Fusion constructs for controlling protein function

Abstract

To provide a simple and effective method for rapidly shutting off the production and / or function of specific proteins in eukaryotes.SOLUTION: Described herein are engineered fusion proteins comprising a variant protease (e.g., an HCV NS3 protease) fused to a polypeptide of interest and a cognate protease cleavage site. The cleavability of the cognate protease cleavage site enables the controllability of one or more functions of the polypeptide of interest. Additionally, disclosed are methods for generating engineered fusion proteins as well as their therapeutic use.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 62 / 797,043, filed January 25, 2019, which is incorporated by reference in its entirety for all purposes.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated by reference in its entirety. The ASCII copy, created on January 17, 2020, is named STB-015WO_SL.txt and is 83,131 bytes in size.

[0003] Technical Field The present disclosure relates generally to the field of protein engineering and methods for controlling protein function. Specifically, the disclosure relates to engineered fusion proteins that include a variant protease (e.g., HCV NS3 protease) fused to a polypeptide of interest and a cognate protease cleavage site, the cleavage of which can be inhibited with a protease inhibitor, such that one or more functions of the polypeptide of interest can be controlled. [Background technology]

[0004] background A technique for rapidly blocking the production and / or function of specific proteins in eukaryotes would be broadly useful as a research tool and for gene or cell therapy applications, but simple and effective methods have not yet been developed.

[0005] Control of protein production through transcriptional repression is slow at the outset, since existing mRNA molecules continue to be translated into proteins after transcriptional inhibition. RNA interference (RNAi) directly induces mRNA destruction, but RNAi is often only partially effective and can exhibit both sequence-independent and sequence-dependent off-target effects (Sigoillot et al. (2011) ACS Chem Biol 6:47-60 (Non-Patent Document 1)). Furthermore, mRNA and protein abundance are not always correlated due to regulation of the translation rate of specific mRNAs (Vogel et al. (2012) Nat Rev Genet 13:227-232 (Non-Patent Document 2), Wu et al. (2013) Nature 499:79-82 (Non-Patent Document 3), Battle et al. (2015) Science 347:664-667 (Non-Patent Document 4)). Finally, both transcriptional repression and RNAi require several days to be reversed (Liu et al. (2008) J Gene Med 10:583-592 (Non-Patent Document 5); Matsukura et al. (2003) Nucleic Acids Res 31:e77 (Non-Patent Document 6)).

[0006] Thus, there remains a need for easy-to-use systems for controlling protein production and function. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Sigoillot et al. (2011) ACS Chem Biol 6:47-60 [Non-Patent Document 2] Vogel et al. (2012) Nat Rev Genet 13:227-232 [Non-Patent Document 3] Wu et al.(2013)Nature 499:79-82 [Non-Patent Document 4] Battle et al.(2015)Science 347:664-667 [Non-Patent Document 5] Liu et al. (2008) J Gene Med 10:583-592 [Non-Patent Document 6] Matsukura et al. (2003) Nucleic Acids Res 31:e77 Summary of the Invention

[0008] overview To meet the above needs, the present disclosure relates to fusion constructs and methods of using them to control protein function and / or production. Specifically, the present disclosure provides engineered fusion proteins containing a variant protease (e.g., HCV NS3 protease) fused to a polypeptide of interest and a cognate protease cleavage site, the cleavage of which can be inhibited with a protease inhibitor, such that one or more functions of the polypeptide of interest are controllable.

[0009] Accordingly, certain aspects of the present disclosure provide a fusion protein having a polypeptide of interest, a variant Hepatitis C virus (HCV) nonstructural protein 3 (NS3) protease, and a cognate protease cleavage site, wherein the variant HCV NS3 protease comprises one or more mutations, which reduce immunogenicity when the fusion protein is expressed in a mammalian cell. In some embodiments, the HCV NS3 protease is derived from an HCV polyprotein comprising an amino acid sequence having at least about 80-100% sequence identity to SEQ ID NO:1 (including any percent identity within this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:1). In some embodiments, the variant NS3 protease is It is derived from HCV NS3 protease having the amino acid sequence of TIFF2025032195000001.tif34160.

