEAIVVIKDIFRKYPNKYESVIATLCENLDSLDEPEARAAMIWIVGEYAERIDNADELLESFLEGFHDESTQVQLQLLTAIVKLFLKKPTETQELVQQVLSLATQDSDNPDLRDRGYIYWRLLSTDPVAAKEVVLAEKPLISEETDLIEPTLLDELICYIGTLASVYHKPPSAFVEGGRGVVHKSLPPRTASSESAESPETAPTGAPPGEQPDVIPAQGDLLGDLLNLDLGPPVSGPPLATSSVQMGAVDLLGGGLDSLIGGTNFVAPPTAAVPANLGAPIGSGLSDLFDLTSGVGTLSGSYVAPKAVGSISMDLQLTNKALQVMTDFAIQFNRNSFGLAPATPLQVHAPLSPNQTVEISLPLSTVGSVMKMEPLNNLQVAVKNNIDVFYFSTLYPLHILFVEDGKMDRQMFLATWKDIPNENEAQFQIRDCPLNAEAASSKLQSSNIFTVAKRNVEGQDMLYQSLKLTNGIWVLAELRIQPGNPSCTLSLKCRAPEVSQHVYQAYETILKN AP-1 complex subunit mu-2 (AP1M2), isoform 1, UniProtKB accession number Q9Y6Q5-1 SEQ ID NO: 223 MSASAVFILDVKGKPLISRNYKGDVAMSKIEHFMPLLVQREEEGALAPLLSHGQVHFLWIKHSNLYLVATTSKNANASLVYSFLYKTIEVFCEYFKELEEESIRDNFVIVYELLDELMDFGFPQTTDSKILQEYITQQSNKLETGKSRVPPTVTNAVSWRSEGIKYKKNEVFIDVIESVNLLVNANGSVLLSEIVGTIKLKVFLSGMPELRLGLNDRVLFELTGRSKNKSVELEDVKFHQCVRLSRFDNDRTISFIPPDGDFELMSYRLSTQVKPLIWIESVIEKFSHSRVEIMVKAKGQFKKQSVANGVEISVPVPSDADSPRFKTSVGSAKYVPERNVVIWSIKSFPGGKEYLMRAHFGLPSVEKEEVEGRPPIGVKFEIPYFTVSGIQVRYMKIIEKSGYQALPWVRYITQSGDYQLRTS AP-1 complex subunit mu-2 (AP1M2), isoform 2, UniProtKB accession number Q9Y6Q5-2 SEQ ID NO: 224 MSASAVFILDVKGKPLISRNYKGDVAMSKIEHFMPLLVQREEEGALAPLLSHGQVHFLWIKHSNLYLVATTSKNANASLVYSFLYKTIEVFCEYFKELEEESIRDNFVIVYELLDELMDFGFPQTTDSKILQEYITQQSNKLETGKSRVPPTVTNAVSWRSEGIKYKKNEVFIDVIESVNLLVNANGSVLLSEIVGTIKLKVFLSGMPELRLGLNDRVLFELTGLSGSKNKSVELEDVKFHQCVRLSRFDNDRTISFIPPDGDFELMSYRLSTQVKPLIWIESVIEKFSHSRVEIMVKAKGQFKKQSVANGVEISVPVPSDADSPRFKTSVGSAKYVPERNVVIWSIKSFPGGKEYLMRAHFGLPSVEKEEVEGRPPIGVKFEIPYFTVSGIQVRYMKIIEKSGYQALPWVRYITQSGDYQLRTS AP-1 complex subunit gamma-2 (AP1G2), isoform 1, UniProtKB accession number O75843-1 SEQ ID NO: 225 MVVPSLKLQDLIEEIRGAKTQAQEREVIQKECAHIRASFRDGDPVHRHRQLAKLLYVHMLGYPAHFGQMECLKLIASSRFTDKRVGYLGAMLLLDERHDAHLLITNSIKNDLSQGIQPVQGLALCTLSTMGSAEMCRDLAPEVEKLLLQPSPYVRKKAILTAVHMIRKVPELSSVFLPPCAQLLHERHHGILLGTITLITELCERSPAALRHFRKVVPQLVHILRTLVTMGYSTEHSISGVSDPFLQVQILRLLRILGRNHEESSETMNDLLAQVATNTDTSRNAGNAVLFETVLTIMDIRSAAGLRVLAVNILGRFLLNSDRNIRYVALTSLLRLVQSDHSAVQRHRPTVVECLRETDASLSRRALELSLALVNSSNVRAMMQELQAFLESCPPDLRADCASGILLAAERFAPTKRWHIDTILHVLTTAGTHVRDDAVANLTQLIGGAQELHAYSVRRLYNALAEDISQQPLVQVAAWCIGEYGDLLLAGNCEEIEPLQVDEEEVLALLEKVLQSHMSLPATRGYALTALMKLSTRLCGDNNRIRQVVSIYGSCLDVELQQRAVEYDTLFRKYDHMRAAILEKMPLVERDGPQADEEAKESKEAAQLSEAAPVPTEPQASQLLDLLDLLDGASGDVQHPPHLDPSPGGALVHLLDLPCVPPPPAPIPDLKVFEREGVQLNLSFIRPPENPALLLITITATNFSEGDVTHFICQAAVPKSLQLQLQAPSGNTVPARGGLPITQLFRILNPNKAPLRLKLRLTYDHFHQSVQEIFEVNNLPVESWQ BTB and CNC homologue 2 (BACH2), isoform 1, UniProtKB accession number Q9BYV9-1 SEQ ID NO: 226 MSVDEKPDSPMYVYESTVHCTNILLGLNDQRKKDILCDVTLIVERKEFRAHRAVLAACSEYFWQALVGQTKNDLVVSLPEEVTARGFGPLLQFAYTAKLLLSRENIREVIRCAEFLRMHNLEDSCFSFLQTQLLNSEDGLFVCRKDAACQRPHEDCENSAGEEEDEEEETMDSETAKMACPRDQMLPEPISFEAAAIPVAEKEEALLPEPDVPTDTKESSEKDALTQYPRYKKYQLACTKNVYNASSHSTSGFASTFREDNSSNSLKPGLARGQIKSEPPSEENEEESITLCLSGDEPDAKDRAGDVEMDRKQPSPAPTPTAPAGAACLERSRSVASPSCLRSLFSITKSVELSGLPSTSQQHFARSPACPFDKGITQGDLKTDYTPFTGNYGQPHVGQKEVSNFTMGSPLRGPGLEALCKQEGELDRRSVIFSSSACDQVSTSVHSYSGVSSLDKDLSEPVPKGLWVGAGQSLPSSQAYSHGGLMADHLPGRMRPNTSCPVPIKVCPRSPPLETRTRTSSSCSSYSYAEDGSGGSPCSLPLCEFSSSPCSQGARFLATEHQEPGLMGDGMYNQVRPQIKCEQSYGTNSSDESGSFSEADSESCPVQDRGQEVKLPFPVDQITDLPRNDFQMMIKMHKLTSEQLEFIHDVRRRSKNRIAAQRCRKRKLDCIQNLECEIRKLVCEKEKLLSERNQLKACMGELLDNFSCLSQEVCRDIQSPEQIQALHRYCPVLRPMDLPTASSINPAPLGAEQNIAASQCAVGENVPCCLEPGAAPPGPPWAPSNTSENCTSGRRLEGTD PGTFSERGPPLEPRSQTVTVDFCQEMTDKCTTDEQPRKDYT Transcription factor 7 (TCF7; also known as T cell factor 1 (TCF-1)), isoform 4L, UniProtKB accession number P36402-1 SEQ ID NO: 227 MPQLDSGGGGAGGGDDLGAPDELLAFQDEGEEQDDKSRDSAAGPERDLAELKSSLVNESEGAAGGAGIPGVPGAGAGARGEAEALGREHAAQRLFPDKLPEPLEDGLKAPECTSGMYKETVYSAFNLLMHYPPPSGAGQHPQPQPPLHKANQPPHGVPQLSLYEHFNSPHPTPAPADISQKQVHRPLQTPDLSGFYSLTSGSMGQLPHTVSWFTHPSLMLGSGVPGHPAAIPHPAIVPPSGKQELQPFDR NLKTQAESKAEKEAKKPTIKKPLNAFMLYMKEMRAKVIAECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSARDNYGKKKRRSREKHQESTTETNWPRELKDGNGQESLSMSSSSSPA Transcription factor 7 (TCF7; also known as T cell factor 1 (TCF-1)), isoform 4S, UniProtKB accession number P36402-2 SEQ ID NO: 228 MYKETVYSAFNLLMHYPPPSGAGQHPQPQPPLHKANQPPHGVPQLSLYEHFNSPHPTPAPADISQKQVHRPLQTPDLSGFYSLTSGSMGQLPHTVSWFTHPSLMLGSGVPGHPAAIPHPAIVPPSGKQELQPFDRNLKTQAESKAEKEAKKPTIKKPLNAFMLYMKEMRAKVIAECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSARDNYGKKKRRSREKHQESTTETNWPRELKDGNGQESLSMSSSSSPA Transcription factor 7 (TCF7; also known as T cell factor 1 (TCF-1)), isoform 1L, UniProtKB accession number P36402-3 SEQ ID NO: 229 MPQLDSGGGGAGGGDDLGAPDELLAFQDEGEEQDDKSRDSAAGPERDLAELKSSLVNESEGAAGGAGIPGVPGAGAGARGEAEALGREHAAQRLFPDKLPEPLEDGLKAPECTSGMYKETVYSAFNLLMHYPPPSGAGQHPQPQPPLHKANQPPHGVPQLSLYEHFNSPHPTPAPADISQKQVHRPLQTPDLSGFYSLTSGSMGQLPHTVSWFTHPSLMLGSGVPGHPAAIPHPAIVPPSGKQELQPFDR NLKTQAESKAEKEAKKPTIKKPLNAFMLYMKEMRAKVIAECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSARDNYGKKKRRSREKHQESTTDPGSPKKCRARFGLNQQTDWCGPCRRKKKCIRYLPGEGRCPSPVPSDDSALGCPGSPAPQDSPSYHLLPRFPTELLTSPAERHLHPQVSPLLSASQPQGPHRPPAAPCRAHRYSNRNLRDRWPSRHRTPGRLQEPTP Transcription factor 7 (TCF7; also known as T cell factor 1 (TCF-1)), isoform 1S, UniProtKB accession number P36402-4 SEQ ID NO: 230 MYKETVYSAFNLLMHYPPPSGAGQHPQPQPPLHKANQPPHGVPQLSLYEHFNSPHPTPAPADISQKQVHRPLQTPDLSGFYSLTSGSMGQLPHTVSWFTHPSLMLGSGVPGHPAAIPHPAIVPPSGKQELQPFDRNLKTQAESKAEKEAKKPTIKKPLNAFMLYMKEMRAKVIAECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSARDNYGKKKRRSREKHQESTTDPGSPKKCRARFGLNQQTDWCGPCRRKKKCIRYLPGEGRCPSPVPSDDSALGCPGSPAPQDSPSYHLLPRFPTELLTSPAERHLHPQVSPLLSASQPQGPHRPPAAPCRAHRYSNRNLRDRWPSRHRTPGRLQEPTP Transcription factor 7 (TCF7; also known as T cell factor 1 (TCF-1)), isoform 5L, UniProtKB accession number P36402-9 SEQ ID NO: 231 MPQLDSGGGGAGGGDDLGAPDELLAFQDEGEEQDDKSRDSAAGPERDLAELKSSLVNESEGAAGGAGIPGVPGAGAGARGEAEALGREHAAQRLFPDKLPEPLEDGLKAPECTSGMYKETVYSAFNLLMHYPPPSGAGQHPQPQPPLHKANQPPHGVPQLSLYEHFNSPHPTPAPADISQKQVHRPLQTPDLSGFYSLTSGSMGQLPHTVSWFTHPSLMLGSGVPGHPAAIPHPAIVPPSGKQELQPFDR NLKTQAESKAEKEAKKPTIKKPLNAFMLYMKEMRAKVIAECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSARDNYGKKKRRSREKHQESTTDNSLHYS Transcription factor 7 (TCF7; also known as T cell factor 1 (TCF-1)), isoform 5S, UniProtKB accession number P36402-10 SEQ ID NO: 232 MYKETVYSAFNLLMHYPPPSGAGQHPQPQPPLHKANQPPHGVPQLSLYEHFNSPHPTPAPADISQKQVHRPLQTPDLSGFYSLTSGSMGQLPHTVSWFTHPSLMLGSGVPGHPAAIPHPAIVPPSGKQELQPFDRNLKTQAESKAEKEAKKPTIKKPLNAFMLYMKEMRAKVIAECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSARDNYGKKKRRSREKHQESTTDNSLHYS Transcription factor 7 (TCF7; also known as T cell factor 1 (TCF-1)), isoform 6L, UniProtKB accession number P36402-11 SEQ ID NO: 233 MPQLDSGGGGAGGGDDLGAPDELLAFQDEGEEQDDKSRDSAAGPERDLAELKSSLVNESEGAAGGAGIPGVPGAGAGARGEAEALGREHAAQRLFPDKLPEPLEDGLKAPECTSGMYKETVYSAFNLLMHYPPPSGAGQHPQPQPPLHKANQPPHGVPQLSLYEHFNSPHPTPAPADISQKQVHRPLQTPDLSGFYSLTSGSMGQLPHTVSWFTHPSLMLGSGVPGHPAAIPHPAIVPPSGKQELQPFDR NLKTQAESKAEKEAKKPTIKKPLNAFMLYMKEMRAKVIAECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSARDNYGKKKRRSREKHQESTTDPGSPKKCRARFGLNQQTDWCGPCRKKKCIRYLPGEGRCPSPVPSDDSALGCPGSPAPQDSPSYHLLPRFPTELLTSPAERHLHPQVSPLLSASQPQGPHRPPAAPCRAHRYSNRNLRDRWPSRHRTPGRLQEPTP Transcription factor 7 (TCF7; also known as T cell factor 1 (TCF-1)), isoform 6S, UniProtKB accession number P36402-12 SEQ ID NO: 234 MYKETVYSAFNLLMHYPPPSGAGQHPQPQPPLHKANQPPHGVPQLSLYEHFNSPHPTPAPADISQKQVHRPLQTPDLSGFYSLTSGSMGQLPHTVSWFTHPSLMLGSGVPGHPAAIPHPAIVPPSGKQELQPFDRNLKTQAESKAEKEAKKPTIKKPLNAFMLYMKEMRAKVIAECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSARDNYGKKKRRSREKHQESTTDPGSPKKCRARFGLNQQTDWCGPCRKKKCIRYLPGEGRCPSPVPSDDSALGCPGSPAPQDSPSYHLLPRFPTELLTSPAERHLHPQVSPLLSASQPQGPHRPPAAPCRAHRYSNRNLRDRWPSRHRTPGRLQEPTP Transcription factor 7 (TCF7; also known as T cell factor 1 (TCF-1)), isoform 7L, UniProtKB accession number P36402-13 SEQ ID NO: 235 MPQLDSGGGGAGGGDDLGAPDELLAFQDEGEEQDDKSRDSAAGPERDLAELKSSLVNESEGAAGGAGIPGVPGAGAGARGEAEALGREHAAQRLFPDKLPEPLEDGLKAPECTSGMYKETVYSAFNLLMHYPPPSGAGQHPQPQPPLHKANQPPHGVPQLSLYEHFNSPHPTPAPADISQKQVHRPLQTPDLSGFYSLTSGSMGQLPHTVSWFTHPSLMLGSGVPGHPAAIPHPAIVPPSGKQELQPFDR NLKTQAESKAEKEAKKPTIKKPLNAFMLYMKEMRAKVIAECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSARDNYGKKKRRSREKHQESTTDGIPACTILSP Transcription factor 7 (TCF7; also known as T cell factor 1 (TCF-1)), isoform 7S, UniProtKB accession number P36402-14 SEQ ID NO: 236 MYKETVYSAFNLLMHYPPPSGAGQHPQPQPPLHKANQPPHGVPQLSLYEHFNSPHPTPAPADISQKQVHRPLQTPDLSGFYSLTSGSMGQLPHTVSWFTHPSLMLGSGVPGHPAAIPHPAIVPPSGKQELQPFDRNLKTQAESKAEKEAKKPTIKKPLNAFMLYMKEMRAKVIAECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSARDNYGKKKRRSREKHQESTTDGIPACTILSP Transcription factor 7 (TCF7; also known as T cell factor 1 (TCF-1)), isoform 8L, UniProtKB accession number P36402-15 SEQ ID NO: 237 MPQLDSGGGGAGGGDDLGAPDELLAFQDEGEEQDDKSRDSAAGPERDLAELKSSLVNESEGAAGGAGIPGVPGAGAGARGEAEALGREHAAQRLFPDKLPEPLEDGLKAPECTSGMYKETVYSAFNLLMHYPPPSGAGQHPQPQPPLHKANQPPHGVPQLSLYEHFNSPHPTPAPADISQKQVHRPLQTPDLSGFYSLTSGSMGQLPHTVSWFTHPSLMLGSGVPGHPAAIPHPAIVPPSGKQELQPFDR NLKTQAESKAEKEAKKPTIKKPLNAFMLYMKEMRAKVIAECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSARDNYGKKKRRSREKHQESTTQLEDWDGWARKP Transcription factor 7 (TCF7; also known as T cell factor 1 (TCF-1)), isoform 2L, UniProtKB accession number P36402-5 SEQ ID NO: 239 MPQLDSGGGGAGGGDDLGAPDELLAFQDEGEEQDDKSRDSAAGPERDLAELKSSLVNESEGAAGGAGIPGVPGAGAGARGEAEALGREHAAQRLFPDKLPEPLEDGLKAPECTSGMYKETVYSAFNLLMHYPPPSGAGQHPQPQPPLHKANQPPHGVPQLSLYEHFNSPHPTPAPADISQKQVHRPLQTPDLSGFYSLTSGSMGQLPHTVSWFTHPSLMLGSGVPGHPAAIPHPAIVPPSGKQELQPFDR NLKTQAESKAEKEAKKPTIKKPLNAFMLYMKEMRAKVIAECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSARDNYGKKKRRSREKHQESTTGGKRNAFGTYPEKAAAPAPFLPMTVL Transcription factor 7 (TCF7; also known as T cell factor 1 (TCF-1)), isoform 2S, UniProtKB accession number P36402-6 SEQ ID NO: 240 MYKETVYSAFNLLMHYPPPSGAGQHPQPQPPLHKANQPPHGVPQLSLYEHFNSPHPTPAPADISQKQVHRPLQTPDLSGFYSLTSGSMGQLPHTVSWFTHPSLMLGSGVPGHPAAIPHPAIVPPSGKQELQPFDRNLKTQAESKAEKEAKKPTIKKPLNAFMLYMKEMRAKVIAECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSARDNYGKKKRRSREKHQESTTGGKRNAFGTYPEKAAAPAPFLPMTVL Transcription factor 7 (TCF7; also known as T cell factor 1 (TCF-1)), isoform 3L, UniProtKB accession number P36402-7 SEQ ID NO: 241 MPQLDSGGGGAGGGDDLGAPDELLAFQDEGEEQDDKSRDSAAGPERDLAELKSSLVNESEGAAGGAGIPGVPGAGAGARGEAEALGREHAAQRLFPDKLPEPLEDGLKAPECTSGMYKETVYSAFNLLMHYPPPSGAGQHPQPQPPLHKANQPPHGVPQLSLYEHFNSPHPTPAPADISQKQVHRPLQTPDLSGFYSLTSGSMGQLPHTVSWFTHPSLMLGSGVPGHPAAIPHPAIVPPSGKQELQPFDR NLKTQAESKAEKEAKKPTIKKPLNAFMLYMKEMRAKVIAECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSARDNYGKKKRRSREKHQESTTDPGSPKKCRARFGLNQQTDWCGPCR Transcription factor 7 (TCF7; also known as T cell factor 1 (TCF-1)), isoform 3S, UniProtKB accession number P36402-8 SEQ ID NO: 242 