[0010] In some embodiments that may be combined with any of the preceding embodiments, the one or more mutations comprise one or more amino acid substitutions. In some embodiments that may be combined with any of the preceding embodiments, the one or more amino acid substitutions correspond to amino acid substitutions in SEQ ID NO: 1. In some embodiments that may be combined with any of the preceding embodiments, the one or more amino acid substitutions are at one or more positions corresponding to positions 1038-1047 of SEQ ID NO: 1, positions 1057-1081 of SEQ ID NO: 1, positions 1073-1081 of SEQ ID NO: 1, positions 1073-1082 of SEQ ID NO: 1, positions 1127-1141 of SEQ ID NO: 1, positions 1131-1138 of SEQ ID NO: 1, positions 1169-1177 of SEQ ID NO: 1, and / or positions 1192-1206 of SEQ ID NO: 1. In some embodiments that may be combined with any of the preceding embodiments, the one or more amino acid substitutions are selected from a position corresponding to 1062 of SEQ ID NO:1, a position corresponding to 1069 of SEQ ID NO:1, a position corresponding to 1070 of SEQ ID NO:1, a position corresponding to 1071 of SEQ ID NO:1, a position corresponding to 1072 of SEQ ID NO:1, a position corresponding to 1074 of SEQ ID NO:1, a position corresponding to 1075 of SEQ ID NO:1, a position corresponding to 1077 of SEQ ID NO:1, a position corresponding to 1078 of SEQ ID NO:1, a position corresponding to 1079 of SEQ ID NO:1, a position corresponding to 1080 of SEQ ID NO:1, a position corresponding to 1031 of SEQ ID NO:1, a position corresponding to 1074 of SEQ ID NO:1, a position corresponding to 1132 of SEQ ID NO:1, a position corresponding to 1133 of SEQ ID NO:1, a position corresponding to 1195 of SEQ ID NO:1, a position corresponding to 1196 of SEQ ID NO:1, a position corresponding to 1201 of SEQ ID NO:1, a position corresponding to 1202 of SEQ ID NO:1, and any combination thereof.In some embodiments that may be combined with any of the preceding embodiments, the one or more amino acid substitutions are selected from an Ile to Leu substitution at a position corresponding to position 1074 of SEQ ID NO:1, an Ile to Met substitution at a position corresponding to position 1074 of SEQ ID NO:1, an Asn to Ala substitution at a position corresponding to position 1075 of SEQ ID NO:1, a Val to Ala substitution at a position corresponding to position 1077 of SEQ ID NO:1, a Cys to Phe substitution at a position corresponding to position 1078 of SEQ ID NO:1, a Trp to Ala substitution at a position corresponding to position 1079 of SEQ ID NO:1, a Thr to Ala substitution at a position corresponding to position 1080 of SEQ ID NO:1, a Val to Ala substitution at a position corresponding to position 1081 of SEQ ID NO:1, a Val to Asn substitution at a position corresponding to position 1081 of SEQ ID NO:1, and any combination thereof. In some embodiments that may be combined with any of the preceding embodiments, the one or more amino acid substitutions include a Thr to Ala substitution at a position corresponding to position 1080 of SEQ ID NO:1. In some embodiments that may be combined with any of the preceding embodiments, the one or more amino acid substitutions include a Thr to Ala substitution at a position corresponding to position 1080 of SEQ ID NO:1, and a Val to Ala substitution at a position corresponding to position 1077 of SEQ ID NO:1. In some embodiments that may be combined with any of the preceding embodiments, the one or more amino acid substitutions include a Thr to Ala substitution at a position corresponding to position 1080 of SEQ ID NO:1, and a Val to Ala substitution at a position corresponding to position 1081 of SEQ ID NO:1.

[0011] In some embodiments that may be combined with any of the preceding embodiments, the fusion protein further comprises an HCV NS4A cofactor. In some embodiments, the NS4A cofactor is It has the amino acid sequence of TIFF2025032195000002.tif4153.

[0012] In some embodiments that may be combined with any of the preceding embodiments, the fusion protein further comprises a degron, wherein the degron is operably linked to the polypeptide of interest. In some embodiments that may be combined with any of the preceding embodiments, the degron is selected from the group consisting of HCV NS4 degron, PEST (two copies of residues 277-307 of human IκBα) (SEQ ID NO: 46), GRR (residues 352-408 of human p105) (SEQ ID NO: 47), DRR (residues 210-295 of yeast Cdc34) (SEQ ID NO: 48), SNS (tandem repeats of SP2 and NB of influenza A or influenza B (SP2-NB-SP2 (SEQ ID NO: 49), RPB (four copies of residues 1688-1702 of yeast RPB) (SEQ ID NO: 50), SPmix (influenza A virus M2 The proteins SP1 and SP2 tandem repeats (SP2-SP1-SP2-SP1-SP2 (SEQ ID NO: 51), NS2 (three copies of residues 79-93 of influenza A virus NS protein) (SEQ ID NO: 52), ODC (residues 106-142 of ornithine decarboxylase) (SEQ ID NO: 53), Nek2A, mouse ODC (residues 422-461), mouse ODC_DA (residues 422-461 of mODC containing D433A and D434A point mutations) (SEQ ID NO: 54), the APC / C degron, COP1 The degron is selected from an E3 ligase-binding degron motif, a CRL4-Cdt2-binding PIP degron, an actinfilin-binding degron, a KEAP1-binding degron, a KLHL2- and KLHL3-binding degron, an MDM2-binding motif, an N-degron, a hydroxyproline modification in hypoxia signaling, a plant hormone-dependent SCF-LRR-binding degron, an SCF ubiquitin ligase-binding phosphodegron, a plant hormone-dependent SCF-LRR-binding degron, a DSGxxS (SEQ ID NO: 55) phospho-dependent degron, a Siah-binding motif, an SPOP SBC docking motif, and a PCNA-binding PIP box.

[0013] In some embodiments that may be combined with any of the preceding embodiments, the variant HCV NS3 protease comprises one or more additional mutations. In some embodiments that may be combined with any of the preceding embodiments, the one or more additional mutations modulate the enzymatic activity of the variant HCV NS3 protease. In some embodiments that may be combined with any of the preceding embodiments, the one or more additional mutations are one or more additional amino acid substitutions. In some embodiments that may be combined with any of the preceding embodiments, the one or more additional amino acid substitutions are at one or more positions corresponding to position 1074 of SEQ ID NO:1, position 1078 of SEQ ID NO:1, and / or position 1079 of SEQ ID NO:1. In some embodiments that may be combined with any of the preceding embodiments, the one or more additional amino acid substitutions are selected from an Ile to Ala substitution at a position corresponding to position 1074 of SEQ ID NO:1, a Trp to Ala substitution at a position corresponding to position 1079 of SEQ ID NO:1, and any combination thereof. In some embodiments that may be combined with any of the preceding embodiments, the one or more additional amino acid substitutions reduce enzymatic activity of the variant HCV NS3 protease. In some embodiments that may be combined with any of the preceding embodiments, the one or more additional amino acid substitutions include a Cys to Ala substitution at a position corresponding to position 1078 of SEQ ID NO: 1. In some embodiments that may be combined with any of the preceding embodiments, the one or more additional amino acid substitutions increase enzymatic activity of the variant HCV NS3 protease.

[0014] In some embodiments that may be combined with any of the preceding embodiments, the cognate protease cleavage site comprises an amino acid sequence selected from any of the amino acid sequences listed in Table 1. In some embodiments that may be combined with any of the preceding embodiments, the cognate protease cleavage site comprises In some embodiments that may be combined with any of the preceding embodiments, the cognate protease cleavage site comprises an amino acid sequence selected from: TIFF2025032195000004.tif12144. In some embodiments that may be combined with any of the preceding embodiments, the cognate protease cleavage site comprises one or more mutations. In some embodiments that may be combined with any of the preceding embodiments, the one or more mutations comprise one or more amino acid substitutions. In some embodiments that may be combined with any of the preceding embodiments, the one or more mutations increase the catalytic rate of cleavage. In some embodiments that may be combined with any of the preceding embodiments, the one or more mutations decrease the catalytic rate of cleavage.

[0015] In some embodiments that may be combined with any of the preceding embodiments, the polypeptide of interest is selected from a membrane protein, a receptor, a hormone, a cytokine, a transport protein, a transcription factor, a cytoskeletal protein, an extracellular matrix protein, a signal transduction protein, and an enzyme. In some embodiments that may be combined with any of the preceding embodiments, the polypeptide of interest comprises a biologically active domain of a protein. In some embodiments that may be combined with any of the preceding embodiments, the biologically active domain is a catalytic domain, a ligand binding domain, or a protein-protein interaction domain. In some embodiments that may be combined with any of the preceding embodiments, the polypeptide of interest is a receptor selected from a T cell receptor (TCR), a chimeric T cell receptor, an artificial T cell receptor, a synthetic T cell receptor, a chimeric immunoreceptor, an antibody-binding T cell receptor (ACTR), a T cell receptor fusion construct (TRUC), and a chimeric antigen receptor (CAR). In some embodiments that may be combined with any of the preceding embodiments, the polypeptide of interest is a chimeric antigen receptor (CAR). In some embodiments that may be combined with any of the preceding embodiments, the polypeptide of interest is a cytokine. In some embodiments that may be combined with any of the preceding embodiments, the cytokine is a proinflammatory cytokine. In some embodiments that may be combined with any of the preceding embodiments, the cognate protease cleavage site is located within a domain of the polypeptide of interest. In some embodiments that may be combined with any of the preceding embodiments, the polypeptide of interest comprises multiple domains. In some embodiments that may be combined with any of the preceding embodiments, the cognate protease cleavage site is located between multiple domains of the polypeptide of interest.

[0016] In some embodiments that may be combined with any of the preceding embodiments, the variant HCV NS3 protease may be inhibited by a protease inhibitor. In some embodiments that may be combined with any of the preceding embodiments, the protease inhibitor is selected from simeprevir, danoprevir, asunaprevir, cilprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and voxiloprevir. In some embodiments that may be combined with any of the preceding embodiments, the fusion protein further comprises a targeting sequence. In some embodiments that may be combined with any of the preceding embodiments, the targeting sequence is selected from a secretory protein signal sequence, a membrane protein signal sequence, a nuclear localization sequence, a nucleolar localization signal sequence, an endoplasmic reticulum localization sequence, a peroxisome localization sequence, a mitochondrial localization sequence, and a protein binding motif sequence.

[0017] Other aspects of the disclosure relate to a polynucleotide encoding the fusion protein of any of the preceding embodiments. Other aspects of the disclosure relate to a vector comprising the polynucleotide of any of the preceding embodiments. Other aspects of the disclosure relate to a cell comprising the fusion protein of any of the preceding embodiments, the polynucleotide of any of the preceding embodiments, or the vector of any of the preceding embodiments. In some embodiments that may be combined with any of the preceding embodiments, the cell is an immune cell or a cell line derived from an immune cell. In some embodiments that may be combined with any of the preceding embodiments, the immune cell is selected from a T cell, a B cell, a NK cell, a NKT cell, an innate lymphoid cell, a mast cell, an eosinophil, a basophil, a macrophage, a neutrophil, a dendritic cell, and any combination thereof. In some embodiments that may be combined with any of the preceding embodiments, the cell is a mesenchymal stromal cell. Other aspects of the disclosure relate to a pharmaceutical composition comprising the fusion protein of any of the preceding embodiments and an excipient. Other aspects of the disclosure relate to a pharmaceutical composition comprising the cell of any of the preceding embodiments and an excipient.