MYKETVYSAFNLLMHYPPPSGAGQHPQPQPPLHKANQPPHGVPQLSLYEHFNSPHPTPAPADISQKQVHRPLQTPDLSGFYSLTSGSMGQLPHTVSWFTHPSLMLGSGVPGHPAAIPHPAIVPPSGKQELQPFDRNLKTQAESKAEKEAKKPTIKKPLNAFMLYMKEMRAKVIAECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSARDNYGKKKRRSREKHQESTTDPGSPKKCRARFGLNQQTDWCGPCR Forkhead box protein P1 (FoxP1), isoform 1, UniProKB accession number Q9H334-1 SEQ ID NO: Forkhead box protein P1 (FoxP1), isoform 3, UniProKB accession number Q9H334-3 SEQ ID NO: MFQCVFSSSVLQPHSTSCLFKHLFYHSATPASQKQPEPIYSKKTEIQRQTVRAPFAKLFIFSALQVARQLLLQQQQQQQVSGLKSPKRNDKQPALQQQQVATQQLAFQQQLLQMQQLQQQHLLSLQRQGLLTIQPGQPALPLQPLAQGMIPTELQQLWKEVTSAHTAEETTGNNHSSLDLTTTCVSSSAPSKTSLIMNPHASTNGQLSVHTPKRESLSHEEHPHSHPLYGHGVCKWPGCEAVCEDFQSFL KHLNSEHALDDRSTAQCRVQMQVVQQLELQLAKDKERLQAMMTHLHVKSTEPKAAPQPLNLVSSVTLSKSASEASPQSLPHTPTTPTAPLTPVTQGPSVITTTSMHTVGPIRRRYSDKYNVPISSADIAQNQEFYKNAEVRPPFTYASLIRQAILESPEKQLTLNEIYNWFTRMFAYFRRNAATWKNAVRHNLSLHKCFVRVENVKGAVWTVDEVEFQKRRPQKISGNPSLIKNMQSSHAYCTPLNAALQ ASMAENSIPLYTTASMGNPTLGNLASAIREELNGAMEHTNSNESDSSPGRSPMQAVHPVHVKEEPLDPEEAEGPLSLVTTANHSPDFDHDRDYEDEPVNEDME Forkhead box protein P1 (FoxP1), isoform 4, UniProKB accession number Q9H334-4 SEQ ID NO: MFQCVFSSSVLQPHSTSCLFKHLFYHSATPASQKQPEPIYSKKTEIQRQTVRAPFAKLFIFSALQVARQLLLQQQQQQQVSGLKSPKRNDKQPALQVPVSVAMMTPQVITPQQMQQILQQQVLSPQQLQVLLQQQQALMLQQQQLQEFYKKQQEQLQLQLLQQQHAGKQPKEQQQVATQQLAFQQQLLQMQQLQQQHLLSLQRQGLLTIQPGQPALPLQPLAQGMIPTELQQLWKEVTSAHTAEETTGNNHSSLDLTTTCVSSSAPSKTSLIMNPHASTNGQLSVHTPKRESLSHEEHPHSHPLYGHGVCKWPGCEAVCEDFQSFLKHLNSEHALDDRSTAQCRVQMQVVQQLELQLAKDKERLQAMMTHLHVKSTEPKAAPQPLNLVSSVTLSKSASEASPQSLPHTPTTPTAPLTPVTQGPSVITTTSMHTVGPIRRRYSDKYNVPISSADIAQNQEFYKNAEVRPPFTYASLIRQAILESPEKQLTLNEIYNWFTRM FAYFRRNAATWKNAVRHNLSLHKCFVRVENVKGAVWTVDEVEFQKRRPQKISGNPSLIKNMQSSHAYCTPLNAALQASMAENSIPLYTTASMGNPTLGNLASAIREELNGAMEHTNSNESDSSPGRSPMQAVHPVHVKEEPLDPEEAEGPLSLVTTANHSPDFDHDRDYEDEPVNEDME Forkhead box protein P1 (FoxP1), isoform 5, UniProKB accession number Q9H334-5 SEQ ID NO: MMQESGTETKSNGSAIQNGSGGSNHLLECGGLREGRSNGETPAVDIGAADLAHAQQQQQQWHLINHQPSRSPSSWLKRLISSPWELEVLQVPLWGAVAETKMSGPVCQPNPSPF Forkhead box protein P1 (FoxP1), isoform 6, UniProKB accession number Q9H334-6 SEQ ID NO: MMQESGTETKSNGSAIQNGSGGSNHLLECGGLREGRSNGETPAVDIGAADLAHAQQQQQQALQVARQLLLQQQQQQQVSGLKSPKRNDKQPALQQQQVATQQLAFQQQLLQMQQLQQQHLLSLQRQGLLTIQPGQPALPLQPLAQGMIPTELQQLWKEVTSAHTAEETTGNNHSSLDLTTTCVSSSAPSKTSLIMNPHASTNGQLSVHTPKRESLSHEEHPHSHPLYGHGVCKWPGCEAVCEDFQSFLKHLNSEHALDDRSTAQCRVQMQVVQQLELQLAKDKERLQAMMTHLHVKSTEPKAAPQPLNLVSSVTLSKSASEASPQSLPHTPTTPTAPLTPVTQGPSVITTTSMHTVGPIRRRYSDKYNVPISSADIAQNQEFYKNAEVRPPFTYASLIRQAILESPEKQLTLNEIYNWFTRMFAYFRRNAATWKNAVRHNLSLHKCFVRVENVKGAVWTVDEVEFQKRRPQKISGNPSLIKNMQSSHAYCTPLNAALQASMAENSIPLYTTASMGNPTLGNLASAIREELNGAMEHTNSNESDSSPGRSPMQAVHPVHVKEEPLDPEEAEGPLSLVTTANHSPDFDHDRDYEDEPVNEDME Forkhead box protein P1 (FoxP1), isoform 7, UniProKB accession number Q9H334-7 SEQ ID NO: MMQESGTETKSNGSAIQNGSGGSNHLLECGGLREGRSNGETPAVDIGAADLAHAQQQQQQALQVARQLLLQQQQQQQVSGLKSPKRNDKQPALQVPVSVAMMTPQVITPQQMQQILQQQVLSPQQLQVLLQQQQALMLQQQQLQEFYKKQQEQLQLQLLQQQHAGKQPKEQQQVATQQLAFQQQLLQMQQLQQQHLLSLQ RQGLLTIQPGQPALPLQPLAQGMIPTELQQLWKEVTSAHTAEETTGNNHSSLDLTTTCVSSSAPSKTSLIMNPHASTNGQLSVHTPKRESLSHEEHPHSHPLYGHGVCKWPGCEAVCEDFQSFLKHLNSEHALDDRSTAQCRVQMQVVQQLELQLAKDKERLQAMMTHLHVKSTEPKAAPQPLNLVSSVTLSKSASEASPQSLPHTPTTPTAPLTPVTQGPSVITTTSMHTVGPIRRRYSDKYNVPISSD IAQNQEFYKNAEVRPPFTYASLIRQAILESPEKQLTLNEIYNWFTRMFAYFRRNAATWKNAVRHNLSLHKCFVRVENVKGAVWTVDEVEFQKRRPQKISGNPSLIKNMQSSHAYCTPLNAALQASMAENSIPLYTTASMGNPTLGNLASAIREELNGAMEHTNSNESDSSPGRSPMQAVHPVHVKEEPLDPEEAEGPLSLVTTANHSPDFDHDRDYEDEPVNEDME Forkhead box protein P1 (FoxP1), isoform 8, UniProKB accession number Q9H334-8 SEQ ID NO: MMQESGTETKSNGSAIQNGSGGSNHLLECGGLREGRSNGETPAVDIGAADLAHAQQQQQQALQVARQLLLQQQQQQQVSGLKSPKRNDKQPALQVPVSVAMMTPQVITPQQMQQILQQQVLSPQQLQVLLQQQQALMLQQQQLQEFYKKQQEQLQLQLLQQQHAGKQPKEQQQVATQQLAFQQQLLQMQQLQQQHLLSLQRQGLLTIQPGQPALPLQPLAQGMIPTELQQLWKEVTSAHTAEETTGNNHSSLDLTTTCVSSSAPSKTSLIMNPHASTNGQLSVHTPKRESLSHEEHPHSHPLYGHGVCKWPGCEAVCEDFQSFLKHLNSEHALDDRSTAQCRVQMQVVQQLELQLAKDKERLQAMMTHLHVKSTEPKAAPQPLNLVSSVTLSKSASEASPQSLPHTPTTPTAPLTPVTQGPSVITTTSMHTVGPIRRRYSDKYNVPISSADIAQNQEFYKNAEVRPPFTYASLIRQAILESPEKQLTLNEIYNWFTRMFAYFRRNAATWKGAIRTNLSLHKCFIRVEDEFGSFWTVDDEEFKRGRHIQRGRPRKYCPDENFDELVAHNPSLIKNMQSSHAYCTPLNAALQASMAENSIPLYTTASMGNPTLGNLASAIREELNGAMEHTNSNESDSSPGRSPMQAVHPVHVKEEPLDPEEAEGPLSLVTTANHSPDFDHDRDYEDEPVNEDME Forkhead box protein P1 (FoxP3), isoform 1, UniProKB accession number Q9BZS1-1 SEQ ID NO: MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEK EKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVES EKGAVWTVDELEFRKKRSQRPSRCSNPTPGP Forkhead box protein P1 (FoxP3), isoform 2, UniProKB accession number Q9BZS1-2 SEQ ID NO: MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGP Forkhead box protein P1 (FoxP3), isoform 3, UniProKB accession number Q9BZS1-3 SEQ ID NO: MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKVSSSEVAVTGMASSAIAAQSGQAWVWAHRHIGEERDVGCWWWLLASEVDAHLLPVPGLPQNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGP Forkhead box protein P1 (FoxP3), isoform 4, UniProKB accession number Q9BZS1-4 SEQ ID NO: MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDEKGRAQCLLQREMVQSLEQQASSDK GSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGP C/EBP-homologous protein (CHOP), isoform 1, UniProKB accession number P35638-1 SEQ ID NO: MAAESLPFSFGTLSSWELEAWYEDLQEVLSSDENGGTYVSPPGNEEEESKIFTTLDPASLAWLTEEEPEPAEVTSTSQSPHSPDSSQSSLAQEEEEEDQGRTRKRKQSGHSPARAGKQRMKEKEQENERKVAQLAEENERLKQEIERLLTREVEATRRALIDRMVNLHQA C/EBP-homologous protein (CHOP), isoform 2, UniProKB accession number P35638-2 SEQ ID NO: MELVPATPHYPADVLFQTDPTAEMAAESLPFSFGTLSSWELEAWYEDLQEVLSSDENGGTYVSPPGNEEEESKIFTTLDPASLAWLTEEEEPEPAEVTSTSQSPHSPDSSQSSLAQEEEEEDQGRTRKRKQSGHSPARAGKQRMKEKEQENERKVAQLAEENERLKQEIERLLTREVEATRRALIDRMVNLHQA Nuclear factor of activated T cells, cytoplasmic 2 (NFATC2), isoform 1, UniProKB accession number Q13469-1 SEQ ID NO: MNAPERQPQPDGGDAPGHEPGGSPQDELDFSILFDYEYLNPNEEEPNAHK VASPPSGPAYPDDVLDYGLKPYSPLASLSGEPPGRFGEPDRVGPQKFLSA AKPAGASGLSPRIEITPSHELIQAVGPLRMRDAGLLVEQPPLAGVAASPR FTLPVPGFEGYREPLCLSPASSGSSASFISDTFSPYTSPCVSPNNGGPDD LCPQFQNIPAHYSPRTSPIMSPRTSLAEDSCLGRHSPVPRPASRSSSPGA KRRHSCAEALVALPPGASPQRSRSPSPQPSSHVAPQDHGSPAGYPPVAGS AVIMDALNSLATDSPCGIPPKMWKTSPDPSPVSAAPSKAGLPRHIYPAVE FLGPCEQGERRNSAPESILLVPPTWPKPLVPAIPICSIPVTASLPPLEWP LSSQSGSYELRIEVQPKPHHRAHYETEGSRGAVKAPTGGHPVVQLHGYME NKPLGLQIFIGTADERILKPHAFYQVHRITGKTVTTTSYEKIVGNTKVLE IPLEPKNNMRATIDCAGILKLRNADIELRKGETDIGRKNTRVRLVFRVHI PESSGRIVSLQTASNPIECSQRSAHELPMVERQDTDSCLVYGGQQMILTG QNFTSESKVVFTEKTTDGQQIWEMEATVDKDKSQPNMLFVEIPEYRNKHI RTPVKVNFYVINGKRKRSQPQHFTYHPVPAIKTEPTDEYDPTLICSPTHG GLGSQPYYPQHPMVAESPSCLVATMAPCQQFRTGLSSPDARYQQQNPAAV LYQRSKSLSPSLLGYQQPALMAAPLSLADAHRSVLVHAGSQGQSSALLHP SPTNQQASPVIHYSPTNQQLRCGSHQEFQHIMYCENFAPGTTRPGPPPVS QGQRLSPGSYPTVIQQQNATSQRAAKNGPPVSDQKEVLPAGVTIKQEQNL DQTYLDDVNEIIRKEFSGPPARNQT Nuclear factor of activated T cells 5 (NFAT5), isoform 1, UniProKB accession number O94916-1 SEQ ID NO: MPSDFISLLSADLDLESPKSLYSRESVYDLLPKELQLPPSRETSVASMSQ TSGGEAGSPPPAVVAADASSAPSSSSMGGACSSFTTSSSPTIYSTSVTDS KAMQVESCSSAVGVSNRGVSEKQLTSNTVQQHPSTPKRHTVLYISPPPED LLDNSRMSCQDEGCGLESEQSCSMWMEDSPSNFSNMSTSSYNDNTEVPRK SRKRNPKQRPGVKRRDCEESNMDIFDADSAKAPHYVLSQLTTDNKGNSKA GNGTLENQKGTGVKKSPMLCGQYPVKSEGKELKIVVQPETQHRARYLTEG SRGSVKDRTQQGFPTVKLEGHNEPVVLQVFVGNDSGRVKPHGFYQACRVT GRNTTPCKEVDIEGTTVIEVGLDPSNNMTLAVDCVGILKLRNADVEARIG IAGSKKKSTRARLVFRVNIMRKDGSTLTLQTPSSPILCTQPAGVPEILKK SLHSCSVKGEEEVFLIGKNFLKGTKVIFQENVSDENSWKSEAEIDMELFH QNHLIVKVPPYHDQHITLPVSVGIYVVTNAGRSHDVQPFTYTPDPAAAGA LNVNVKKEISSPARPCSFEEAMKAMKTTGCNLDKVNIIPNALMTPLIPSS MIKSEDVTPMEVTAEKRSSTIFKTTKSVGSTQQTLENISNIAGNGSFSSP SSSHLPSENEKQQQIQPKAYNPETLTTIQTQDISQPGTFPAVSASSQLPN SDALLQQATQFQTRETQSREILQSDGTVVNLSQLTEASQQQQQSPLQEQA QTLQQQISSNIFPSPNSVSQLQNTIQQLQAGSFTGSTASGSSGSVDLVQQ VLEAQQQLSSVLFSAPDGNENVQEQLSADIFQQVSQIQSGVSPGMFSSTE PTVHTRPDNLLPGRAESVHPQSENTLSNQQQQQQQQQQVMESSAAMVMEM QQSICQAAAQIQSELFPSTASANGNLQQSPVYQQTSHMMSALSTNEDMQM QCELFSSPPAVSGNETSTTTTQQVATPGTTM FQTSSSGDGEETGTQAKQI QNSVFQTMVQMQHSGDNQPQVNLFSSTKSMMSVQNSGTQQQGNGLFQQGN EMMSLQSGNFLQQSSHSQAQLFHPQNPIADAQNLSQETQGSLFHSPNPIV HSQTSTTSSEQMQPPMFHSQSTIAVLQGSSVPQDQQSTNIFLSQSPMNNL QTNTVAQEAFFAAPNSISPLQSTSNSEQQAAFQQQAPISHIQTPMLSQEQ AQPPQQGLFQPQVALGSLPPNPMPQSQQGTMFQSQHSIVAMQSNSPSQEQ QQQQQQQQQQQQQQQQSILFSNQNTMATMASPKQPPPNMIFNPNQNPMAN QEQQNQSIFHQQSNMAPMNQEQQPMQFQSQSTVSSLQNPGPTQSESSQTP LFHSSPQIQLVQGSPSSQEQQVTLFLSPASMSALQTSINQQDMQQSPLYS PQNNMPGIQGATSSPQPQATLFHNTAGGTMNQLQNSPGSSQQTSGMFLFG IQNNCSQLLTSGPATLPDQLMAISQPGQPQNEGQPPVTTLLSQQMPENSP LASSINTNQNIEKIDLLVSLQNQGNNLTGSF Nuclear factor of activated T cells, cytoplasmic 1 (NFATC1), isoform 1, UniProKB accession number O95644-1 SEQ ID NO: MPSTSFPVPSKFPLGPAAAVFGRGETLGPAPRAGGTMKSAEEEHYGYASS NVSPALPLPTAHSTLPAPCHNLQTSTPGIIPPADHPSGYGAALDGGPAGY FLSSGHTRPDGAPALESPRIEITSCLGLYHNNNQFFHDVEVEDVLPSSKR SPSTATLSLPSLEAYRDPSCLSPASSLSSRSCNSEASSYESNYSYPYASP QTSPWQSPCVSPKTTDPEEGFPRGLGACTLLGSPRHSPSTSPRASVTEES WLGARSSRPASPCNKRKYSLNGRQPPYSPHHSPTPSPHGSPRVSVTDDSW LGNTTQYTSSAIVAAINALTTDSSLDLGDGVPVKSRKTTLEQPPSVALKV EPVGEDLGSPPPPADFAPEDYSSFQHIRKGGFCDQYLAVPQHPYQWAKPK PLSPTSYMSPTLPALDWQLPSHSGPYELRIEVQPKSHHRAHYETEGSRGA VKASAGGHPIVQLHGYLENEPLMLQLFIGTADDRLLRPHAFYQVHRITGK TVSTTSHEAILSNTKVLEIPLLPENSMRAVIDCAGILKLRNSDIELRKGE TDIGRKNTRVRLVFRVHVPQPSGRTLSLQVASNPIECSQRSAQELPLVEK QSTDSYPVVGGKKMVLSGHNFLQDSKVIFVEKAPDGHHVWEMEAKTDRDL CKPNSLVVEIPPFRNQRITSPVHVSFYVCNGKRKRSQYQRFTYLPANVPI IKTEPTDDYEPAPTCGPVSQGLSPLPRPYYSQQLAMPPDPSSCLVAGFPP CPQRSTLMPAAPGVSPKLHDLSPAAYTKGVASPGHCHLGLPQPAGEAPAV QDVPRPVATHPGSPGQPPPALLPQQVSAPPSSSCPPGLEHSLCPSSPSPP LPPATQEPTCLQPCSPACPPATGRPQHLPSTVRRDESPTAGPRLLPEVHE DGSPNLAPIPVTVKREPEELDQLYLDDVNEIIRNDLSSTSTHS Nuclear factor of activated T cells, cytoplasmic 4 (NFATC4), isoform 1, UniProKB accession number Q14934-1 SEQ ID NO: MGAASCEDEELEFKLVFGEEKEAPPLGAGGLGEELDSEDAPPCCRLALGE PPPYGAAPIGIPRPPPPRPGMHSPPPRPAPSPGTWESQPARSVRLGGPGG GAGGAGGGRVLECPSIRITSISPTPEPPAALEDNPDAWGDGSPRDYPPPE GFGGYREAGGQGGGAFFSPSPGSSSLSSWSFFSDASDEAALYAACDEVES ELNEAASRFGLGSPLPSPRASPRPWTPEDPWSLYGPSPGGRGPEDSWLLL SAPGPTPASPRPASPCGKRRYSSSGTPSSASPALSRRGSLGEEGSEPPPP PPLPLARDPGSPGPFDYVGAPPAESIPQKTRRTSSEQAVALPRSEEPASC NGKLPLGAEESVAPPGGSRKEVAGMDYLAVPSPLAWSKARIGGHSPIFRT SALPPLDWPLPSQYEQLELRIEVQPRAHHRAHYETEGSRGAVKAAPGGHP VVKLLGYSEKPLTLQMFIGTADERNLRPHAFYQVHRITGKMVATASYEAV VSGTKVLEMTLLPENNMAANIDCAGILKLRNSDIELRKGETDIGRKNTRV RLVFRVHVPQGGGKVVSVQAASVPIECSQRSAQELPQVEAYSPSACSVRG GEELVLTGSNFLPDSKVVFIERGPDGKLQWEEEATVNRLQSNEVTLTLTV PEYSNKRVSRPVQVYFYVSNGRRKRSPTQSFRFLPVICKEEPLPDSSLRG FPSASATPFGTDMDFSPPRPPYPSYPHEDPACETPYLSEGFGYGMPPLYP QTGPPPSYRPGLRMFPETRGTTGCAQPPAVSFLPRPFPSDPYGGRGSSFS LGLPFSPPAPFRPPPLPASPPLEGPFPSQSDVHPLPAEGYNKVGPGYGPG EGAPEQEKSRGGYSSGFRDSVPIQGITLEEVSEIIGRDLSGFPAPPGEEP PA Nuclear factor of activated T cells, cytoplasmic 3 (NFATC3), isoform 1, UniProKB accession number