[0018] Another aspect of the present disclosure relates to a method of treating a subject in need thereof comprising administering a pharmaceutical composition of any of the preceding embodiments.

[0019] Another aspect of the present disclosure relates to a method of modulating the activity of a protein of interest, comprising: a) providing a population of cells comprising a fusion protein of any of the preceding embodiments, a polynucleotide of any of the preceding embodiments, or a vector of any of the preceding embodiments; and b) contacting the population of cells with a protease inhibitor. In some embodiments that may be combined with any of the preceding embodiments, the method further comprises removing the protease inhibitor from the population of cells. In some embodiments that may be combined with any of the preceding embodiments, the method further comprises administering the population of cells to a subject in need of a cell-based therapy.

[0020] Another aspect of the present disclosure relates to a method of treating a subject in need of a cell-based therapy, comprising administering to the subject a population of cells comprising a fusion protein of any of the preceding embodiments, a polynucleotide of any of the preceding embodiments, or a vector of any of the preceding embodiments. In some embodiments that may be combined with any of the preceding embodiments, the population of cells has been cultured in the presence of a protease inhibitor capable of inhibiting the inhibitory protease. In some embodiments that may be combined with any of the preceding embodiments, the population of cells has been cultured in the absence of a protease inhibitor capable of inhibiting the inhibitory protease. In some embodiments that may be combined with any of the preceding embodiments, the method further comprises administering to the subject a protease inhibitor capable of inhibiting the inhibitory protease. In some embodiments that may be combined with any of the preceding embodiments, the method further comprises discontinuing the use of a protease inhibitor capable of inhibiting the inhibitory protease in the subject.

[0021] In another aspect, the disclosure includes a fusion protein comprising: a) a polypeptide of interest; b) a degron, the degron operably linked to the polypeptide of interest when the fusion protein is in an uncleaved state, such that the fusion protein promotes degradation of the polypeptide of interest in a cell; and c) a variant protease, the variant protease that can be inhibited by contacting the fusion protein with a protease inhibitor; and c) a cleavable linker located between the polypeptide of interest and the degron, the variant protease comprising a cognate cleavage site recognized by the protease, such that cleavage of the cleavable linker by the protease releases the polypeptide of interest from the fusion protein, such that the degron no longer controls degradation of the polypeptide of interest when the fusion protein is in a cleaved state.

[0022] In some embodiments, the degron may be linked to the C-terminus of a polypeptide of interest in a fusion protein. In certain embodiments, the fusion protein comprises the following components: a) the polypeptide of interest, b) a cleavable linker, c) a variant protease, and d) a degron arranged N-terminal to C-terminal in an uncleaved state.

[0023] Alternatively, the degron may be linked to the N-terminus of the polypeptide of interest in a fusion protein. In certain embodiments, the fusion protein comprises the following components arranged N-terminus to C-terminus in an uncleaved state: a) a variant protease, b) a degron, c) a cleavable linker, and d) a polypeptide of interest. Exemplary targeting sequences include secretory protein signal sequences, membrane protein signal sequences, nuclear localization sequences, nucleolar localization signal sequences, endoplasmic reticulum localization sequences, peroxisome localization sequences, mitochondrial localization sequences, and protein binding motif sequences.

[0024] In certain embodiments, the fusion protein further comprises a tag. Exemplary tags include a His tag, a Strep tag, a TAP tag, an S tag, an SBP tag, an Arg tag, a calmodulin-binding peptide tag, a cellulose-binding domain tag, a DsbA tag, a c-myc tag, a glutathione S-transferase tag, a FLAG tag, a HAT tag, a maltose-binding protein tag, a NusA tag, and a thioredoxin tag.

[0025] In certain embodiments, the fusion protein further comprises a detectable label. The detectable label can include any molecule that can be detected. For example, the detectable label can be a fluorescent label, a bioluminescent label, a chemiluminescent label, a colorimetric label, or an isotopic label. In certain embodiments, the detectable label is a fluorescent protein or a bioluminescent protein.

[0026] In certain embodiments, the polypeptide of interest in the fusion protein is a membrane protein, a receptor, a hormone, a transport protein, a transcription factor, a cytoskeletal protein, an extracellular matrix protein, a signal transduction protein, an enzyme, or any other protein of interest. The polypeptide of interest may include an entire protein, or a biologically active domain (e.g., a catalytic domain, a ligand binding domain, or a protein-protein interaction domain), or a polypeptide fragment of the selected protein of interest.

[0027] In another aspect, the disclosure includes a polynucleotide encoding the fusion protein described herein. In one embodiment, the polynucleotide is a recombinant polynucleotide comprising a polynucleotide encoding a fusion protein operably linked to a promoter. The recombinant polynucleotide may comprise an expression vector, for example, a bacterial plasmid vector or a viral expression vector. Exemplary viral vectors include measles virus, vesicular stomatitis virus, adenovirus, retrovirus (e.g., gamma-retrovirus and lentivirus), poxvirus, adeno-associated virus, baculovirus, or herpes simplex virus vectors.