Q12968-1 SEQ ID NO: MTTANCGAHDELDFKLVFGEDGAPAPPPPGSRPADLEPDDCASIYIFNVD PPPSTLTTPLCLPHHGLPSHSSVLSPSFQLQSHKNYEGTCEIPESKYSPL GGPKPFECPSIQITSISPNCHQELDAHEDDLQINDPEREFLERPSRDHLY LPLEPSYRESSLSPSPASSISSRSWFSDASSCESLSHIYDDVDSELNEAA ARFTLGSPLTSPGGSPGGCPGEETWHQQYGLGHSLSPRQSPCHSPRSSVT DENWLSPRPASGPSSRPTSPCGKRRHSSAEVCYAGSLSPHHSPVPSPGHS PRGSVTEDTWLNASVHGGSGLGPAVFPFQYCVETDIPLKTRKTSEDQAAI LPGKLELCSDDQGSLSPARETSIDDGLGSQYPLKKDSCGDQFLSVPSPFT WSKPKPGHTPIFRTSSLPPLDWPLPAHFGQCELKIEVQPKTHHRAHYETE GSRGAVKASTGGHPVVKLLGYNEKPINLQMFIGTADDRYLRPHAFYQVHR ITGKTVATASQEIIIASTKVLEIPLLPENNMSASIDCAGILKLRNSDIEL RKGETDIGRKNTRVRLVFRVHIPQPSGKVLSLQIASIPVECSQRSAQELP HIEKYSINSCSVNGGHEMVVTGSNFLPESKIIFLEKGQDGRPQWEVEGKI IREKCQGAHIVLEVPPYHNPAVTAAVQVHFYLCNGKRKKSQSQRFTYTPV LMKQEHREEIDLSSVPSLPVPHPAQTQRPSSDSGCSHDSVLSGQRSLICS IPQTYASMVTSSHLPQLQCRDESVSKEQHMIPSPIVHQPFQVTPTPPVGS SYQPMQTNVVYNGPTCLPINAASSQEFDSVLFQQDATLSGLVNLGCQPLS SIPFHSSNSGSTGHLLAHTPHSVHTLPHLQSMGYHCSNTGQRSLSSPVAD QITGQPSSQLQPITYGPSHSGSATTASPAASHPLASSPLSGPPSPQLQPM PYQSPSSGTASSPSPATRMHSGQHSTQAQST GQGGLSAPSSLICHSLCDP ASFPPDGATVSIKPEPEDREPNFATIGLQDITLDDVNEIIGRDMSQISVS QGAGVSRQAPLPSPESLDLRSDGL Von Hippel-Lindau tumor suppressor (VHL), isomer 1, UniProKB accession number P40337-1 SEQ ID NO: MPRRAENWDEAEVGAEEAGVEEYGPEEDGGEESGAEESGPEESGPEELGA EEEMEAGRPRPVLRSVNSREPSQVIFCNRSPRVVLPVWLNFDGEPQPYPT LPPGTGRRIHSYRGHLWLFRDAGTHDGLLVNQTELFVPSLNVDGQPIFAN ITLPVYTLKERCLQVVRSLVKPENYRRLDIVRSLYEDLEDHPN Cytokine-induced SH2-containing protein (CISH), isoform 1, UniProKB accession number Q9NSE2-1 SEQ ID NO: MVLCVQGPRPLLAVERTGQRPLWAPSLELPKPVMQPLPAGAFLEEVAEGT PAQTESEPKVLDPEEDLLCIAKTFSYLRESGWYWGSITASEARQHLQKMP EGTFLVRDSTHPSYLFTLSVKTTRGPTNVRIEYADSSFRLDSNCLSRPRI LAFPDVVSLVQHYVASCTADTRSDSPDPAPTPALPMPKEDAPSDPALPAP PPATAVHLKLVQPFVRRSSARSLQHLCRLVINRLVADVDCLPLPRRMADY LRQYPFQL Suppressor of cytokine signaling 1 (SOCS1), isoform 1, UniProKB accession number O15524-1 SEQ ID NO: MVAHNQVAADNAVSTAAEPRRRPEPSSSSSSSPAAPARPRPCPAVPPAPAPGDTHFRTFRSHADYRRITRASALLDACGFYWGPLSVHGAHERLRAEPVGTFFLVRDSRQRNCFFALSVKMASGPTSIRVHFQAGRFHLDGSRESFDCLFELLEHYVAAPRRMLGAPLRQRRVRPLQELCRQRIVATVGRENLARIPLNPVLRDYLSS Suppressor of cytokine signaling 2 (SOCS2), isoform 1, UniProKB accession number O14508-1 SEQ ID NO: MTLRCLEPSGNGGEGTRSQWGTAGSAEEPSPQAARLAKALRELGQTGWYWGSMTVNEAKEKLKEAPEGTFLIRDSSSHSDYLLTISVKTSAGPTNLRIEYQDGKFRLDSIICVKSKLKQFDSVVHLIDYYVQMCKDKRTGPEAPRNGTVHLYLTKPLYTSAPSLQHLCRLTINKCTGAIWGLLEPLPTRLKFDY Suppressor of cytokine signaling 3 (SOCS3), isoform 1, UniProKB accession number O14543-1 SEQ ID NO: MVTHSKFPAAAGMSRPLDTSLRLKTFSSKSEYQLVVNAVRKLQESGFYWSAVTGGEANLLLSAEPAGTFLIRDSSDQRHFFTLSVKTQSGTKNLRIQCEGGSFSLQSDPRSTQPVPRFDCVLKLVHHYMPPPGAPSFPSPPTEPSSEVPEQPSAQPLPGSPPRRAYYIYSGGEKIPLVLSRPLSSNVATLQHLCRYPLKETVKNGHLT Suppressor of cytokine signaling 4 (SOCS4), isoform 1, UniProKB accession number Q8WXH5-1 SEQ ID NO: MAENNENISKNVDVRPKTSRSRSADRKDGYVWSGKKLSWSKKSESYSDAETVNGIEKTEVSLRNQERKHSCSSIELDLDHSCGHRFLGRSLKQKLQDAVGQCFPIKNCSSRHSSGLPSKRKIHISELMLDKCPFPPRSDLAFRWHFIKRHTAPINSKSDEWVSTDLSQTELRDGQLKRRNMEENINCFSHTNVQPCVITTDNALCREGPMTGSVMNLVSNNSIEDSDMDSDDEILTLCTSSRKRNKPKWDLDDEILQLETPPKYHTQIDYVHCLVPDLLQINNNPCYWGVMDKYAAEALLEGKPEGTFLLRDSAQEDYLFSVSFRRYSRSLHARIEQWNHNFSFDAHDPCVFHSPDITGLLEHYKDPSACMFFEPLLSTPLIRTFPFSLQHICRTVICNCTTYDGIDALPIPSSMKLYLKEYHYKSKVRVLRIDAPEQQC Suppressor of cytokine signaling 5 (SOCS5), isoform 1, UniProKB accession number O75159-1 SEQ ID NO: MDKVGKMWNNFKYRCQNLFGHEGGSRSENVDMNSNRCLSVKEKNISIGDSTPQQQSSPLRENIALQLGLSPSKNSSRRNQNCATEIPQIVEISIEKDNDSCVTPGTRLARRDSYSRHAPWGGKKKHSCSTKTQSSLDADKKFGRTRSGLQRRERRYGVSSVHDMDSVSSRTVGSRSLRQRLQDTVGLCFPMRTYSKQSKPLFSNKRKIHLSELMLEKCPFPAGSDLAQKWHLIKQHTAPVSPHSTFFDTFDPSLVSTEDEEDRLRERRRLSIEEGVDPPPNAQIHTFEATAQVNPLYKLGPKLAPGMTEISGDSSAIPQANCDSEEDTTTLCLQSRRQKQRQISGDSHTHVSRQGAWKVHTQIDYIHCLVPDLLQITGNPCYWGVMDRYEAEALLEGKPEGTFLLRDSAQEDYLFSVSFRRYNRSLHARIEQWNHNFSFDAHDPCVFHSSTVTGLLEHYKDPSSCMFFEPLLTISLNRTFPFSLQYICRAVICRCTTYDGIDGLPLPSMLQDFLKEYHYKQKVRVRWLEREPVKAK Suppressor of cytokine signaling 6 (SOCS6), isoform 1, UniProKB accession number O14544-1 SEQ ID NO: MKKISLKTLRKSFNLNKSKEETDFMVVQQPSLASDFGKDDSLFGSCYGKDMASCDINGEDEKGGKNRSKSESLMGTLKRRLSAKQKSKGKAGTPSGSSADEDTFSSSSAPIVFKDVRAQRPIRSTSLRSHHYSPAPWPLRPTNSEETCIKMEVRVKALVHSSSPSPALNGVRKDFHDLQSETTCQEQANSLKSSASHNGDLHLHLDEHVPVVIGLMPQDYIQYTVPLDEGMYPLEGSRSYCLDSSSPMEVSAVPPQVGGRAFPEDESQVDQDLVVAPEIFVDQSVNGLLIGTTGVMLQSPRAGHDDVPPLSPLLPPMQNNQIQRNFSGLTGTEAHVAESMRCHLNFDPNSAPGVARVYDSVQSSGPMVVTSLTEELKKLAKQGWYWGPITRWEAEGKLANVPDGSFLVRDSSDDRYLLSLSFRSHGKTLHTRIEHSNGRFSFYEQPDVEGHTSIVDLIEHSIRDSENGAFCYSRSRLPGSATYPVRLTNPVSRFMQVRSLQYLCRFVIRQYTRIDLIQKLPLPNKMKDYLQEKHY Suppressor of cytokine signaling 7 (SOCS7), isoform 1, UniProKB accession number O14512-1 SEQ ID NO: MVFRNVGRPPEEEDVEAAPEPGPSELLCPRHRCALDPKALPPGLALERTWGPAAGLEAQLAALGLGQPAGPGVKTVGGGCCPCPCPPQPPPPQPQPPAAAPQAGEDPTETSDALLVLEGLESEAESLETNSCSEEELSSPGRGGGGGGRLLLQPPGPELPPVPFPLQDLVPLGRLSRGEQQQQQQQQPPPPPPPPGPLRPLAGPSRKGSFKIRLSRLFRTKSCNGGSGGGDGTGKRPSGELAASAASLTDMGGSAGRELDAGRKPKLTRTQSAFSPVSFSPLFTGETVSLVDVDISQRGLTSPHPPTPPPPPRRSLSLLDDISGTLPTSVLVAPMGSSLQSFPLPPPPPPHAPDAFPRIAPIRAAESLHSQPPQHLQCPLYRPDSSSFAASLRELEKCGWYWGPMNWEDAEMKLKGKPDGSFLVRDSSDPRYILSLSFRSQGITHHTRMEHYRGTFSLWCHPKFEDRCQSVVEFIKRAIMHSKNGKFLYFLRSRVPGLPPTPVQLLYPVSRFSNVKSLQHLCRFRIRQLVRIDHIPDLPLPKPLISYIRKFYYYDPQEEVYLSLKEAQLISKQKQEVEPST Protein tyrosine phosphatase non-receptor type 2 (PTPN2), isoform 1, UniProKB accession number P17706-1 SEQ ID NO: MPTTIEREFEELDTQRRWQPLYLEIRNESHDYPHRVAKFPENRNRNRYRDVSPYDHSRVKLQNAENDYINASLVDIEEAQRSYILTQGPLPNTCCHFWLMVWQQKTKAVVMLNRIVEKESVKCAQYWPTDDQEMLFKETGFSVKLLSEDVKSYYTVHLLQLENINSGETRTISHFHYTTWPDFGVPESPASFLNFLFKVRESGSLNPDHGPAVIHCSAGIGRSGTFSLVDTCLVLMEKGDDINIKQVLLNMRKYRMGLIQTPDQLRFSYMAIIEGAKCIKGDSSIQKRWKELSKEDLSPAFDHSPNKIMTEKYNGNRIGLEEEKLTGDRCTGLSSKMQDTMEENSESALRKRIREDRKATTAQKVQQMKQRLNENERKRKRWLYWQPILTKMGFMSVILVGAFVGWTLFFQQNAL Protein Tyrosine Phosphatase SHP-1 (SHP1), Isomer 1, UniProKB Accession No. P29350-1 SEQ ID NO: MVRWFHRDLSGLDAETLLKGRGVHGSFLARPSRKNQGDFSLSVRVGDQVTHIRIQNSGDFYDLYGGEKFATLTELVEYYTQQQGVLQDRDGTIIHLKYPLNCSDPTSERWYHGHMSGGQAETLLQAKGEPWTFLVRESLSQPGDFVLSVLSDQPKAGPGSPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHFKKTGIEEASGAFVYLRQPYYATRVNAADIENRVLELNKKQESEDTAKAGFWEEFESLQKQEVKNLHQRLEGQRPENKGKNRYKNILPFDHSRVILQGRDSNIPGSDYINANYIKNQLLGPDENAKTYIASQGCLEATVNDFWQMAWQENSRVIVMTTREVEKGRNKCVPYWPEVGMQRAYGPYSVTNCGEHDTTEYKLRTLQVSPLDNGDLIREIWHYQYLSWPDHGVPSEPGGVLSFLDQINQRQESLPHAGPIIVHCSAGIGRTGTIIVIDMLMENISTKGLDCDIDIQKTIQMVRAQRSGMVQTEAQYKFIYVAIAQFIETTKKKLEVLQSQKGQESEYGNITYPPAMKNAHAKASRTSSKHKEDVYENLHTKNKREEKVKKQRSADKEKSKGSLKRK Protein Tyrosine Phosphatase (SHP2), Isoform 1, UniProKB Accession No. Q06124-2 SEQ ID NO: MTSRRWFHPNITGVEAENLLLTRGVDGSFLARPSKSNPGDFTLSVRRNGA VTHIKIQNTGDYYDLYGGEKFATLAELVQYYMEHHGQLKEKNGDVIELKY PLNCADPTSERWFHGHLSGKEAEKLLTEKGKHGSFLVRESQSHPGDFVLS VRTGDDKGESNDGKSKVTHVMIRCQELKYDVGGGERFDSLTDLVEHYKKN PMVETLGTVLQLKQPLNTTRINAAEIESRVRELSKLAETTDKVKQGFWEE FETLQQQECKLLYSRKEGQRQENKNKNRYKNILPFDHTRVVLHDGDPNEP VSDYINANIIMPEFETKCNNSKPKKSYIATQGCLQNTVNDFWRMVFQENS RVIVMTTKEVERGKSKCVKYWPDEYALKEYGVMRVRNVKESAAHDYTLRE LKLSKVGQGNTERTVWQYHFRTWPDHGVPSDPGGVLDFLEEVHHKQESIM DAGPVVVHCSAGIGRTGTFIVIDILIDIIREKGVDCDIDVPKTIQMVRSQ RSGMVQTEAQYRFIYMAVQHYIETLQRRIEEEQKSKRKGHEYTNIKYSLA DQTSGDQSPLPPCTPTPPCAEMREDSARVYENVGLMQQQKSFR GRB2-related adapter protein 2 (GRAP2; also known as GADS), isoform 1, UniProKB accession number O75791-1 SEQ ID NO: MEAVAKFDFTASGEDELSFHTGDVLKILSNQEEWFKAELGSQEGYVPKNF IDIQFPKWFHEGLSRHQAENLLMGKEVGFFIIRASQSSPGDFSISVRHED DVQHFKVMRDNKGNYFLWTEKFPSLNKLVDYYRTNSISRQKQIFLRDRTR EDQGHRGNSLDRRSQGGPHLSGAVGEEIRPSMNRKLSDHPPTLPLQQHQH QPQPPQYAPAPQQLQQPPQQRYLQHHHFHQERRGGSLDINDGHCGTGLGS EMNAALMHRRHTDPVQLQAAGRVRWARALYDFEALEDDELGFHSGEVVEV LDSSNPSWWTGRLHNKLGLFPANYVAPMTR Growth factor receptor binding protein 2 (Grb2), isoform 1, UniProKB accession number P62993-1 SEQ ID NO: MEAIAKYDFKATADDELSFKRGDILKVLNEECDQNWYKAELNGKDGFIPK NYIEMKPHPWFFGKIPRAKAEEMLSKQRHDGAFLIRESESAPGDFSLSVK FGNDVQHFKVLRDGAGKYFLWVVKFNSLNELVDYHRSTSVSRNQQIFLRD IEQVPQQPTYVQALFDFDPQEDGELGFRRGDFIHVMDNSDPNWWKGACHG QTGMFPRNYVTPVNRNV 1-phosphatidylinositol 4,5-diphosphate phosphodiesterase gamma-1 (PLC gamma 1), isoform 1, UniProKB accession number P19174-1 SEQ ID NO: MAGAASPCANGCGPGAPSDAEVLHLCRSLEVGTVMTLFYSKKSQRPERKT FQVKLETRQITWSRGADKIEGAIDIREIKEIRPGKTSRDFDRYQEDPAFR PDQSHCFVILYGMEFRLKTLSLQATSEDEVNMWIKGLTWLMEDTLQAPTP LQIERWLRKQFYSVDRNREDRISAKDLKNMLSQVNYRVPNMRFLRERLTD LEQRSGDITYGQFAQLYRSLMYSAQKTMDLPFLEASTLRAGERPELCRVS LPEFQQFLLDYQGELWAVDRLQVQEFMLSFLRDPLREIEEPYFFLDEFVT FLFSKENSVWNSQLDAVCPDTMNNPLSHYWISSSHNTYLTGDQFSSESSL EAYARCLRMGCRCIELDCWDGPDGMPVIYHGHTLTTKIKFSDVLHTIKEH AFVASEYPVILSIEDHCSIAQQRNMAQYFKKVLGDTLLTKPVEISADGLP SPNQLKRKILIKHKKLAEGSAYEEVPTSMMYSENDISNSIKNGILYLEDP VNHEWYPHYFVLTSSKIYYSEETSSDQGNEDEEEPKEVSSSTELHSNEKW FHGKLGAGRDGRHIAERLLTEYCIETGAPDGSFLVRESETFVGDYTLSFW MRLSEPVPQTNAHESKEWYHASLTRAQAEHMLMRVPRDGAFLVRKRNEPN SYAISFRAEGKIKHCRVQQEGQTVMLGNSEFDSLVDLISYYEKHPLYRKM KLRYPINEEALEKIGTAEPDYGALYEGRNPGFYVEANPMPTFKCAVKALF DYKAQREDELTFIKSAIIQNVEKQEGGWWRGDYGGKKQLWFPSNYVEEMV NPVALEPEREHLDENSPLGDLLRGVLDVPACQIAIRPEGKNNRLFVFSIS MASVAHWSLDVAADSQEELQDWVKKIREVAQTADARLTEGKIMERRKKIA LELSELVVYCRPVPFDEEKIGTERACYRDMSSFPETKAEKYVNKAKGKKF LQYNRLQLSRIYPKGQRLDSSNYDPLPMWIC GSQLVALNFQTPDKPMQMN QALFMTGRHCGYVLQPSTMRDEAFDPFDKSSLRGLEPCAISIEVLGARHL PKNGRGIVCPFVEIEVAGAEYDSTKQKTEFVVDNGLNPVWPAKPFHFQIS NPEFAFLRFVVYEEDMFSDQNFLAQATFPVKGLKTGYRAVPLKNNYSEDL ELASLLIKIDIFPAKENGDLSPFSGTSLRERGSDASGQLFHGRAREGSFE SRYQQPFEDFRISQEHLADHFDSRERRAPRRTRVNGDNRL Linker for priming T cell family member 1 (LAT), isoform 1, UniProKB accession number O43561-1 SEQ ID NO: MEEAILVPCVLGLLLLPILAMLMALCVHCHRLPGSYDSTSSDSLYPRGIQ FKRPHTVAPWPPAYPPVTSYPPLSQPDLLPIPRSPQPLGGSHRTPSSRRD SDGANSVASYENEGASGIRGAQAGWGVWGPSWTRLTPVSLPPEPACEDAD EDEDDYHNPGYLVVLPDSTPATSTAAPSAPALSTPGIRDSAFSMESIDDY VNVPESGESAEASLDGSREYVNVSQELHPGAAKTEPAALSSQEAEEVEEE GAPDYENLQELN SH2 domain-containing 76 kDa leukocyte protein (SLP76), isoform 1, UniProKB accession number Q13094-1 SEQ ID NO: MALRNVPFRSEVLGWDPDSLADYFKKLNYKDCEKAVKKYHIDGARFLNLT ENDIQKFPKLRVPILSKLSQEINKNEERRSIFTRKPQVPRFPEETESHEE DNGGWSSFEEDDYESPNDDQDGEDDGDYESPNEEEEAPVEDDADYEPPPS NDEEALQNSILPAKPFPNSNSMYIDRPPSGKTPQQPPVPPQRPMAALPPP PAGRNHSPLPPPQTNHEEPSRSRNHKTAKLPAPSIDRSTKPPLDRSLAPF DREPFTLGKKPPFSDKPSIPAGRSLGEHLPKIQKPPLPPTTERHERSSPL PGKKPPVPKHGWGPDRRENDEDDVHQRPLPQPALLPMSSNTFPSRSTKPS PMNPLPSSHMPGAFSESNSSFPQSASLPPYFSQGPSNRPPIRAEGRNFPL PLPNKPRPPSPAEEENSLNEEWYVSYITRPEAEAALRKINQDGTFLVRDS SKKTTTNPYVLMVLYKDKVYNIQIRYQKESQVYLLGTGLRGKEDFLSVSD IIDYFRKMPLLLIDGKNRGSRYQCTLTHAAGYP Tyrosine protein kinase Lck (Lck), isoform 1, UniProKB accession number P06239-1 SEQ ID NO: MGCGCSSHPEDDWMENIDVCENCHYPIVPLDGKGTLLIRNGSEVRDPLVT YEGSNPPASPLQDNLVIALHSYEPSHDGDLGFEKGEQLRILEQSGEWWKA QSLTTGQEGFIPFNFVAKANSLEPEPWFFKNLSRKDAERQLLAPGNTHGS