[0028] In another aspect, the disclosure includes a host cell comprising a recombinant polynucleotide encoding a fusion protein operably linked to a promoter. In one embodiment, the host cell is a eukaryotic cell. In another embodiment, the host cell is a mammalian cell. In certain embodiments, the host cell is a stem cell (e.g., an embryonic stem cell or an adult stem cell). The host cell can be cultured as a single cell or a multicellular entity (e.g., a tissue, an organ, or an organoid comprising the recombinant vector). The promoter can be an endogenous or exogenous promoter. In certain embodiments, the recombinant polynucleotide encoding the fusion protein is present in an extrachromosomal plasmid or vector. In other embodiments, the recombinant polynucleotide encoding the fusion protein is integrated into the cell genome. For example, the recombinant polynucleotide can be integrated into the cell genome at a position where the polynucleotide sequence encoding the fusion protein is operably linked to an endogenous promoter of a gene. In another embodiment, the disclosure includes the progeny of the host cell, wherein the progeny inherits the recombinant polynucleotide encoding the fusion protein.

[0029] In another embodiment, the present disclosure includes an organoid comprising a recombinant polynucleotide encoding a fusion protein operably linked to a promoter. The promoter can be an endogenous or exogenous promoter. In certain embodiments, the recombinant polynucleotide encoding the fusion protein is present in an extrachromosomal plasmid or vector. In other embodiments, the recombinant polynucleotide encoding the fusion protein is integrated into the organoid genome. For example, the recombinant polynucleotide can be integrated into the organoid genome at a position where the polynucleotide sequence encoding the fusion protein is operably linked to the endogenous promoter of a gene. In another embodiment, the present disclosure includes a recombinant animal comprising a recombinant polynucleotide encoding a fusion protein operably linked to a promoter. The promoter can be an endogenous or exogenous promoter. In certain embodiments, the recombinant polynucleotide encoding the fusion protein is present in an extrachromosomal plasmid or vector. In other embodiments, the recombinant polynucleotide encoding the fusion protein is integrated into the genome of a recombinant animal. For example, the recombinant polynucleotide can be integrated into the genome at a position where the polynucleotide sequence encoding the fusion protein is operably linked to the endogenous promoter of a gene. In another embodiment, the disclosure includes progeny of the recombinant animal, wherein the progeny inherit the recombinant polynucleotide encoding the fusion protein.

[0030] In another aspect, the disclosure includes a method for producing a fusion protein, the method comprising transforming a host cell with a recombinant polynucleotide encoding the fusion protein operably linked to a promoter, culturing the transformed host cell under conditions in which the fusion protein is expressed, and isolating the fusion protein from the host cell.

[0031] In another aspect, the disclosure includes a method for controlling production of a polypeptide of interest, the method comprising: a) transforming a host cell with a recombinant polynucleotide encoding a fusion protein described herein, b) culturing the transformed host cell under conditions in which the fusion protein is expressed, and c) contacting the cell with a protease inhibitor that inhibits a protease of the fusion protein when production of the polypeptide of interest is no longer desired. The protease inhibitor can be removed when it is desired to resume production of the polypeptide of interest.

[0032] The recombinant polynucleotide encoding the fusion protein can preferably provide efficient production of a polypeptide of interest with biological activity comparable to that of the wild-type polypeptide. Additionally, production of the polypeptide of interest from the recombinant polynucleotide can preferably be rapidly and almost completely inhibited in the presence of a protease inhibitor. For example, a protease inhibitor can reduce production of the polypeptide of interest by at least 80%, 90%, or 100%, or any amount therebetween, compared to the level of the polypeptide in the absence of the protease inhibitor. In certain embodiments, production of the polypeptide of interest by the recombinant polynucleotide in a host cell in the presence of a protease inhibitor is inhibited by at least about 90% to 100%, including any percent identity within this range, such as 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%.

[0033] In certain embodiments, the fusion protein used to control the production of the polypeptide of interest comprises HCV NS3 protease.NS3 protease inhibitors that can be used in the practice of the present disclosure include, but are not limited to, simeprevir, danoprevir, asunaprevir, cilprevir, boceprevir, sovaprevir, paritaprevir, and telaprevir.

[0034] In another aspect, the disclosure includes a method for controlling the production of a polypeptide of interest in a subject, the method comprising: a) administering to the subject a recombinant polynucleotide encoding a fusion protein such that the fusion protein is expressed in the subject; and b) administering to the subject a protease inhibitor that inhibits the protease of the fusion protein when production of the polypeptide of interest is undesirable. The method may further comprise ceasing administration of the protease inhibitor when it is desired to resume production of the polypeptide of interest in the subject. The recombinant polynucleotide may comprise an expression vector, for example, a viral expression vector, such as, but not limited to, an adenovirus, a retrovirus (e.g., gamma-retrovirus and lentivirus), a poxvirus, an adeno-associated virus, a baculovirus, or a herpes simplex virus vector. In one embodiment, the recombinant polynucleotide comprises a polynucleotide sequence encoding a fusion protein operably linked to an exogenous promoter. In another embodiment, the recombinant polynucleotide is integrated into the genome of the subject. For example, the recombinant polynucleotide can be integrated into the genome at a position where the polynucleotide sequence encoding the fusion protein is operably linked to an endogenous promoter of a gene in the subject.