FLIRESESTAGSFSLSVRDFDQNQGEVVKHYKIRNLDNGGFYISPRITFP GLHELVRHYTNASDGLCTRLSRPCQTQKPQKPWWEDEWEVPRETLKLVER LGAGQFGEVWMGYYNGHTKVAVKSLKQGSMSPDAFLAEANLMKQLQHQRL VRLYAVVTQEPIYIITEYMENGSLVDFLKTPSGIKLTINKLLDMAAQIAE GMAFIEERNYIHRDLRAANILVSDTLSCKIADFGLARLIEDNEYTAREGA KFPIKWTAPEAINYGTFTIKSDVWSFGILLTEIVTHGRIPYPGMTNPEVI QNLERGYRMVRPDNCPEELYQLMRLCWKERPEDRPTFDYLRSVLEDFFTA TEGQYQPQP Interleukin 2 inducible T cell kinase (Itk), isoform 1, UniProKB accession number Q08881-1 SEQ ID NO: MNNFILLEEQLIKKSQQKRRTSPSNFKVRFFVLTKASLAYFEDRHGKKRT LKGSIELSRIKCVEIVKSDISIPCHYKYPFQVVHDNYLLYVFAPDRESRQ RWVLALKEETRNNNSLVPKYHPNFWMDGKWRCCSQLEKLATGCAQYDPTK NASKKPLPPTPEDNRRPLWEPEETVVIALYDYQTNDPQELALRRNEEYCL LDSSEIHWWRVQDRNGHEGYVPSSYLVEKSPNNLETYEWYNKSISRDKAE KLLLDTGKEGAFMVRDSRTAGTYTVSVFTKAVVSENNPCIKHYHIKETND NPKRYYVAEKYVFDSIPLLINYHQHNGGGLVTRLRYPVCFGRQKAPVTAG LRYGKWVIDPSELTFVQEIGSGQFGLVHLGYWLNKDKVAIKTIREGAMSE EDFIEEAEVMMKLSHPKLVQLYGVCLEQAPICLVFEFMEHGCLSDYLRTQ RGLFAAETLLGMCLDVCEGMAYLEEACVIHRDLAARNCLVGENQVIKVSD FGMTRFVLDDQYTSSTGTKFPVKWASPEVFSFSRYSSKSDVWSFGVLMWE VFSEGKIPYENRSNSEVVEDISTGFRLYKPRLASTHVYQIMNHCWKERPE DRPAFSRLLRQLAEIAESGL B cell progenitor kinase (Btk), isoform 1, UniProKB accession number Q06187-1 SEQ ID NO: MAAVILESIFLKRSQQKKKTSPLNFKKRLFLLTVHKLSYYEYDFERGRRG SKKGSIDVEKITCVETVVPEKNPPPERQIPRRGEESSEMEQISIIERFPY PFQVVYDEGPLYVFSPTEELRKRWIHQLKNVIRYNSDLVQKYHPCFWIDG QYLCCSQTAKNAMGCQILENRNGSLKPGSSHRKTKKPLPPTPEEDQILKK PLPPEPAAAPVSTSELKKVVALYDYMPMNANDLQLRKGDEYFILEESNLP WWRARDKNGQEGYIPSNYVTEAEDSIEMYEWYSKHMTRSQAEQLLKQEGK EGGFIVRDSSKAGKYTVSVFAKSTGDPQGVIRHYVVCSTPQSQYYLAEKH LFSTIPELINYHQHNSAGLISRLKYPVSQQNKNAPSTAGLGYGSWEIDPK DLTFLKELGTGQFGVVKYGKWRGQYDVAIKMIKEGSMSEDEFIEEAKVMM NLSHEKLVQLYGVCTKQRPIFIITEYMANGCLLNYLREMRHRFQTQQLLE MCKDVCEAMEYLESKQFLHRDLAARNCLVNDQGVVKVSDFGLSRYVLDDE YTSSVGSKFPVRWSPPEVLMYSKFSSKSDIWAFGVLMWEIYSLGKMPYER FTNSETAEHIAQGLRLYRPHLASEKVYTIMYSCWHEKADERPTFKILLSN ILDVMDEES BET family Ten-eleven translocation 1 (TET1), isoform 1, UniProKB accession number Q8NFU7-1 SEQ ID NO: MSRSRHARPSRLVRKEDVNKKKKNSQLRKTTKGANKNVASVKTLSPGKLKQLIQERDVKKKTEPKPPVPVRSLLTRAGAARMNLDRTEVLFQNPESLTCNGFTMALRSTSLSRRLSQPPLVVAKSKKVPLSKGLEKQHDCDYKILPALGVKHSENDSVPMQDTQVLPDIETLIGVQNPSLLKGKSQETTQFWSQRVEDSKINIPTHSGPAAEILPGPLEGTRCGEGLFSEETLNDTSGSPKMFAQDTVCAPFPQRATPKVTSQGNPSIQLEELGSRVESLKLSDSYLDPIKSEHDCYPTSSLNKVIPDLNLRNCLALGGSTSPTSVIKFLLAGSKQATLGAKPDHQEAFEATANQQEVSDTTSFLGQAFGAIPHQWELPGADPVHGEALGETPDLPEIPGAIPVQGEVFGTILDQQETLGMSGSVVPDLPVFLPVPPNPIATFNAPSKWPEPQSTVSYGLAVQGAIQILPLGSGHTPQSSSNSEKNSLPPVMAISNVENEKQVHISFLPANTQGFPLAPERGLFHASLGIAQLSQAGPSK SDRGSSQVSVTSTVHVVNTTVVTMPVPMVSTSSSSYTTLLPTLEKKKRKRCGVCEPCQQKTNCGECTYCKNRKNSHQICKKRKCEELKKKPSVVVPLEVIKENKRPQREKKPKVLKADFDNKPVNGPKSESMDYSRCGHGEEQKLELNPHTVENVTKNEDSMTGIEVEKWTQNKKSQLTDHVKGDFSANVPEAEKSKNSEVDKKRTKSPKLFVQTVRNGIKHVHCLPAETNVSFKKFNIEEFGKTLENNSYKFLKDTANHKNAMSSVATDMSCDHLKGRSNVLVFQQPGFNCSSIPHSSHSIINHHASIHNEGDQPKTPENIPSKEPKDGSPVQPSLLSLMKDRRLTLEQVVAIEALTQLSEAPSENSSPSKSEKDEESEQRTASLLNSCKAILYTVRKDLQDPNLQGEPPKLNHCPSLEKQSSCNTVVFNGQTTTLSNSHINSATNQASTKSHEYSKV TNSLSLFIPKSNSSKIDTNKSIAQGIITLDNCSNDLHQLPPRNNEVEYCNQLLDSSKKLDSDDLSCQDATHTQIEEDVATQ LTQLASIIKINYIKPEDKKVESTPTSLVTCNVQQKYNQEKGTIQQKPPSSVHNNHGSSLTKQKNPTQKKTKSTPSRDRRKKKPTVVSYQENDRQKWEKLSYMYGTICDIWIASKFQNFGQFCPHDFPTVFGKISSSTKIWKPLAQTRSIMQPKTVFPPLTQIKLQRYPESAEEKVKVEPLDSLSLFHLKTESNGKAFTDKAYNSQVQLTVNANQKAHPLTQPSSPPNQCANVMAGDDQIRFQQVVKEQLMHQRLPTLPGISHETPLPESALTLRNVNVVCSGGITVVSTKSEEEVCSSSFGTSEFSTVDSAQKNFNDYAMNFFTNPTKNLVSITKDSELPTCSCLDRVIQKDKGPYYTHLGAGPSVAAVREIMENRYGQKGNAIRIEIVVYTGKEGKSSHGCPIAKWVLRRSSDEEKVLCLVRQRTGHHCPTAVMVVLIMVWDGIPLPMADRLYTELTENLKSYNGHPTDRRCTLNENRTCTCQGIDPETCGASFSFGCSWSMYFNGCKFGRSPSPRRFRIDPSSPLHEKNLEDNLQSLA TRLAPIYKQYAPVAYQNQVEYENVARECRLGSKEGRPFSGVTACLDFCAHPHRDIHNMNNGSTVVCTLTREDNRSLGVIPQDEQLHVLPLYKLSDTDEFGSKEGMEAKIKSGAIEVLAPRRKKRTCFTQPVPRSGKKRAAMMTEVLAHKIRAVEKKPIPRIKRKNNSTTTNNSKPSSLPTLGSNTETVQPEVKSETEPHFILKSSDNTKTYSLMPSAPHPVKEASPGFSWSPKTASATPAPLKNDATASCGFSERSSTPHCTMPSGRLSGANAAAADGPGISQLGEVAPLPTLSAPVMEPLINSEPSTGVTEPLTPHQPNHQPSFLTSPQDLASSPMEEDEQHSEADEPPSDEPLSDDPLSPAEEKLPHIDEYWSDS EHIFLDANIGGVAIAPAHGSVLIECARRELHATTPVEHPNRNHPTRLSLVFYQHKNLNKPQHGFELNKIKFEAKEAKNKKMKASEQKDQAANEGPEQSSEVNELNQIPSHKALTLTHDNVVTVSPYALTHVAGPYNHWV Ten-eleven translocation 2 (TET2), isoform 1, UniProKB accession number Q6N021-1 SEQ ID NO: MEQDRTNHVEGNRLSPFLIPSPPICQTEPLATKLQNGSPLPERAHPEVNGDTKWHSFKSYYGIPCMKGSQNSRVSPDFTQESRGYSKCLQNGGIKRTVSEPSLSGLLQIKKLKQDQKANGERRNFGVSQERNPGESSQPNVSDLSDKKESVSSVAQENAVKDFTSFSTHNCSGPENPELQILNEQEGKSANYHDKNIVLLKNKAVLMPNGATVSASSVEHTHGELLEKTLSQYYPDCVSIAVQKTTSHINAINSQATNELSCEITHPSHTSGQINSAQTSNSELPPKPAAVVSEACDADDADNASKLAAMLNTCSFQKPEQLQQQKSVFEICPSPAENNIQGTTKLASGEEFCSGSSSNLQAPGGSSERYLKQNEMNGAYFKQSSVFTKDSFSATTTPPPPSQLLLSPPPPLPQVPQLPSEGKSTLNGGVLEEHHHYPNQSNTTLLREVKIEGKPEAPPSQSPNPSTHVCSPSPMLSERPQNNCVNRNDIQTAGTMTVPLCSEKTRPMSEHLKHNPPIFGSSGELQDNCQQLMRNKEQELKGRDKEQTRDLVPPTQHYLKPGWIELKAPRFHQAESHLKRNEASLPSILQYQPNLSNQMTSKQYTGNSNMPGGLPRQAYTQKTTQLEHKSQMYQVEMNQGQSQGTVDQHLQFQKPSHQVHFSKTDHLPKAHVQSLCGTRFHFQQRADSQTEKLMSPVLKQHLNQQASETEPFSNSHLLQHKPHKQAAQTQPSQSSHLPQNQQQQQKLQIKNKEEILQTFPHPQSNNDQQREGSFFGQTKVEECFHGENQYSKSSEFETHNVQMGLEEVQNINRRNSPYSQTMKSSACKIQVSCSNNTHLVSENKEQTTHPELFAGNKTQNLHHMQYFPNNVIPKQDLLHRCFQEQEQKSQQASVLQGYKNRNQDMSGQQAAQLAQQRYLIHNHANVFPVPDQGGSHTQTPPQKDTQKHAALRWHLLQKQEQQQTQQPQTESCHSQMHRPIKVEPGCKPHACMHTAPPENK TWKKVTKQENPPASCDNVQQKSIIETMEQHLKQFHAKSLFDHKALTLKSQKQVKVEMSGPVTVLTRQTTAAELDSHTPALEQQTTSSEKTPTKRTAASVLNNFIESPSKLLDTPIKNLLDTPVKTQYDFPSCRCVEQIIEKDEGPFYTHLGAGPNVAAIREIMEERFGQKGKAIRIERVIYTGKEGKSSQGCPIAKWVVRRSSSEEKLLCLVRERAGHTCEAAVIVILILVWEGIPLSLADKLYSELTETLRKYGTLTNRRCALNEERTCACQGLDPETCGASFSFGCSWSMYYNGCKFARSKIPRKFKLLGDDPKEEEKLESHLQNLSTLMAPTYKKLAPDAYNNQIEYEHRAPECRLGLKEGRPFSGVTACLDFCAHAHRDLHNMQNGSTLVCTLTREDNREFGGKPEDEQLHVLPLYKVSDVDEFGSVEAQEEKKRSGAIQVLSSFRRKVRMLAEPVKTCRQRKLEAKKAAAEKLSSLENSSNKNEKEKSAPSRTKQTENASQAKQLAELLRLSGPVMQQSQQPQPLQKQPPQPQQQQRPQQQQPHHPQTESVNSYSASGSTNPYMRRPNPVSPYPNSSHTSDIYGSTSPMNFYSTSSQAAGSYLNSSNPMNPYPGLLNQNTQYPSYQCNGNLSVDNCSPYLGSYSPQSQPMDLYRYPSQDPLSKLSLPPIHTLYQPRFGNSQSFTSKYLGYGNQNMQGDGFSSCTIRPNVHHVGKLPPYPTHEMDGHFMGATSRLPPNLSNPNMDYKNGEHHSPSHIIHNYSAAPGMFNSSLHALHLQNKENDMLSHTANGLSKMLPALNHDRTACVQGGLHKLSDANGQEKQPLALVQGVASGAEDNDEVWSDSEQSFLDPDIGGVAVAPTHGSILIECAKRELHATTPLKNPNRNHPTRISLVFYQHKSMNEPKHGLALWEAKMAEKAREKEEECEKYGPDYVPQKSHGKKVKREPAEPHETSEPTYLRFIKSLAERTMSVTTDSTVTTSPYAFTRVTGPYNRY I (DNA(cytosine-5)-methyltransferase 3A) (DNMT3a), isoform 1, UniProKB accession number Q9Y6K1-1 SEQ ID NO: MPAMPSSGPGDTSSSAAEREEDRKDGEEQEEPRGKEERQEPSTTARKVGRPGRKRKHPPVESGDTPKDPAVISKSPSMAQDSGASELLPNGDLEKRSEPQPEEGSPAGGQKGGAPAEGEGAAETLPEASRAVENGCCTPKEGRGAPAEAGKEQKETNIESMKMEGSRGRLRGGLGWESSL RQRPMPRLTFQAGDPYYISKRKRDEWLARWKREAEKKAKVIAGMNAVEENQGPGESQKVEEASPPAVQQPTDPASPTVATTPEPVGSDAGDKNATKAGDDEPEYEDGRGFGIGELVWGKLRGFSWWPGRIVSWWMTGRSRAAEGTRWVMWFGDGKFSVVCVEKLMPLSSFCSAFHQATYNKQPMYRKAIYEVLQVASSRAGKLFPVCHDSDESDTAKAVEVQNKPMIEWALGGFQPSGPKGLEPPEEEKNPYKEVYTDMWVEPEAAAYAPPPPAKKPRKSTAEKPKVKEIIDERTRERLVYEVRQKCRNIEDICISCGSLNVTLEHPLFVGGMCQNCKNCFLECAYQYDDDGYQSYCTICCGGREVLMCGNNNCCRCFCVECVDLLVGPGAAQAAIKEDPWNCYMCGHKGTYGLLRRREDWPSRLQMFFANNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACV (DNA(cytosine 5)-methyltransferase 3B) (DNMT3b), isoform 1, UniProKB accession number Q9UBC3-1 SEQ ID NO: MKGDTRHLNGEEDAGGREDSILVNGACSDQSSDSPPILEAIRTPEIRGRRSSSRLSKREVSSLLSYTQDLTGDGDGEDGDGSDTPVMPKLFRETRTRSESPAVRTRNNNSVSSRERHRPSPRSTRGRQGRNHVDESPVEFPATRSLRRRATASAGTPWPSPPSSYLTIDLTDDTEDTHGTPQSSSTPYARLAQDSQQGGMESPQVEADSGDGDSSEYQDGKEFGIGDLVWGKIKGFSWWPAMVVSWKATSKRQAMSGMRWVQWFGDGKFSEVSADKLVALGLFSQHFNLATFNKLVSYRKAMYHALEKARVRAGKTFPSSPGDSLEDQLKPMLEWAHGGFKPTGIEGLKPNNTQPVVNKSKVRRAGSRKLESRKYENKTRRRTADDSATSDYCPAPKRLKTNCYNNGKDRGDEDQSREQMASDVANNKSSLEDGCLSCGRKNPVSFHPLFEGGLCQTCRDRFLELFYMYDDDGYQSYCTVCCEGRELLLCSNTSCCRCFCVECLEVLVGTGTAAEAKLQEPWSCYMCLPQRCHGVLRRRK DWNVRLQAFFTSDTGLEYEAPKLYPAIPAARRRPIRVLSLFDGIATGYLVLKELGIKVGKYVASEVCEESIAVGTVKHEGNIKYVNDVRNITKKNIEEWGPFDLVIGGSPCNDLSNVNPARKGLYEGTGRLFFEFYHLLNYSRPKEGDDRPFFWMFENVVAMKVGDKRDISRFLECNPVMIDAIKVSAAHRARYFWGNLPGMNRPVIASKNDKLELQDCLEYNRIAKLKKVQTITTKSNSIKQGKNQLFPVVMNGKEDVLWCTELERIFGFPVHYTDVSNMGRGARQKLLGRSWSVPVIRHLFAPLKDYFACE Neurogenic locus Notch homolog 1 (NOTCH1), isoform 1, UniProKB accession number Q9UBC3-1 SEQ ID NO: MPPLLAPLLCLALLPALAARGPRCSQPGETCLNGGKCEAANGTEACVCGGAFVGPRCQDPNPCLSTPCKNAGTCHVVDRRGVADYACSCALGFSGPLCLTPLDNACLTNPCRNGGTCDLLTLTEYKCRCPPGWSGKSCQQADPCASNPCANGGQCLPFEASYICHCPPSFHGPTCRQDVNECGQKPGLCRHGGTCHNEVGSYRCVCRATHTGPNCERPYVPCSPSPCQNGGTCRPTGDVTHECACLPGFTGQNCEENIDDCPGNNCKNGGACVDGVNTYNCRCPPEWTGQYCTEDVDECQLMPNACQNGGTCHNTHGGYNCVCVNGWTGEDCSENIDDCASAACFHGATCHDRVASFYCECPHGRTGLLCHLNDACISNPCNEGSNCDTNPVNGKAICTCPSGYTGPACSQDVDECSLGANPCEHAGKCINTLGSFECQCLQGYTGPRCEIDVNECVSNPCQNDATCLDQIGEFQCICMPGYEGVHCEVNTDECASSPCLHNGRCLDKINEFQCECPTGFTGHLCQYDVDECASTPCKNG AKCLDGPNTYTCVCTEGYTGTHCEVDIDECDPDPCHYGSCKDGVATFTCLCRPGYTGHHCETNINECSSQPCRHGGTCQDRDNAYLCFCLKGTTGPNCEINLDDCASSPCDSGTCLDKIDGYECACEPGYTGSMCNINIDECAGNPCHNGGTCEDGINGFTCRCPEGYHDPTCLSEVNECNSNPCVHGACRDSLNGYKCDCDPGWSGTNCDINNNECESNPCVNGGTCKDMTSGYVCTCREGFSGPNCQTNINECASNPCLNQGTCIDDVAGYKCNCLLPYTGATCEVVLAPCAPSPCRNGGECRQSEDYESFSCVCPTGWQGQTCEVDINECVLSPCRHGASCQNTHGGYRCHCQAGYSGRNCETDIDDCRPNPCHNGGSCTDGINTAFCDCLPGFRGTFCEEDINECASDPCRNGANCTDCVDSYTCTCPAGFSGIHCENNTPDCTESSCFNGGTCV DGINSFTCLCPPGFTGSYCQHDVNECDSQPCLHGGTCQDGCGSYRCTCPQGYTGPNCQNLVHWCDSSPCKNGGKCWQTHTQYRCECPSGWTGLYCDVPSVSCEVAAQRQGVDVARLCQHGGLCVDAGNTHHCRCQAGYTGSYCEDLVDECSPSPCQNGATCTDYLGGYSCKCVAGYHGVNCSEEIDECLSHPCQNGGTCLDLPNTYKCSCPRGTQGVHCEINVDDCNPPVDPVSRSPKCFNNGTCVDQVGGYSCTCPPGFVGERCEGDVNECLSNPCDARGTQNCVQRVNDFHCECRAGHTGRRCESVINGCKGKPCKNGGTCAVASNTARGFICKCPAGFEGATCENDARTCGSLRCLNGGTCISGPRSPTCLCLGPFTGPECQFPASSPCLGGNPCYNQGTCEPTSESPFYRCLCPAKFNGLLCHILDYSFGGGAGRDI PPPLIEEACELPECQEDAGNKVCSLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFSDGHCDSQCNSAGCLFDGFDCQRAEGQCNPLYDQYCKDHFSDGHCDQGCNSAECEWDGLDCAEHVPERLAAGTLVVVVLMPPEQLRNSSFHFLRELSRVLHTNVVFKRDAHGQQMIFPYYGREEELRKHPIKRAAEGWAAPDALLGQVKASLLPGGSEGGRRRRELDPMDVRGSIVYLEIDNRQCVQASSQCFQSATDVAAFLGALASLGSLNIPYKIEAVQSETVEPPPPAQLHFMYVAAAAFVLLFFVGCGVLLSRKRRRQHGQLWFPEGFKVSEASKKKRREPLGEDSVGLKPLKNASDGALMDDNQNEWGDEDLETKKFRFEEPVVLPDLDDQTDHRQWTQQHLDAADLRMSAMAPTPPQGEVDADCMDVNVRGPDGFTPLMIASCSGGGLETGNSEEEEDAPAVISDFIYQGASLHNQTDRTGETALHLAARYSRSDAAKRLLEASADANIQDNMGRTPLHAAVSADAQGVFQIL IRNRATDLDARMHDGTT PLILAARLAVEGMLEDLINSHADVNAVDDLGKSALHWAAAVNNVDAAVVLLKNGANKDMQNNREETPLFLAAREGSYETAKVLLDHFANRDITDHMDRLPRDIAQERMHHDIVRLLDEYNLVRSPQLHGAPLGGTPTLSPPLCSPNGYLGSLKPGVQGKKVRKPSSKGLACGSKEAKDLKARRKKSQDGKGCLLDSSGMLSPVDSLESPHGYLSDVASPPLLPSPFQQSPSVPLNHLPGMPDTHLGIGHLNVAAKPEMAALGGGGRLAFETGPPRLSHLPVASGTSTVLGSSSGGALNFTVGGSTSLNGQCEWLSRLQSGMVPNQYNPLRGSVAPGPLSTQAPSLQHGMVGPLHSSLAASALSQMMSYQGLPSTRLATQPHLVQTQQVQPQNLQMQQQNLQPANIQQQQSLQPPPPPQPHLGVSSAASGHLGRSFLSGEPSQADVQPLGPSSLAVHTILPQESPALPTSLPSSLVPPVTAAQFLTPPSQHSYSSPVDNTPSHQLQVPEHPFLTPSPESPDQWSSSSPHSNVSDWSEGVSSPPTSMQSQIARIPEAFK

Table 14. E3 ligase motifs

E3 ligase motif APC/C (DBOX) .R..L..[LIVM].APC/C (KEN) .KEN.APC/C (ABBA) [FIVL].[ILMVP][FHY].[DE ].{0,3}[DEST] APCC_TPR_1 .[ILM]R$ CBL (PTK) [DN]. Y [ST]..P CBL (MET) D Y R COP1 [DE][DE].{2, 3}VP[DE] CRL4_CDT2_1 [NQ]{0,1}..[ILMV][ST][DEN][FY][FY].{2,3}[KR]{2,3}[^DE] CRL4_CDT2_2 [NQ]{0,1}..[ILMV]T[DEN][HMFY][FMY].{2,3}[KR]{2,3}[^DE] Kelch_KEAP1_1 [DNS].[DES] [TNS]GE Kelch_KEAP1_2 QD.DLGV Kelch_actinfilin [AP]P[MV][IM]V Kelch_KLHL3 E.EE.E[AV]DQH MDM2_SWIB F[^P]{3}W[^P]{2,3}[ VIL] Nend_Nbox_1 ^M{0,1}[FYLIW][^P] Nend_UBRbox_1 ^M{0,1}[RK][^P]. Nend_UBRbox_2 ^M{0,1}([ED]). Nend_UBRbox_3 ^M {0,1}([NQ]).Nend_UBRbox_4 ^M{0,1}(C).ODPH_VHL_1 [IL]A( P ).{6,8}[FLIVM].[FLIVM] SCF_COI1_1 ..[RK] [RK].SL..F[FLM].[RK]R[HRK].[RK]. SCF_FBW7_1 [LIVMP].{0,2}( T )P..([ ST ]) SCF_FBW7_2 [LIVMP]. {0,2}( T )P..E SCF_SKP2-CKS1_1 ..[DE].( T )PK SCF_TIR1_1 .[VLIA][VLI]GWPP[VLI]...R. SCF-TRCP1 D( S )G .{2,3}([ ST ]) SIAH .PAVP[^P] SPOP [AVP].[ST][ST][ST]

For the E3 ligases in Table 14, the motif patterns use the following nomenclature: "." designates any amino acid type, "[X]" designates the amino acid types allowed at that position, and "^X" starts with the amino acid type in the specified sequence of the pattern. X, '[^X]' indicates that the position can have any amino acid except type X, and the numbers are specified as follows 'X{x,y}', where x and y specify the 'X' amino acids required for the position The minimum and maximum number of types. The "$" symbol indicates the C-terminus of the protein chain. Bold shows conserved residue positions within the primary apogroup of known post-translational modifications (eg, phosphorylation and proline hydroxylation) (PTM data from UniProt61).

Example

DHD-A and DHD-B binding element pair

To evaluate pairs of DHD-A and DHD-B binding elements with reference to the design described in Table 1, an enzyme-linked immunosorbent assay (ELISA) was performed according to a standard protocol. Briefly, 96-well Maxisorp plates (Nunc-Immuno, Sigma-Aldrich) were coated with NeutrAvidin (10 μg/ml) overnight at 4°C and subsequently blocked with BSA (2% w/v) at 20°C1 Hour. Two (2) nM biotinylated MBP-DHD-B were captured on neutralin-coated wells for 30 min, then added in ELISA buffer (PBS, pH 7.4, 0.05% Tween-20, 0.2% BSA) FLAG-labeled DHD-A diluted in medium for 30 minutes. Bound DHD-A protein was then detected using horseradish peroxidase (HRP)-conjugated anti-FLAG M2 monoclonal antibody (Sigma Aldrich) and TMB substrate. For analysis, the Graphpad Prism software was used to fit curves to a single-site specific binding model with Hill coefficients.

Figure 8A is a schematic diagram showing the Kd determination of a competitive binding ELISA assay of DHD-A:DHD-B interaction from DHD-A (SEQ ID NO: 11) and DHD-B (SEQ ID NO: 13) binding elements. Figure 8B is a Kd assay showing a competitive binding ELISA assay of DHD-A:DHD-B interaction from binding elements DHD-A (SEQ ID NO: 11) and DHD-B-Ntrunc (SEQ ID NO: 15). schematic diagram. The concentration of bound DHD-B competitive inhibitor is plotted against the total concentration of DHD-B competitive inhibitor. Binding curves were constructed and Kd values were determined by nonlinear curve fitting using Graphpad Prism software. The data demonstrate that the designed binding elements have high affinity with Kd in the nanomolar range.

In anticipation of autoubiquitination of the binding element upon fusion to the E3 ligase domain, the lysine (K) residue in the DHD-B protein (SEQ ID NO:13) was changed to arginine (R) to generate DHD37-short-B-KtoR (SEQ ID NO: 16). Figure 9 is a schematic diagram showing the comparison of DHD-B (SEQ ID NO: 13) and DHD37-short-B-KtoR (SEQ ID NO: 16) in a direct binding ELISA assay with DHD-A (SEQ ID NO: 11) . ELISA absorbance at 450 nm was plotted against the added DHD-A concentration. Binding curves were constructed by nonlinear curve simulation using Graphpad Prism software. The data show that the affinity of the binding element is essentially unchanged following the lysine to arginine mutation. Often, surface lysine residues in the designed sequence can be mutated to arginine without altering structure and function (except for the expected effect of preventing autoubiquitination).

E3 ligase screening and evaluation of expression constructs

Functional E3 ligase domain and ubiquitin variants that could be used to generate expression constructs for targeted protein degradation were evaluated using lentiviral transduction of T cells and flow cytometric analysis (see Table 2 above ).

Jurkat, SupT1, A549, and Jeko1 T cell lines were obtained from the American Type Culture Collection (Manassas VA). SupT1 and Jeko1 cells were maintained in RPMI 1640 medium in Glutamax (Gibco) containing 10% heat-inactivated fetal bovine serum (Gibco). For lentiviral transduction, SupT1 and Jurkat cells were fed with fresh medium 4-16 hours prior to transduction and then incubated with lentivirus in complete medium + LentiBOOST at the manufacturer's recommended concentration (Sirion Biotech). 18 hours after transfection, dilute lentivirus and LentiBOOST by adding 1 volume of fresh medium.

Obtain preselected, cryopreserved primary human CD4 and CD8 T cells from Bloodworks (Seattle WA) or Stemcell (Vancouver BC), or pooled CD4/CD8+ T cells from normal donors. Human T cells were supplemented with immune cell serum exchange (Thermo-Fisher), 2mM L-glutamine (Gibco), 2mM glutamine (Gibco), 200IU/ml IL-2 (culture system), 120 IU/ ml IL-7 (culture system) and 20 IU/ml IL-15 (culture system) in OpTmizer medium (Thermo-Fisher).

For lentiviral transduction, T cells were stimulated for 30 h with a 1:100 dilution of T cell TransAct (Miltenyi). Viruses are then added to T cells for 18-24 hours. In some cases, virus was added to T cells 24 hours after stimulation and a second lentivirus was added 30 hours after stimulation. Stimulation and virus infection were then terminated by adding 7 volumes of fresh medium without TransAct, and cells were cultured for an additional 3-7 days before analysis. For danoprevir-induced dimerization of binding elements, cells were treated with 500 nM danoprevir (RG7227, Product No. A4024) or an equivalent amount of dimethylsulfoxide for 18-24 hours prior to analysis. To stimulate CD3/CD28 in cells expressing E3 ligase domain and SH2 or SH3 domain fusion constructs, T cells were incubated with a 1:100 dilution of T Cell TransAct for 4 hours prior to analysis. For CD3 priming, precoat a 24-well plate with monoclonal CD3 agonist antibody (OKT3) in PBS overnight at 4°C. The dishes were washed with PBS and ~2x106 transduced cells were incubated in each well of the dish for 1–2 hr before collection for flow cytometry and immunoblotting.