[0035] In another aspect, the present disclosure includes a method for controlling the production of a polypeptide of interest in a recombinant animal, the method comprises: a) administering a recombinant polynucleotide encoding a fusion protein to the recombinant animal, so that the fusion protein is expressed in the recombinant animal; and b) administering a protease inhibitor to the recombinant animal, which inhibits the protease of the fusion protein, when the production of the polypeptide of interest is not desired.In another aspect, the present disclosure includes a method for controlling the production of a polypeptide of interest in an organoid, the method comprises: a) introducing a recombinant polynucleotide encoding a fusion protein according to claim 4 into the organoid; b) culturing the organoid under the condition that the fusion protein is produced in the organoid; and c) contacting the organoid with a protease inhibitor, which inhibits the protease of the fusion protein, when the production of the polypeptide of interest is no longer desired.

[0036] In another aspect, the present disclosure includes a method for measuring the turnover of a polypeptide of interest, the method comprising: a) introducing into a cell a recombinant polynucleotide encoding a fusion protein described herein; b) measuring the amount of the polypeptide of interest in the cell before and after contacting the cell with a protease inhibitor that inhibits the protease of the fusion protein; and c) calculating the turnover of the polypeptide of interest based on the amount of the polypeptide of interest in the cell before and after adding the protease inhibitor. Additionally, the half-life of the polypeptide of interest in the cell can be calculated. The amount of the polypeptide of interest in the cell can be measured either continuously or periodically over a period of time.

[0037] In another aspect, the disclosure includes a conditionally replicating viral vector comprising a modified genome of a virus such that production of a polypeptide required for efficient viral replication is controllable, the viral vector comprising a nucleic acid encoding a fusion protein comprising: i) a polypeptide required for efficient viral replication; ii) a degron, operably linked to the polypeptide required for efficient viral replication when the fusion protein is in an uncleaved state, such that the degron promotes degradation of the polypeptide within the cell; iii) a protease, which can be inhibited by contacting the fusion protein with a protease inhibitor; and iv) a cleavable linker located between the polypeptide required for efficient viral replication and the degron, which comprises a cleavage site recognized by the protease, such that cleavage of the cleavable linker by the protease releases the polypeptide required for efficient viral replication from the fusion protein, such that when the fusion protein is in a cleaved state, the degron no longer controls degradation of the polypeptide required for efficient viral replication. In certain embodiments, the virus is an RNA virus (e.g., measles virus or vesicular stomatitis virus). In another embodiment, the conditionally replicating viral vector is a plasmid. The viral vector may further comprise a multiple cloning site, a transcription promoter, a transcription enhancer element, a transcription termination signal, a polyadenylation sequence, or an exogenous nucleic acid, or any combination thereof.

[0038] In another aspect, the disclosure includes a method of controlling viral production, the method comprising: a) introducing into a host cell a conditionally replicating viral vector as described herein, b) culturing the host cell under conditions suitable for viral production, and c) contacting the host cell with a protease inhibitor when viral production is no longer desired, such that polypeptides required for efficient viral replication are degraded. The protease inhibitor can be removed when it is desired to resume viral production.

[0039] Conditionally replicating viral vectors can preferably provide efficient production of virus in host cells in the absence of protease inhibitors, which is comparable to the level of virus produced by wild-type viral genome.In certain embodiments, the level of virus produced by conditionally replicating viral vectors in the absence of protease inhibitors is at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, or any amount therebetween, compared to the level of virus produced by wild-type viral genome.

[0040] Additionally, the production of virus from the conditionally replicating viral vector can preferably be almost completely suppressed in the presence of a protease inhibitor.For example, the protease inhibitor can reduce the production of virus by 80%, 90%, or 100%, or any amount therebetween, compared to the level of virus in the absence of the protease inhibitor.In certain embodiments, the production of virus by the conditionally replicating viral vector in host cells in the presence of a protease inhibitor is suppressed at least about 90% to 100%, including any percent identity within this range, such as 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%.

[0041] In certain embodiments, the conditionally replicating viral vector used to control virus production expresses a fusion protein containing HCV NS3 protease, and the addition of NS3 protease inhibitors can be used to suppress virus production.The NS3 protease inhibitors that can be used include, but are not limited to, simeprevir, danoprevir, asunaprevir, cilprevir, boceprevir, sovaprevir, paritaprevir, and telaprevir.

[0042] In another aspect, the disclosure includes a recombinant virion comprising a conditionally replicating viral vector described herein.

[0043] In another aspect, the present disclosure includes a kit for preparing or using fusion protein according to the method described herein.Such a kit may include one or more fusion proteins, nucleic acids encoding such fusion proteins, expression vectors, conditionally replicating viral vectors, cells, or other reagents for preparing or using fusion proteins as described herein.The kit may further include a protease inhibitor, such as an HCV NS3 protease inhibitor, including, for example, simeprevir, danoprevir, asunaprevir, silprevir, boceprevir, sovaprevir, paritaprevir, or telaprevir.