Flow cytometry was performed on a Ze5 cytometer (Biorad). To determine expression of cell surface markers, 1x105-2x105 total cells were transferred into V-bottom 96-well dishes (Corning). Cells were washed twice with Flow Cytometry Staining Buffer (eBioscience) and then stained with relevant reagents in Flow Cytometry Staining Buffer in a total volume of 50 µL for 30 min on ice. After staining, cells were washed twice with flow cytometry staining buffer, fixed in FluoroFix buffer (Biolegend), and kept at 4oC in the dark until analysis. For experiments involving intracellular BACH2 staining, cells were surface stained as described above, then fixed, permeabilized, and labeled according to the manufacturer's protocol of the FoxP3 transcription factor staining buffer set (eBioscience, #00-5523-00). Purified recombinant ROR1 ectodomain linked to human Ig Fc was produced in-house and conjugated to Alexa 647 dye for detection. During primary antibody staining, eFluor 780 fixable reactive dye (eBioscience) was added at a 1:8000 dilution. Flow cytometry data were analyzed using FlowJo 10 (Tree Star). The list of antibodies used is shown in Table 8. Table 8. Antibodies used for performance analysis Target fluorescent dye copy source CD19 BV 421 HIB19 Biolegend FLAG PE L5 Biolegend CD3 BV 421 SK7 Biolegend TCR α/β BV 421 IP26 Biolegend PD1 PE-Cy7 J105 Thermo Fisher LAG3 SB702 3DS223H Thermo Fisher TIM3 SB708 F38-2E2 Thermo Fisher CD39 BV 605 A1 Biolegend

A panel of designed DHD-E3/Ub constructs (see Table 2) were screened to efficiently degrade a ROR1-specific CAR (ROR1 CAR-DHD-A, SEQ ID NO: 115), see Table 3, which was proposed as a target for degrading membrane proteins. model target. The set of membrane-associated DHD-E3 ligases screened was as follows: RNF43-v1 (SEQ ID NO: 74), RNF43-v2 (SEQ ID NO: 77), RNF43-v3 (SEQ ID NO: 76), ZNRF3-v1 (SEQ ID NO: 76), ID NO: 84), ZNRF3-v2 (SEQ ID NO: 84) NO: 85), ZNRF3-v3 (SEQ ID NO: 86), MARCH8-v1 (SEQ ID NO: 87), MARCH8-v2 (SEQ ID NO : 88), MARCH8-v3 (SEQ ID NO: 89), RNF128-v1 (SEQ ID NO: 78), RNF128-v2 (SEQ ID NO: 79), RNF128-v3 (SEQ ID NO: 80). The set of cytoplasmic DHD-E3 ligases screened was as follows: ELOC (SEQ ID NO: 97), FBW1A (SEQ ID NO: 98), FBXW7 (SEQ ID NO: 96), LNX1 (SEQ ID NO: 90), CHIP (SEQ ID NO: 95), NEDD4 (SEQ ID NO: 92), SOCS2 (SEQ ID NO: 94), SPOP (SEQ ID NO: 93), VHL (SEQ ID NO: 91), RNF4 (SEQ ID NO: 99 ), TRAF6 (SEQ ID NO: 100), 3xUb (SEQ ID NO: 81), 3xUbK48R (SEQ ID NO: 82) and 3xUbK63R (SEQ ID NO: 83). Briefly, SupT1 cells were co-transduced with lentivirus for each member of the expressed panel and the ROR1 CAR-DHD-A (black bars) or a control CAR lacking the dimerization domain (binding element; white bars), and cells were treated with ROR1-ECD-Fc Alexa Fluor 647 conjugate staining, and the surface expression of CAR-DHD detected by flow cytometry. The gMFI ratio for each construct was calculated by normalizing with the gMFI measured in cells transduced with the CAR construct only. Functional constructs were determined as normalized gMFI with a cutoff below 0.25.

10A and 10B are schematic diagrams showing normalized gMFI of membrane-associated DHD-E3 ligase and cytoplasmic DHD-E3 ligase, respectively. New E3 ligase designs are indicated with an asterisk (*). We identified a membrane-associated DHD-E3 design (Fig. 10A) and a cytoplasmic DHD-E3 design (Fig. 10B) capable of degrading CAR targets. The design exhibiting the greatest degradative activity was: a triple tandem linear chain of RNF43, RNF4, LNX1, TRAF6 and a ubiquitin K48R mutant (3xUb ^K48R ). When CAR-DHD was co-expressed with these synthetic degraders, the geometric mean fluorescence intensity (gMFI) of CAR-DHD expression was reduced to about 10-20% of that of control CAR.

The panel of cytoplasmic DHD-E3 constructs (SEQ ID NO, see above with reference to Figure 10) was screened for efficient degradation of 3xFLAG-tagged DHD-B-BACH2 as a model target of cytoplasmic proteins. Briefly, SupT1 cells were co-transduced with lentivirus expressing DHD-B-BACH2 (SEQ ID NO: 120) (see Table 3) and each member of the panel, then stained with anti-FLAG Brilliant Violet 421 antibody conjugate, Ectopic expression of DHD-BACH2 was detected by intracellular flow cytometry. The gMFI ratio for each construct was calculated by normalizing to the gMFI measured in cells transduced with the wild-type BACH2 construct. Functional constructs were identified as having a normalized gMFI around or below the 0.25 cut-off point.

Figure 11 is a graph showing the normalized gMFI of cytoplasmic DHD-E3 ligases screened for degradation of 3xFLAG-tagged DHD-B-BACH2. Intracellular flow cytometric analysis of FLAG-tagged BACH2 levels demonstrates efficient degradation of exogenously expressed BACH2 to negative controls when DHD-B fusions of LNX1, SOCS2, SPOP, VHL, and RNF4 E3 domains are co-expressed with DHD-BACH2 10-20% of that.

Referring now to Figure 10A, Figure 10B and Figure 11, the results of these two screening experiments allowed us to identify DHD-E3 candidate structures for optimal degradation of membrane receptors and cytoplasmic target proteins.

Since the DHD subunit contains lysine residues that are potential sites for autoubiquitination, we hypothesized that the binding domain could be replaced with its lysine-depleted variant DHD-KtoR (SEQ ID NO: 16) , to stabilize binding element-E3 fusions and improve degradation of protein targets. In addition to lysine to arginine mutagenesis (i.e., lysine (K) to arginine (R); KtoR), the use of a C-terminal endocytic motif was explored to enhance CAR degradation. The endocytic motif (indicated by "endo") contains a double leucine motif [D/E]xxxLL and a C-terminal valine residue, enabling efficient endoplasmic reticulum export (Kozik, P. et al. , Traffic (2010) doi:10.1111/j.1600-0854.2010.01056.x, the entire contents of which are incorporated herein by reference). Table 4 shows the amino acid sequence of the optimized RNF43-DHD-B construct that exhibits enhanced target degradation.

To test these hypotheses, primary CD4+ T cells were co-transfected with CAR-DHD-expressing and RNF43-DHD, RNF43-DHD-endo (SEQ ID NO: 122) or RNF43-DHD-KtoR variants (SEQ ID NO: 121) guide (see Table 4). CD4+ T cells were transduced with CAR-DHD alone and CD4+ T cells were co-transduced with CAR control and RNF43 DHD-KtoR as controls. Untransduced CD4+ T cells were used as a background CAR expression level control. The level of CAR expression in transduced and nontransduced cells was assessed using CAR surface staining and flow cytometry.

Figure 12 is a graph showing CAR surface staining and geometric mean fluorescent intensity (gMFI) levels in transduced and control CD4+ T cells. Panel A is a table showing the "CAR" and "E3" expressing constructs used for each CD4+ T cell transduction (i.e., row 1: CAR-DHD only; row 2: CAR-DHD+RNF43-DHD; Row 3: CAR-DHD+RNF43 DHD endo; Row 4: CAR-DHD+RNF43 DHD KtoR; Row 5: CAR control+RNF43 DHD KtoR, and Row 6: non-transduced cells (-,-). Panel B is an overlay of histograms showing CAR surface staining levels for each CD4+ T cell transduced and non-transduced cells. Panel C is a histogram showing CAR gMFI for each CD4+ T cell transduced and nontransduced cells. The histograms shown in panels B and C are aligned with the corresponding expression constructs (CAR and E3) shown in panel A. Flow cytometry analysis indicated that CAR surface expression in CAR-DHD + RNF43-DHD-KtoR transduced cells decreased to the same level as non-transduced control cells (-,-). The data also showed that the lysine-depleted variant RNF43-DHD-KtoR did not affect the surface expression of the CAR control (row 5: CAR control + RNF43 -DHD-KtoR). Similar to the RNF43-DHD-KtoR variant, the construct RNF43-DHD-endo containing the endocytic motif induced degradation of CAR-DHD to background levels observed in non-transduced controls. We also observed to similar amounts of degradation as the other two endocytic motifs (data not shown). gMFI measurements confirmed that when RNF43-DHD-KtoR or RNF43-DHD-endo were co-transduced with CAR-DHD constructs, CAR expression was reduced to near background levels.The results of these two optimization methods show that the cellular pool of DHD-E3 ligase is increased by minimizing autoubiquitination and enhancing CAR endocytosis by using endocytic motifs , can further promote the degradation of CAR.

Small molecule concentrated peptide

The DHD degradation system does not require the addition of small molecules to control dimerization of binding element pairs. However, the functional E3 ligase domain discovered in our screen could be manipulated by small molecules by using "chemically induced dimer" (CID) instead of DHD. An example of a small molecule controlled heterodimer system is described above with reference to FIG. 5 . Table 5 shows the amino acid sequences of the DNCR-E3 and CAR-NS3a constructs used to evaluate the use of small molecule focused binding elements in degradation systems.

To assess the use of small molecule binding elements in degradation systems, lentiviral constructs encoding CAR-NS3a (SEQ ID NO: 126) and DNCR2-E3 ligase, including the E3 ligase domain RNF43, DNCR2-RNF4 (SEQ ID NO: 128) and LNX1-DNCR2 (SEQ ID NO: 129) (see Table 5) were generated and evaluated for their ability to promote CAR degradation in response to danoprevir. Briefly, SUP-T1 cells were co-transduced with lentivirus expressing CAR-NS3a and each DNCR-E3 ligase. The gMFI of surface CAR expression was measured by flow cytometry 24 hours after addition of danoprevir. SUP-T1 cells transduced with CAR-NS3a only were used as controls. Functional constructs had gMFI values lower than 10% of cells transduced with CAR-NS3a alone.

Figure 13 is a schematic diagram showing the inducible degradation of CAR-NS3a with LNX1-DNCR2, DNCR2-RNF4 and RNF43-DNCR2 in the presence of 500 nM danoprevir. Flow cytometry analysis of CAR surface expression showed that when cells were transduced with RNF43-DNCR2 and DNCR2-RNF4, CAR-NS3a was degraded to less than 10% of the original CAR level upon addition of danoprevir.

We further optimized the design by introducing a lysine-arginine mutation (KtoR) and an endocytic motif (endo) in a single lentiviral construct tagged with a P2A self-cleaving peptide-linked transduction CAR-NS3a and RNF43-DNCR2-KtoR-endo (SEQ ID NO: 132) variants. A single lentiviral construct encoding RNF43-DNCR2 served as a control. Figure 14 is a schematic diagram of a single vector construct encoding CAR-NS3a linked by a P2A self-cleaving peptide and RNF43-DNCR2 (i) and RNF43-DNCR2-KtoR-endo (ii).

SUP-T1 cells were transfected with lentivirus expressing control RNF43-DNCR2 control construct (i) or DNCR2 KtoR endo (ii) construct and incubated with 500 nM of danoprevir. The gMFI of surface CAR expression was measured by flow cytometry 24 hours after addition of danoprevir.

Figure 15 is a schematic diagram showing CAR surface staining and gMFI levels in SUP-T1 cells transduced with lentiviruses expressing control RNF43-DNCR negative control (i) or RNF43-DNCR-KtoR-endo (ii) constructs. Panel A is a table showing the control (i) DNCR-KtoR endo (ii) with and without the addition of danoprevir (Dano) for each experimental condition. Panel B is an overlay of histograms showing the level of CAR surface staining for each SUP-T1 cell transduced and non-transduced (-,-). Panel C is a histogram showing the gMFI of CAR for each SUP-T1 cell transduced and non-transduced cell (NTD). The histogram shown in panel B aligns with the corresponding representational structures (i and ii) shown in panel A. Flow cytometry analysis of transduced cells showed that addition of 500 nM danoprevir reduced the CAR gMFI of cells transduced with construct (i) to approximately 10% below the negative control. As expected from a single lentiviral format, the level of expression of DNCR2-E3 may be limited, so the observed CAR degradation after drug treatment was only ~32% of the original level. With the RNF43-DNCR2-KtoR variant, the level of CAR degradation was substantially reduced by four-fold, approximately 8% of the original level. The data suggest that these optimized designs may be useful in applications that require external control over targeted degradation.

Native motif-binding elements for concentrating E3 ligases

In addition to using DHD and small-molecule-focused peptide-binding elements, it is also possible to control degradation in our system using the focus of E3 ligases to endogenous target protein domains, including domains corresponding to post-translational modifications (as referenced above described in Figure 6A and Figure 6B). We generated sequences to test our identified E3 ligase domains against other protein recognition modules, including transmembrane domains targeting TCRs and protein binders targeting phosphorylation sites. The amino acid sequences of TMCD3z-, SH2ZAP70-, SH3NCK1-E3 fusion constructs are shown in Table 6.

To target the endogenous TCR complex, constructs encoding the CD3𝜁 transmembrane domain (TMCD3z) and the E3 ligase domain of RNF43 were generated. Jurkat cells were transduced with lentiviruses expressing these TMCD3z-RNF43 fusion proteins, and the surface expression of TCR complexes was measured by flow cytometry.

Figure 16 is a schematic diagram showing overlaid histograms of TCR staining and gMFI levels for cells transduced with TMCD3z-RNF43 construct, GFP control, and non-transduced cells using anti-TCRα/β antibody (BV421), respectively. In this example, data are shown for four versions of the TMCD3z-RNF43 construct: v1 (SEQ ID NO: 133), v2 (SEQ ID NO: 134), v3 (SEQ ID NO: 135) and v4 (SEQ ID NO: : 136). Flow cytometry analysis revealed that gMFI degraded endogenous TCR proteins in 10-20% of non-transduced control cells. Furthermore, using the SH2 domain from ZAP70 and the SH3 domain from NCK1, which are known to interact with CD3𝜁 and TCR-CD3𝜀, respectively, were also tested in activated TCR complexes. Jurkat cells were transduced with a lentivirus expressing a fusion construct of a phosphoresponse domain and an E3 ligase domain, and stimulated for 4 hours with a polymer nanomatrix conjugated to a recombinant humanized CD3 and CD28 agonist (TransAct-1). Surface representation of TCR complexes was measured by flow cytometry. Constructs tested were: LNX1-nSH2-cSH2 (SEQ ID NO: 139), nSH2-cSH2 and LNGFR (see Table 6).

Figure 17A is an overlay of histograms showing the level of endogenous TCR staining on cells expressing LNX1-nSH2-cSH2, nSH2-cSH2, and LNGFR control cells using anti-TCRα/β antibodies. Figure 17B is a bar graph showing gMFI values in cells expressing LNX1-nSH2-cSH2, nSH2-cSH2 and LNGFR. Flow cytometric analysis revealed that levels of endogenous TCR proteins were downregulated by approximately 70% in unstimulated cells expressing an E3 fusion protein with a tandem SH2 domain (nSH2-cSH2). After 4 hours of stimulation with polymer nanomatrixes conjugated to humanized recombinant CD3 and CD28 agonist antibodies, endogenous TCR levels were further downregulated to approximately 5% and remained at a higher level than in untransduced cells or cells expressing tandem SH2 domains The levels observed in the cells were approximately 3-fold lower. These results suggest that these fusion constructs can be used to efficiently modulate TCR signaling in CAR-T applications such as CAR-T cell therapy.

CAR Regulation in Primary T Cells

To confirm that our designed DHD-E3 ligase can regulate CAR activity in primary T cells, a 1:1 mixture of CD4+/CD8+ primary cells was co-infected with lentivirus expressing ROR1 CAR-DHD (SEQ ID NO: 115). Transductions (designated "CAR-DHD") and RNF43-DHD-KtoR (SEQ ID NO: 121) (designated "DHD-E3R") or LNX1-DHD-KtoR (designated "DHD-E3L") were as follows: Not transduced (mock-T), transduced with CAR or CAR-DHD alone, or double transduced with CAR-DHD and DHD-E3R or DHD-ESL or GFP control. Cytokine secretion, immune cell killing, and marker expression of cells were measured.

To test the ability of DHD-E3 to centrally regulate acute CAR activation, we challenged non-transduced (Mock-T), single-transduced or double-transduced T cells against co-cultured Jeko1 target cells or ROR1 knockout Jeko1 cells. Cells were co-cultured at an effector-to-target ratio of 1:4 Jeko1 cells or control Jeko1 cells lacking the CAR target antigen (i.e., Jeko1-KO). After 24 h of co-culture, supernatants from cultured T cells were collected, centrifuged at 300xg for 5 min to remove cells and debris, and then frozen at -80°C until analysis. Secretion of the cytokines interleukin 2 (IL2) and interferon-gamma (IFNγ) was measured using a custom V-plex human pro-inflammatory kit (human IFNγ, IL2, TNFα; Meso Scale Discovery (MSD), Rockville, MD) . For cytokine assays, supernatants were diluted 1:5 with MDS calibration diluent and analyzed according to the manufacturer's instructions.

Figure 18 is two graphs showing the concentrations of IL-2 and IFNγ present in the supernatant after 24 hours of co-cultivation, respectively. The data showed that co-expression of the DHD-E3 construct modulated a significant reduction in CAR-T cell antigen-driven cytokine production compared to cells transduced with CAR-DHD alone or CAR without DHD.

To examine CAR-mediated cytolytic activity, the number of target cells in T cell:Jeko-1 co-cultures was measured over time. Briefly, CAR T cells were mixed with NucLight Red (NLR)-ROR1+ target cells or NLR-ROR1-JeKo1 KO target cells or A549 Nuclight in Corning 96-well plates at a ratio of 1:1 or 1:5 effector cells to target cells Red (NLR) target cells were seeded. Plates were incubated for 72 hours in the Incucyte system (Sartorius; Basic Biosciences, Ann Arbor, MI). Tumor cell (JeKo1) injury to total NLR+ cells/well was determined over time by Incucyte measurements compared to tumor cells alone.

Figure 19 is a schematic diagram showing the killing of Jeko1 target cells by T cells co-cultured at an effector to target ratio of 1:4. The data showed that co-expression of the DHD-E3 construct effectively blocked CAR antigen-driven killing of target cells, with results close to those observed in control Mock T co-cultures. These results suggest that expression of DHD-E3 fusion protein effectively blocks acute CAR activity.

We next sought to determine whether intrinsic control of CAR expression could prevent exhaustion caused by chronic CAR signaling. To examine chronic signaling-induced exhaustion in vitro, T cells were co-cultured with the A549 lung adenocarcinoma cell line (target cells) at an effector-to-target ratio of 1:3 under constant antigen exposure for 7 days, and then T cells were detected by flow cytometry. Surface markers of cellular depletion (CD39, PD-1 and Lag3).

Figure 20 is a set of schematic diagrams showing the surface expression of CD39, PD-1 and Lag3 depletion markers by gMFI on single or double transduced CAR-T cells co-cultured with A549 target cells. The data showed that co-expression of the CAR-DHD and DHD-E3 constructs reduced the expression of the exhaustion markers CD39, PD1 and Lag3 compared to CAR, CAR-DHD or CAR-DHD + eGFP cultures. These results suggest that modular expression of DHD-E3 fusions regulates both acute and chronic signals from paired CAR-DHD constructs.

Since regulated degradation of the CAR is triggered by the expression of paired E3 fusions that converge to the CAR, dynamic regulatory patterns can be generated by placing engineered E3 modules under the control of inducible promoters that respond to cell-intrinsic or environmental stimuli. In one embodiment, expression of the DHD-E3 module is placed under a calcium-responsive NFAT-driven promoter (see Table 7). In this example, priming T cells with CAR signaling would induce calcium flux and expression of the regulatory DHD-E3 module, thereby reducing CAR expression and terminating signaling. Since T cell depletion is considered a major factor limiting the anticancer efficacy of chimeric antigen receptor (CAR) T cells, we hypothesized that this intrinsic CAR degradation system regulated by our engineered E3 ligase fusion would render CAR signaling transiently stops, and allows the exhausted cells to resume their antitumor functions and differentiate into a memory state.

Figure 1 is a schematic diagram showing a pair of 3+1 heterodimeric binding elements.

Figure 2 is an exemplary schematic diagram showing a pair of small molecule-mediated polypeptide binding modules.

Figure 3 is a schematic illustration of the modulation of an endogenous protein target using a binding element ("B") specific to a native motif ("A") on the target.

Figure 4A shows a schematic diagram of the use of 3+1 DHD pairs as binding elements for targeted degradation of transmembrane proteins.

Figure 4B shows a schematic of the use of 3+1 DHD pairs as binding elements for targeted degradation of cytoplasmic proteins.

Figure 5 shows a schematic diagram of the degradation of chimeric receptors using a small molecule-mediated degradation system.

Figure 6A shows a schematic diagram of the targeted degradation of chimeric transmembrane receptors using transmembrane receptor domains as binding elements.

Figure 6B shows a schematic of the use of phosphotyrosine binding domains (PYBDs) as binders targeting degradation of post-translationally phosphorylated ("P") transmembrane receptors.

Figure 7A shows a schematic diagram of a unidirectional forward configuration for encoding an inducible polynucleotide component and a constitutive polynucleotide component on a single vector.

Figure 7B is a schematic diagram illustrating a two-vector system for encoding an inducible polynucleotide component and a constitutive polynucleotide component.

Figure 8A is a Kd assay showing a competitive binding ELISA assay of DHD-A:DHD-B interaction from dimerization domains DHD-A (SEQ ID NO: 11) and DHD-B (SEQ ID NO: 13) schematic diagram.

Figure 8B shows the Kd for dimerization domains DHD-A (SEQ ID NO: 11) and DHD-B-Ntrunc (SEQ ID NO: 15) from a competitive binding ELISA assay of DHD-A:DHD-B interaction Schematic diagram of the assay.

Figure 9 shows a schematic comparison of DHD-B (SEQ ID NO: 13) and DHD37-short-B-KtoR (SEQ ID NO: 16) in a direct binding ELISA assay with DHD-A (SEQ ID NO: 11) .

Figure 10A and Figure 10B show schematic diagrams of normalized gMFI of membrane-associated DHD-E3 ligase and cytoplasmic DHD-E3 ligase, respectively.

Figure 11 is a schematic diagram showing normalized gMFI of cytoplasmic DHD-E3 ligases screened for degradation of 3xFLAG-tagged DHD-B-BACH2.

Figure 12 shows a schematic diagram of CAR surface staining and geometric mean fluorescence intensity (gMFI) levels in CD4+ T cells from transduced and control groups.

Figure 13 shows a schematic diagram of the induced degradation of CAR-NS3a and LNX1-DNCR2, DNCR2-RNF4 and RNF43-DNCR2 in the presence of 500 nM danoprevir.

Figure 14 shows a schematic diagram of a single vector construct encoding CAR-NS3a and RNF43-DNCR2 (i) and RNF43-DNCR2-KtoR-endo (ii) connected by a P2A self-cleaving peptide.

Figure 15. Schematic panel showing CAR surface staining and gMFI levels in SUP-T1 cells transfected with lentiviruses expressing control RNF43-DNCR negative control (i) or RNF43-DNCR-KtoR-endo (ii) constructs guide.

Figure 16 is two graphs showing overlaid bars of TCR staining and gMFI levels of cells transduced with TMCD3z-RNF43 construct, GFP control, and non-transduced cells using anti-TCRα/β antibody (BV421), respectively.

Figure 17A is an overlay histogram showing the level of endogenous TCR staining on cells expressing LNX1-nSH2-cSH2, nSH2-cSH2, and LNGFR control cells using anti-TCRα/β antibodies.

Figure 17B shows a bar graph of gMFI values in cells expressing LNX1-nSH2-cSH2, nSH2-cSH2, and LNGFR.

Figure 18 is two graphs showing the concentrations of IL-2 and IFNγ, respectively, present in the supernatant after 24 hours of co-cultivation.

Figure 19 is a schematic diagram showing the killing of Jeko1 target cells by T cells co-cultured at an effector to target ratio of 1:4.

Figure 20 is a set of graphs showing gMFI surface expressed CD39, PD-1 and Lag3 depletion markers on single or double transduced CAR-T cells co-cultured with A549 target cells.

21A-21C are a series of schematic diagrams showing that adding DHD to CAR does not change the surface appearance or function of CAR. As shown in the figure, the label "CAR" is a CAR without any DHD, the label "CAR-DHD_S_R2K" is a CAR fused with SEQ ID NO: 170, and the label "CAR-DHD_L_R2K" is a CAR fused with SEQ ID NO: 172, And the label "CAR-DHD_L" is a CAR fused with SEQ ID NO:173. (A) Comparison of CAR surface representation in unmodified CAR and CAR-DHD constructs. Screening of a panel of designed Her2 CAR-DHD constructs for Her2 CAR surface expression. Primary T cells were transduced with lentivirus expressing only Her2 CAR or a set of Her2 CAR-DHD constructs or not (analog T), and cells were stained with Her2 Alexa-Fluor 647 conjugate, and CAR- Surface manifestations of DHD. Measurement of surface expression was performed by comparing transduced marker-positive Her2 CAR gMFI between constructs. (B) Cytokine release after challenge with tumor cells in unmodified CAR and CAR-DHD constructs. CAR T cells were co-cultured with A549 cells at a 1:1 ratio of effector to target. The concentrations of IL2 (right Y-axis) and interferon-γ (left Y-axis) in supernatants were measured after 24 h of co-culture. (C) Measurement of T cell cytotoxicity in unmodified CAR and CAR-DHD constructs. CAR T cells were co-cultured at a 1:1 effector-to-target ratio in 96-well plates with A549 labeled with NucLight Red (NLR) cells positive for Her2 targets. Plates were incubated in incubation cells (Sartorius) for 72 hours. Tumor cell killer was determined by Incucyte measuring total NLR+ cells/well over time compared to tumor cells alone.

22A-22B are a series of schematic diagrams showing that designed degraders facilitate efficient degradation of protein targets. (A) A panel of designed DHD-E3/Ub constructs were screened to efficiently degrade Her2-specific CARs as model targets for degrading membrane proteins. Primary T cells were co-transduced with lentivirus and Her2 CAR-DHD for each member of the expression panel, and cells were stained with Her2-Alexa Fluor 647 conjugate to detect surface expression of CAR-DHD by flow cytometry. The gMFI ratio for each construct was calculated by normalizing with the gMFI measured in cells transduced with the CAR construct only. As shown, left to right, designed DHD-E3/Ub constructs (sequences provided in Table 10): F43 native RNF43 1X DHD-B; DAP10-V5 CD8TMD LNX1 2X DHD-B; DAP10-V5 CD8TMD DAP10-V5 CD8TMD LNX1 4X DHD-B; DAP10-V5 CD8TMD LNX1 KtoR 2X DHD-B; DAP10-V5 CD8TMD LNX1 KtoR 2X DHD-B; DAP10-V5 CD8TMD LNX1 KtoR 3X DHD-B; DAP10 -V5 CD8TMD LNX1 KtoR 4X DHD-B; LNX1 1X DHD-B; LNX1 2X DHD-B; LNX1 3X DHD-B; LNX1 KtoR 1X DHD-B; LNX1 KtoR 2X DHD-B; -V5 CD8TMD RNF4 KtoR 1X DHD-B; DAP10-V5 CD8TMD RNF4 KtoR 2X DHD-B; DAP10-V5 CD8TMD RNF4 3X DHD-B; DAP10-V5 CD8TMD RNF4 4X DHD-B; DAP10-V5 CD8TMD RNF4 1X DHD-B ;DAP10-V5 CD8TMD RNF4 2X DHD-B; DAP10-V5 CD8TMD RNF4 3X DHD-B; DAP10-V5 CD8TMD RNF4 4X DHD-B; RNF4 2X DHD-B; RNF4 3X DHD-B; 2X DHD-B; RNF4 3X DHD-B; RNF4 4X DHD-B; DAP10-V5 RNF43 1X DHD-B; DAP10-V5 RNF43 2X DHD-B; DAP10-V5 RNF43 3X DHD-B; -B. (B) In primary T cells from two normal donors, functional structure was determined with a normalized gMFI below the 0.25 cutoff. Bar graph showing CAR gMFI in single-transduced (CAR-DHD only) and double-transduced (+DHD-E3) cells. As shown in this figure, the terms "1X", "2X", "3X" and "4X" refer to the number of repeats containing the G4S linker (where a single or "1X" repeat has the sequence "GGGGS") (see Table 10) . As shown in this figure, the term "K2R" is interchangeable with the term "KtoR" and does not indicate an amino acid substitution at the second position of any degradant sequence shown. As shown in the figure, the label "DHD37B" refers to "DHD37-short-B-KtoR" (SEQ ID NO: 16).

Figure 23 is a schematic diagram demonstrating that RtoK mutations increase CAR downregulation. Comparison of CAR surface expression in CAR-DHD and CAR-DHD RtoK constructs. Primary T cells were co-transduced with lentiviruses expressing DHD-e3 constructs with a range of degradative activities and Her2 CAR-DHD or Her2 CAR-DHD with RtoK mutation, and cells were stained with Her2 Alexa-Fluor 647 conjugate, The surface expression of CAR-DHD was detected by flow cytometry. The gMFI ratio for each construct was calculated by normalizing to the gMFI measured in cells transduced with the CAR construct only, and the effect of the RtoK mutation was assessed using the Wilcoxon paired signed-rank test. As shown in this figure, the term "R2K" is interchangeable with the term "RtoK" and does not indicate an amino acid substitution at the second position of any degradant sequence shown.

Figures 24A-24C are a series of schematic diagrams showing that C-terminal fusions of coil-coil domains can maintain CAR activity and enable targeted modulation by paired coil-coil domain-E3 fusions. A) Cytolytic activity of CART cells challenged with Jeko1 target cells showing control T cells, (mock), parental CAR, and CAR constructs fused to the indicated coil-coil domains. B) Cytokine secretion by CAR constructs. C) Modulation of surface CAR expression by co-transduction of Coil-Coil-E3 fusions as indicated.

Figures 25A-25C are a series of schematic diagrams showing that modulation of CAR-DHD with a DHD-e3 construct improves survival and prevents the acquisition of priming/depletion markers in a tumor cell co-culture model. A) Experimental design: T cells were transduced with a single lentiviral construct encoding CAR-DHD (CAR only), encoding 2 different CAR-DHD designs (CAR_DHD_L and CAR_DHD_S), or with CAR-DHD and DHD-E3 constructs co-transduction. CAR T cells were cultured repeatedly with A549 adenocarcinoma cells for 4 days, followed by flow cytometry analysis. B) Expression patterns of CD39, PD1, and Lag3 depletion/activation markers on CAR-transduced cells. C) Percentage of CD3+ cells that were CAR+ under the indicated transduction conditions after co-culture compared to input cells. As shown in this figure, the label "DHD\U S" is the serial number 171, and the label "DHD\U L" is the serial number 173.

Claims (144)