[0044] [The present invention 1001] A fusion protein comprising: A polypeptide of interest; a variant hepatitis C virus (HCV) nonstructural protein 3 (NS3) protease; Cognate protease cleavage sites and Including, the variant HCV NS3 protease comprises one or more mutations, the one or more mutations reduce immunogenicity of the fusion protein when expressed in a mammalian cell. The fusion protein. [The present invention 1002] The fusion protein of the present invention, wherein said variant HCV NS3 protease is derived from an HCV polyprotein comprising the amino acid sequence of SEQ ID NO:1. [The present invention 1003] The fusion protein of invention 1001 or 1002, wherein said one or more mutations comprise one or more amino acid substitutions. [The present invention 1004] The fusion protein of the invention 1003, wherein said one or more amino acid substitutions correspond to amino acid substitutions in SEQ ID NO:1. [The present invention 1005] The fusion protein of the present invention 1004, wherein the one or more amino acid substitutions are at one or more positions corresponding to positions 1038 to 1047 of SEQ ID NO:1, positions 1057 to 1081 of SEQ ID NO:1, positions 1073 to 1081 of SEQ ID NO:1, positions 1073 to 1082 of SEQ ID NO:1, positions 1127 to 1141 of SEQ ID NO:1, positions 1131 to 1138 of SEQ ID NO:1, positions 1169 to 1177 of SEQ ID NO:1, and / or positions 1192 to 1206 of SEQ ID NO:1. [The present invention 1006] The fusion protein of the present invention, wherein the one or more amino acid substitutions are selected from the group consisting of a position corresponding to position 1062 of SEQ ID NO:1, a position corresponding to position 1069 of SEQ ID NO:1, a position corresponding to position 1070 of SEQ ID NO:1, a position corresponding to position 1071 of SEQ ID NO:1, a position corresponding to position 1072 of SEQ ID NO:1, a position corresponding to position 1074 of SEQ ID NO:1, a position corresponding to position 1075 of SEQ ID NO:1, a position corresponding to position 1077 of SEQ ID NO:1, a position corresponding to position 1078 of SEQ ID NO:1, a position corresponding to position 1079 of SEQ ID NO:1, a position corresponding to position 1080 of SEQ ID NO:1, a position corresponding to position 1031 of SEQ ID NO:1, a position corresponding to position 1132 of SEQ ID NO:1, a position corresponding to position 1133 of SEQ ID NO:1, a position corresponding to position 1195 of SEQ ID NO:1, a position corresponding to position 1196 of SEQ ID NO:1, a position corresponding to position 1201 of SEQ ID NO:1, a position corresponding to position 1202 of SEQ ID NO:1, and any combination thereof. [The present invention 1007] The fusion protein of the present invention 1005, wherein the one or more amino acid substitutions are selected from the group consisting of a substitution of Ile to Leu at a position corresponding to position 1074 of SEQ ID NO:1, a substitution of Ile to Met at a position corresponding to position 1074 of SEQ ID NO:1, a substitution of Asn to Ala at a position corresponding to position 1075 of SEQ ID NO:1, a substitution of Val to Ala at a position corresponding to position 1077 of SEQ ID NO:1, a substitution of Cys to Phe at a position corresponding to position 1078 of SEQ ID NO:1, a substitution of Trp to Ala at a position corresponding to position 1079 of SEQ ID NO:1, a substitution of Thr to Ala at a position corresponding to position 1080 of SEQ ID NO:1, a substitution of Val to Ala at a position corresponding to position 1081 of SEQ ID NO:1, a substitution of Val to Asn at a position corresponding to position 1081 of SEQ ID NO:1, and any combination thereof. [The present invention 1008] The fusion protein of the present invention 1005, wherein said one or more amino acid substitutions comprises a Thr to Ala substitution at a position corresponding to position 1080 of SEQ ID NO:1. [The present invention 1009] The fusion protein of the present invention 1005, wherein the one or more amino acid substitutions include a substitution of Thr to Ala at a position corresponding to position 1080 of SEQ ID NO:1, and a substitution of Val to Ala at a position corresponding to position 1077 of SEQ ID NO:1. [The present invention 1010] The fusion protein of the present invention 1005, wherein the one or more amino acid substitutions include a substitution of Thr to Ala at a position corresponding to position 1080 of SEQ ID NO:1, and a substitution of Val to Ala at a position corresponding to position 1081 of SEQ ID NO:1. [The present invention 1011] The fusion protein of any one of claims 1001 to 1010, further comprising an HCV NS4A cofactor. [The present invention 1012] The fusion protein of any of claims 1001 to 1011, further comprising a degron, said degron being operably linked to said polypeptide of interest. [The present invention 1013] The degron may be an HCV NS4 degron, PEST (two copies of residues 277-307 of human IκBα) (SEQ ID NO: 46), GRR (residues 352-408 of human p105) (SEQ ID NO: 47), DRR (residues 210-295 of yeast Cdc34) (SEQ ID NO: 48), SNS (tandem repeats of SP2 and NB of influenza A or influenza B (SP2-NB-SP2) (SEQ ID NO: 49), RPB (four copies of residues 1688-1702 of yeast RPB) (SEQ ID NO: 50), SPmix (influenza A virus M2 tandem repeats of proteins SP1 and SP2 (SP2-SP1-SP2-SP1-SP2) (SEQ ID NO: 51), NS2 (three copies of residues 79-93 of influenza A virus NS protein) (SEQ ID NO: 52), ODC (residues 106-142 of ornithine decarboxylase) (SEQ ID NO: 53), Nek2A, mouse ODC (residues 422-461), mouse ODC_DA (residues 422-461 of mODC containing D433A and D434A point mutations) (SEQ ID NO: 54), APC / C degron, COP1 The fusion protein of the present invention 1012 is selected from the group consisting of E3 ligase binding degron motif, CRL4-Cdt2 binding PIP degron, actinfilin binding degron, KEAP1 binding degron, KLHL2 and KLHL3 binding degron, MDM2 binding motif, N-degron, hydroxyproline modification in hypoxia signaling, plant hormone dependent SCF-LRR binding degron, SCF