A fusion protein comprising: a binding element and a degradation initiator, wherein the binding element selectively binds a target molecule, and wherein the degradation initiator comprises a sequence isolated or derived from an E3 ligase. The fusion protein as described in Item 1 of the scope of the patent application, wherein: (a) the E3 ligase comprises LNX1, RNF4, RNF43, RNF128, XNRF3, MARCH8, LRG1, NEDD4, SOCS2, CHIP, SPOP, FBXW7, FBXW1A, ELOC, TRAF6, VHL or any functional fragment thereof; or (b) the E3 ligase comprises LNX1, RNF4, RNF43 or any functional fragment thereof; or (c) the E3 ligase comprises LNX1 or any functional fragment thereof; or (d) the E3 ligase comprises RNF4 or any functional fragment thereof; or (e) the E3 ligase comprises RNF43 or any functional fragment thereof; or (f) A sequence isolated or derived from the E3 ligase, comprising a sequence of Table 10, a functional fragment thereof, or having at least 70%, 75%, 80%, 85%, 90%, or a sequence of Table 10 or a functional fragment thereof Sequences that are 95%, 97% or 99% homologous; or (g) a sequence isolated or derived from the E3 ligase, including the E3 ligase sequences of SEQ ID NOs: 74-114; or (h) a sequence isolated or derived from the E3 ligase, comprising any one of SEQ ID NOs: 159-163; or (i) The E3 ligase comprising the motifs of Table 14. The fusion protein as described in item 1 or item 2 of the patent application, wherein, in the cells capable of expressing the fusion protein, the target molecule is an endogenous molecule. The fusion protein as described in claim 3 of the patent application, wherein the target molecule is a naturally occurring molecule. The fusion protein as described in any one of items 1 to 4 of the patent application, wherein the binding element includes DNA sequence, RNA sequence, amino acid sequence or any combination thereof. The fusion protein as described in item 5 of the patent claims, wherein the sequence of the binding element and the sequence of the target molecule form a nucleic acid duplex. The fusion protein as described in item 6 of the patent claims, wherein the binding element selectively binds an epitope of the target molecule. The fusion protein as described in item 6 or item 7 of the patent application, wherein the target molecule includes the sequence in the sequence list file "016-TNP023PCT_SeqList_v2", which was created on February 2, 2022 and has a size of 52, 267 KB, which is incorporated herein by reference in its entirety, or includes the nucleic acid sequence encoding the molecule of interest. The fusion protein according to any one of claims 1 to 8, wherein the fusion protein or the binding element includes a dimerization domain. The fusion protein as described in claim 9 of the patent application, wherein the dimerization domain includes a designed heterodimer (DHD) polypeptide. The fusion protein described in any one of items 1 to 10 of the scope of the patent application, wherein the target molecule or a sequence encoding the target molecule is modified to: (a) comprising a binding element capable of forming a heterodimer with the dimerization domain of the fusion protein; or (b) operably linked to a binding element capable of forming heterodimers with the dimerization domain of the fusion protein. The fusion protein as described in item 11 of the patent application, wherein the binding element includes a dimerization domain. The fusion protein according to claim 12 of the patent application, wherein the dimerization domain includes a designed heterodimer (DHD) polypeptide. The fusion protein according to any one of claims 11 to 13, wherein the binding element of the fusion protein is a first binding element and the binding element of the target molecule is a second binding element; as well as (a) wherein the first binding element or the second binding element comprises a single helix; or (b) wherein the first binding element or the second binding element comprises at least a double helix; or (c) wherein the first binding element or the second binding element comprises 3, 4, 5, 6, 7 or 8 helices; or (d) the first binding element comprises a single helix and the second binding element comprises three helices; or (e) the first binding element comprises three helices and the second binding element comprises a single helix. The fusion protein as described in any one of the 11th to 13th items of the patent application, wherein: (a) the first binding element comprises DHD-A and the second binding element comprises DHD-B; or (b) the first binding element comprises DHD-B and the second binding element comprises DHD-A. The fusion protein as described in item 15 of the patent application, wherein the DHD-B includes a sequence of DHD37-short-B-KtoR (SEQ ID NO: 16). The fusion protein as described in claim 11 to claim 16, wherein the first binding element or the second binding element comprises a non-helical element. The fusion protein as described in item 11 to item 17 of the patent application, wherein the heterodimer includes a non-helical element. The fusion protein as described in claim 17 or claim 18, wherein the non-helical element includes a small molecule. A composition comprising: (a) a first fusion protein comprising a first binding element; and (b) a second fusion protein comprising a second binding element; in: (1) The first fusion protein also includes a degradation initiator or a functional variant thereof, and the second fusion protein also includes a target molecule; or (2) The first fusion protein also includes a target molecule, and the second fusion protein also includes a degradation initiator or a functional variant thereof. The composition of claim 20, wherein the first binding element and the second binding element form a heterodimer. The composition of claim 20 or claim 21, wherein the first binding element or the second binding element comprises a single helix. The composition of claim 20 or claim 21, wherein the first binding element or the second binding element comprises at least a double helix. The composition of claim 20 or claim 21, wherein the first binding element or the second binding element comprises at least a double helix. The composition of claim 20 or claim 21, wherein the first binding element or the second binding element comprises 3, 4, 5, 6, 7 or 8 helices. The composition described in item 20 or item 21 of the scope of the patent application, in: (c) the first binding element comprises DHD-A and the second binding element comprises DHD-B; or (d) the first binding element comprises DHD-B and the second binding element comprises DHD-A. The composition according to any one of claims 21 to 26, wherein the first binding element or the second binding element comprises a non-helical element. The composition of any one of claims 21 to 27, wherein the heterodimer includes a non-helical element. The composition of claim 27 or claim 28, wherein the non-helical element comprises a small molecule. The composition according to any one of claims 20 to 29, wherein the first binding element comprises a sequence of DHD37-short-B-KtoR (SEQ ID NO: 16). The composition of claim 30, wherein the first binding element further comprises a sequence isolated or derived from an E3 ligase. The composition as described in item 31 of the scope of application, wherein: (j) the E3 ligase comprises LNX1, RNF4, RNF43, RNF128, XNRF3, MARCH8, LRG1, NEDD4, SOCS2, CHIP, SPOP, FBXW7, FBXW1A, ELOC, TRAF6, VHL or any functional fragment thereof; or (k) the E3 ligase comprises LNX1, RNF4, RNF43 or any functional fragment thereof; or (l) the E3 ligase comprises LNX1 or any functional fragment thereof; or (m) the E3 ligase comprises RNF4 or any functional fragment thereof; or (n) the E3 ligase comprises RNF43 or any functional fragment thereof; or (o) a sequence isolated or derived from the E3 ligase, comprising a sequence of Table 10, a functional fragment thereof, or having at least 70%, 75%, 80%, 85%, 90%, or a sequence of Table 10 or a functional fragment thereof Sequences that are 95%, 97% or 99% homologous; or (p) a sequence isolated or derived from the E3 ligase, including the E3 ligase sequence of SEQ ID NO: 74-114; or (q) a sequence isolated or derived from the E3 ligase, comprising any one of SEQ ID NOs: 159-163; or (r) E3 ligase comprising the motifs of Table 14. The composition as described in any one of claims 20 to 32, wherein the first binding element comprises the sequence of SEQ ID NO: 174. The composition according to any one of claim 20 to claim 29, wherein the first binding element comprises any one of SEQ ID NO: 1-73 or 121-125. The composition according to any one of claims 20 to 29, wherein the second binding element comprises the sequence of DHD37-short-B-KtoR (SEQ ID NO: 16). The composition according to claim 35, wherein the first binding element further comprises a sequence isolated or derived from E3 ligase. The composition according to claim 36, wherein the sequence isolated or derived from E3 ligase comprises any one of SEQ ID NO: 159-163. The composition according to any one of claims 20-29 or 35-37, wherein the first binding element comprises the sequence of SEQ ID NO:174. The composition as described in any one of claim 20 to claim 29, wherein the second binding element comprises the sequence of any one of SEQ ID NO: 1-73 or 121-125. The composition of any one of claims 21 to 29, wherein a small molecule regulates the formation of heterodimers. The composition according to claim 40, wherein the first binding element or the second binding element binds to the small molecule. The composition according to claim 40, wherein the first binding element and the second binding element bind to the small molecule. The composition as described in claim 41 or claim 42, wherein the first binding element and the second binding element are not directly bound to each other. The composition according to any one of claims 40 to 43, wherein the small molecule increases the formation of heterodimers. The composition according to any one of claims 40 to 43, wherein the small molecule reduces the formation of heterodimers. The composition as described in any one of the 40th to 43rd items in the scope of the patent application, wherein a first small molecule and a second small molecule regulate the formation of a heterodimer, wherein the second small molecule is passed with The first small molecule competes to bind the first binding element or the second binding element to reduce heterodimer formation with the first small molecule. The second small molecule reduces binding of the first small molecule to the heterodimer by competing with the first small molecule for binding to the first binding element or the second binding element. The composition as described in any one of items 21-29 or 40-43 of the scope of patent application, wherein: (a) the first binding element comprises an NS3a sequence and the second binding element comprises a DNCR2 sequence or a GNCR1 sequence; or (b) the first binding element comprises a DNCR2 sequence or a GNCR1 sequence and the second binding element comprises an NS3a sequence. The composition as described in claim 29 or any one of claims 40-47, wherein the small molecule includes danoprevir or its analogues. The composition as described in claim 48, wherein the first small molecule or the second small molecule comprises danoprevir or an analog thereof. The composition as described in claim 29 or any one of claims 40-47, wherein the small molecule includes glazoprevir or its analogues. The composition as described in claim 50, wherein the first small molecule or the second small molecule comprises glazoprevir or an analogue thereof. The composition as described in any one of claims 20 to 51, wherein the target molecule includes a synthetic molecule or an exogenous molecule. The composition of claim 52, wherein the synthetic molecule or the foreign molecule comprises a protein. The composition according to claim 52, wherein the synthetic molecule or the foreign molecule comprises a chimeric protein, a chimeric receptor or a chimeric antigen receptor. The composition as described in item 54 of the patent application, wherein the chimeric receptor includes an extracellular domain, and the extracellular domain includes an antigen sensing domain, a transmembrane domain and an intracellular domain. The composition as described in item 55 of the patent application, wherein the antigen sensing domain includes one or more of: (a) a set of three complementarity determining regions (CDRs) of a heavy chain variable region; (b) a set of three complementarity determining regions (CDRs) of a heavy chain variable region and a set of three complementarity determining regions (CDRs) of a light chain variable region; (c) a fibronectin-based scaffold; Wherein the antigen sensing domain specifically binds to a target antigen. The composition as described in claim 55 or claim 56, wherein the antigen sensing domain comprises one or more sequences isolated or derived from a mammalian sequence. The composition of any one of claims 55 to 57, wherein the antigen sensing region comprises one or more sequences isolated or derived from a human sequence. The composition as described in any one of item 55 to item 58 of the patent application, wherein the antigen sensing region comprises humanized or fully human antibodies. The composition according to any one of claims 55 to 59, wherein the antigen sensing region comprises a single-chain variable fragment (scFv). The composition as described in any one of claim 55 to claim 60, wherein the extracellular domain further comprises one or more of a hinge region, a spacer sequence or a safety switch. The composition according to claim 61, wherein the hinge region comprises a sequence isolated or derived from a CD4 (cluster of differentiation 4) polypeptide, a CD8 (cluster of differentiation 8) polypeptide or a CD28 (cluster of differentiation 28) polypeptide. The composition of claim 62, wherein the hinge region comprises a sequence isolated or derived from a human sequence. The composition as described in claim 61, wherein the spacer sequence comprises a sequence isolated or derived from a CD4 polypeptide, a CD8 polypeptide, or a CD28 polypeptide. The composition of claim 64, wherein the spacer sequence comprises a sequence isolated or derived from a human sequence. The composition of claim 61, wherein the safety switch comprises a sequence isolated or derived from an epidermal growth factor receptor (EGFR) polypeptide. The composition according to claim 66, wherein the safety switch comprises a truncated EGFR (EGFRt) polypeptide. The composition of claim 66 or claim 67, wherein the safety switch comprises a sequence isolated or derived from a human sequence. The composition according to any one of items 55 to 68 of the patent application, wherein the transmembrane domain comprises a sequence isolated or derived from a CD4 polypeptide, a CD8 polypeptide, or a CD28 polypeptide. The composition of claim 69, wherein the transmembrane domain comprises a sequence isolated or derived from a human sequence. The composition according to any one of claim 55 to claim 70, wherein the intracellular domain comprises one or more co-stimulatory domains. The composition of claim 71, wherein the one or more co-stimulatory domains comprise a sequence isolated or derived from a CD3ζ (cluster of differentiation 3 zeta) polypeptide. The composition as described in item 71 or item 72 of the scope of patent application, wherein the one or more co-stimulatory domains include CD28 polypeptide, 4-1BB (cluster of differentiation 137) polypeptide, ICOS (inducible T cell co-stimulatory domain) A sequence isolated or derived from a CD27 (cluster of differentiation 27) polypeptide, OX40 polypeptide or CD27 (cluster of differentiation 27) polypeptide. The composition as described in item 71 or item 72 of the patent application, wherein the one or more co-stimulatory domains include: (a) a first co-stimulatory domain comprising a sequence isolated or derived from a CD28 polypeptide, 4-1BB polypeptide or ICOS polypeptide; and (b) a second co-stimulatory domain comprising a sequence isolated or derived from a 4-1BB polypeptide, an OX40 polypeptide or a CD27 polypeptide. The composition of any one of claims 71 to 74, wherein the one or more co-stimulatory domains comprise a sequence isolated or derived from a human sequence. The composition as described in any one of claim 55 to claim 75, wherein the chimeric receptor includes an intracellular domain, and the intracellular domain further includes an inducible cytokine domain. The composition as described in claim 76, wherein the inducible cytokine domain includes a nuclear factor of activated T cell (NFAT) polypeptide capable of inducing the expression of IL-12 cytokine. The composition according to any one of claims 55 to 75, wherein the chimeric receptor includes an intracellular domain, and the intracellular domain further includes an intracellular structure of a cytokine receptor area. The composition as described in claim 78, wherein the cytokine receptor intracellular domain includes IL-2 receptor beta (IL-2Rβ) chain fragment. The composition as described in item 78 or item 79 of the scope of application, wherein the chimeric receptor includes an intracellular domain, and the intracellular domain further includes a signal transducer and a transcriptional promoter (STAT3/5 ) binding motif. The composition according to any one of claims 55 to 80, wherein the chimeric receptor includes an intracellular domain, and the intracellular domain further includes at least one immunoreceptor-based tyrosine Initiation motif (ITAM) sequence. A nucleic acid sequence comprising: encoding: (a) the fusion protein described in any one of items 1 to 19 of the patent application; or (b) One or more elements of (a). A nucleic acid sequence comprising: encoding: (a) the first fusion protein described in any one of items 20 to 81 of the patent application; or (b) the second fusion protein described in any one of items 20 to 81 of the scope of patent application; or (c) one or more elements of (a); or (d) One or more elements of (b). A nucleic acid sequence, comprising: encoding the first fusion protein and the second fusion protein described in any one of the 20th to 81st items in the scope of the patent application. The nucleic acid sequence described in any one of the 82nd to 84th items of the scope of the patent application also includes one or the Various. The nucleic acid sequence described in any one of the 82nd to 84th claims of the patent application further includes at least one promoter capable of driving the expression of the nucleic acid sequence in a mammalian cell. The nucleic acid sequence described in any one of the 82nd to 84th claims of the patent application further comprises at least one promoter capable of driving the expression of the nucleic acid sequence in a human cell. The nucleic acid sequence according to claim 86 or claim 87, wherein at least one promoter includes a constitutive promoter. The nucleic acid sequence as described in item 88 of the scope of patent application, wherein the constitutive promoter comprises MND promoter, hPGK promoter, CMV promoter, CAG promoter, SFFV promoter, EF1α promoter, UBC promoter and CD43 A sequence isolated or derived from one or more of the promoters. The nucleic acid sequence of claim 86 or claim 87, wherein at least one promoter includes an inducible promoter. The nucleic acid sequence as described in item 90 of the scope of the patent application, wherein the inducible promoter comprises YB_TATA promoter, human beta globulin (huBG) promoter, minIL2 promoter, minimalCMV (minCMV) promoter and TRE3G promoter An isolated or derived sequence from one or more of . The nucleic acid sequence as described in claim 90 or claim 91, wherein the inducible promoter includes a minimal sequence. The nucleic acid sequence according to any one of the 90th to 92nd claims, wherein the inducible promoter further includes a transcription factor specific recognition sequence. The nucleic acid sequence according to claim 93, wherein the transcription factor-specific recognition sequence includes a transcription factor-specific response element. The nucleic acid sequence according to claim 93, wherein the transcription factor specific response element comprises a sequence isolated or derived from the NFAT sequence. The nucleic acid sequence according to any one of claim 93 to claim 95, wherein the transcription factor-specific recognition sequence includes at least one repeating segment of the transcription factor-specific response element. The nucleic acid sequence as described in any one of the 93rd to 95th items of the patent application, wherein the transcription factor-specific recognition sequence includes at least 2, 3, 4, 5, 6, 7 of the transcription factor-specific response element , 8, 9 or 10 repeats. The nucleic acid sequence as described in any one of the 86th to 97th items of the patent application, including: a first promoter drives expression of the first fusion protein; or A second promoter drives expression of the second fusion protein. The nucleic acid sequence according to claim 98, wherein the first promoter and the second promoter are identical. The nucleic acid sequence of claim 98, wherein the first promoter and the second promoter are different. The nucleic acid sequence according to any one of claims 85 to 100, wherein the separation element comprises a ribosomal skipping sequence. The nucleic acid sequence according to claim 101, wherein the separation element comprises at least two ribosomal skipping sequences. The nucleic acid sequence as described in claim 101 or claim 102, wherein the ribosomal skipping sequence includes a P2a sequence or a T2a sequence. The nucleic acid sequence according to any one of item 101 to item 103 of the patent application, wherein the ribosomal skipping sequence includes a T2a-RFP-P2a sequence, a P2a-T2a sequence or a T2a-P2a sequence. The nucleic acid sequence of any one of claims 85 to 100, wherein the polycistronic element includes an internal ribosome entry site (IRES) sequence. A carrier, comprising: the nucleic acid described in any one of the 85th to 100th items of the patent application. A vector, comprising: a first vector and a second vector; wherein: the first vector comprises a nucleic acid encoding the first fusion protein described in any one of the 20th to 81st items in the scope of the patent application; the first vector The second vector includes a nucleic acid encoding the second fusion protein described in any one of the 20th to the 81st items of the patent application. The vector as described in claim 106 or claim 107, wherein the vector comprises an expression vector for expressing a nucleic acid in a mammalian cell. The vector as described in claim 108, wherein the vector comprises a plasmid. The vector according to claim 106 or claim 107, wherein the vector comprises a delivery vector for introducing a nucleic acid into a mammalian cell. The carrier as described in item 110 of the patent application, wherein the delivery carrier includes one or more of viral vectors, non-viral vectors, liposomes, micelles, polymers and nanoparticles. The vector according to claim 111, wherein the vector comprises a sequence isolated or derived from a virus or a viral vector. The vector described in item 112 of the scope of the patent application, wherein the viral vector includes adenoviral vector, lentiviral vector, baculoviral vector, human herpes virus vector, Bartholin virus (parvovirus) vector, vaccinia virus vector, A sequence isolated or derived from one or more of herpes simplex virus vectors, adeno-associated virus (AAV) vectors and hepatitis B virus vectors. The vector according to any one of claims 106 to 110, wherein the vector comprises a sequence isolated or derived from a transposition system. The vector as described in item 114 of the scope of the patent application, wherein the vector comprises isolated or derived from piggyBAC transposition system, sleeping beauty transposition system, Tc1/mariner transposition system, Tol2 transposition system, helraiser transposition system and Tn7 transposition system A sequence of one or more of the seat systems. The vector according to any one of items 106 to 115 of the patent application, wherein the vector includes one or more sequences regulating homology-directed repair. A cell comprising the fusion protein described in any one of items 1 to 19 of the patent application. A cell, including the composition described in any one of the 20th to 81st items in the scope of the patent application. A cell comprising the nucleic acid sequence described in any one of items 82 to 105 of the patent application. A cell, including the carrier described in any one of the 106th to 116th items in the scope of the patent application. The cell described in any one of items 117 to 120 of the patent application, wherein the cell stably expresses the fusion protein described in any one of items 1 to 19 of the patent application. The cell described in any one of the 117th to 120th items of the patent application, wherein the cell stably expresses the first fusion protein or the second fusion protein described in any one of the 20th to 81st of the patent application protein. The cell as described in any one of claims 117 to 122, wherein the cell is a eukaryotic cell. The cell as described in any one of claims 117 to 122, wherein the cell is a mammalian cell. The cell as described in any one of claims 117 to 122, wherein the cell is a human cell. The cell as described in any one of claims 117 to 125, wherein the cell is a stem cell. The cell according to any one of claims 117 to 126, wherein the stem cell is a hematopoietic cell. The cell according to any one of claims 117 to 125, wherein the stem cell is a mesenchymal cell. The cell as described in claim 127 or claim 128, wherein the cell is an immune cell. The cell according to claim 129, wherein the immune cell is a T cell, a natural killer (NK) cell or an innate lymphocyte (ILC). A composition comprising: (a) a fusion protein as described in any one of items 1 to 19 of the scope of the patent application; or (b) a composition as described in any one of claims 20 to 81; or (c) the nucleic acid sequence described in any one of claims 82 to 105; or (d) a carrier as described in any one of claims 106 to 116; or (e) The cells described in any one of claims 117 to 130 of the patent application. A pharmaceutical composition, comprising the composition of item 131 of the patent application and a pharmaceutically acceptable carrier. The fusion protein as described in any one of the 1st to 19th items of the patent application, the use of the composition described in any one of the 20th to 81st items of the patent application, such as the 82nd of the patent application The nucleic acid sequence described in any one of items 1 to 105, the carrier described in any one of items 106 to 116 of the scope of application, any one of items 117 to 130 of the scope of application The cells or the pharmaceutical composition as described in item 132 of the patent application are used to prepare a medicine for treating a disease or a disease. The fusion protein as described in any one of the 1st to 19th items of the patent application, the use of the composition described in any one of the 20th to 81st items of the patent application, such as the 82nd of the patent application The nucleic acid sequence described in any one of items 1 to 105, the carrier described in any one of items 106 to 116 of the scope of application, any one of items 117 to 130 of the scope of application The cell or the pharmaceutical composition as described in item 132 of the patent application is used for treating a disease or a disease. Use as described in claim 133 or 134, wherein the disease or condition comprises one or more autoimmune diseases or conditions; inflammatory diseases or conditions; immunodeficiency diseases or disorders; ischemic diseases or conditions Diseases or conditions of the blood; Diseases or conditions of the bone; Diseases or conditions of the nervous system; Diseases or conditions of the heart; Diseases or conditions of blood vessels; diseases or conditions; liver diseases or conditions; kidney diseases or conditions; hearing diseases or disorders; ophthalmic diseases or conditions; and proliferative diseases or conditions. The use as described in claim 133 or claim 134, wherein the disease or disease includes cancer. The use as described in any one of items 133 to 136 of the patent application, wherein the disease or disease includes infection or a disease or disease caused by an infectious disease. The use as described in any one of items 133 to 136 of the patent claims, wherein the disease or disorder includes a genetic disease or disorder. A method for treating a disease or an illness, comprising: administering to a subject an effective dose of the fusion protein as described in any one of items 1 to 19 of the scope of application, such as item 20 of the scope of application to the composition described in any one of the 81st or 131st items, the nucleic acid sequence as described in any one of the 82nd to 105th items of the scope of application, such as the 106th to 116th items of the patent scope of the application The carrier according to any one of the claims, the cell according to any one of the 117th to 130th items of the patent application, or the pharmaceutical composition according to the 132nd item of the patent application, wherein the disease or the disease The severity of the signs or symptoms is reduced, thereby treating the disease or the condition. A method for preventing a disease or a disease, comprising: administering to a subject an effective dose of the fusion protein as described in any one of items 1 to 19 of the scope of application, such as item 20 of the scope of application to the composition described in any one of the 81st or 131st items, the nucleic acid sequence as described in any one of the 82nd to 105th items of the scope of application, such as the 106th to 116th items of the patent scope of the application The carrier according to any one of the claims, the cell according to any one of the 117th to 130th items of the patent application, or the pharmaceutical composition according to the 132nd item of the patent application, wherein the disease or the disease The onset or recurrence of a sign or symptom is delayed or inhibited, thereby preventing the disease or the condition. The method of claim 139 or claim 140, wherein the disease or condition comprises one or more of an autoimmune disease or condition; an inflammatory disease or condition; an immunodeficiency disease or disorder; an ischemic disease or condition Diseases or conditions of the blood; Diseases or conditions of the bones; Diseases or conditions of the nervous system; Diseases or conditions of the heart; Diseases or conditions of blood vessels; diseases or conditions; liver diseases or conditions; kidney diseases or conditions; hearing diseases or disorders; ophthalmic diseases or conditions; and proliferative diseases or conditions. The method of claim 139 or 140, wherein the disease or condition comprises cancer. The method as described in claim 139 or claim 142, wherein the disease or condition comprises an infection or a disease or condition caused by an infectious disease. The method of claim 139 or claim 142, wherein the disease or condition comprises a genetic disease or condition.

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