ubiquitin ligase binding phosphodegron, plant hormone dependent SCF-LRR binding degron, DSGxxS (SEQ ID NO: 55) phospho-dependent degron, Siah binding motif, SPOP SBC docking motif, and PCNA binding PIP box. [The present invention 1014] The fusion protein of any of claims 1001 to 1013, wherein the variant HCV NS3 protease comprises one or more additional mutations. [The present invention 1015] The fusion protein of the invention, wherein said one or more additional mutations modulate the enzymatic activity of said variant HCV NS3 protease. [The present invention 1016] The fusion protein of the present invention 1014 or 1015, wherein said one or more additional mutations are one or more additional amino acid substitutions. [The present invention 1017] The fusion protein of the present invention 1016, wherein the one or more additional amino acid substitutions are at one or more positions corresponding to position 1074 of SEQ ID NO:1, position 1078 of SEQ ID NO:1, and / or position 1079 of SEQ ID NO:1. [The present invention 1018] The fusion protein of the present invention, wherein the one or more additional amino acid substitutions are selected from the group consisting of an Ile to Ala substitution at a position corresponding to position 1074 of SEQ ID NO:1, a Trp to Ala substitution at a position corresponding to position 1079 of SEQ ID NO:1, and any combination thereof. [The present invention 1019] The fusion protein of the invention, wherein said one or more additional amino acid substitutions reduce the enzymatic activity of said variant HCV NS3 protease. [The present invention 1020] The fusion protein of the present invention, wherein the one or more additional amino acid substitutions comprise a Cys to Ala substitution at a position corresponding to position 1078 of SEQ ID NO:1. [The present invention 1021] The fusion protein of the invention 1020, wherein said one or more additional amino acid substitutions increase the enzymatic activity of said variant HCV NS3 protease. [The present invention 1022] The fusion protein of any of claims 1001 to 1021, wherein said cognate protease cleavage site comprises an amino acid sequence selected from the group consisting of any of the amino acid sequences listed in Table 1. [The present invention 1023] The cognate protease cleavage site is A fusion protein according to any one of claims 1001 to 1021, comprising an amino acid sequence selected from the group consisting of TIFF2025032195000005.tif19140. [The present invention 1024] The cognate protease cleavage site is A fusion protein according to any one of claims 1001 to 1021, comprising an amino acid sequence selected from the group consisting of TIFF2025032195000006.tif12144. [The present invention 1025] The fusion protein of any of claims 1022 to 1024, wherein said cognate protease cleavage site comprises one or more mutations. [The present invention 1026] The fusion protein of the invention 1025, wherein said one or more mutations comprise one or more amino acid substitutions. [The present invention 1027] The fusion protein of the invention 1025 or 1026, wherein said one or more mutations increase the catalytic rate of cleavage. [The present invention 1028] The fusion protein of invention 1025 or invention 1026, wherein said one or more mutations reduce the catalytic rate of cleavage. [The present invention 1029] The fusion protein of any of claims 1001 to 1028, wherein the target polypeptide is selected from the group consisting of membrane proteins, receptors, hormones, cytokines, transport proteins, transcription factors, cytoskeletal proteins, extracellular matrix proteins, signal transduction proteins, and enzymes. [The present invention 1030] The fusion protein of any of claims 1001 to 1028, wherein the target polypeptide comprises a biologically active domain of a protein. [The present invention 1031] The fusion protein of the present invention 1030, wherein said biologically active domain is a catalytic domain, a ligand binding domain, or a protein-protein interaction domain. [The present invention 1032] The fusion protein of any of claims 1001 to 1031, wherein the target polypeptide is a receptor selected from the group consisting of a T cell receptor (TCR), a chimeric T cell receptor, an artificial T cell receptor, a synthetic T cell receptor, a chimeric immunoreceptor, an antibody-binding T cell receptor (ACTR), a T cell receptor fusion construct (TRUC), and a chimeric antigen receptor (CAR). [The present invention 1033] The fusion protein of any of claims 1001 to 1031, wherein the target polypeptide is a chimeric antigen receptor (CAR). [The present invention 1034] The fusion protein of any one of claims 1001 to 1028, wherein the target polypeptide is a cytokine. [The present invention 1035] The fusion protein of the present invention, wherein said cytokine is a proinflammatory cytokine. [The present invention 1036] The fusion protein of any of claims 1001 to 1035, wherein said cognate protease cleavage site is localized within a domain of said polypeptide of interest. [The present invention 1037] The fusion protein of any one of claims 1001 to 1035, wherein the target polypeptide comprises multiple domains. [The present invention 1038] The fusion protein of the present invention, wherein said cognate protease cleavage site is located between said domains of said polypeptide of interest. [The present invention 1039] The fusion protein of any of claims 1001 to 1038, wherein the variant HCV NS3 protease can be inhibited by a protease inhibitor. [The present invention 1040] The fusion protein of the present invention, wherein the protease inhibitor is selected from the group consisting of simeprevir, danoprevir, asunaprevir, cilprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and voxiloprevir. [The present invention 1041] The fusion protein of any one of claims 1001 to 1040, further comprising a targeting sequence. [The present invention 1042] The fusion protein of the present invention 1041, wherein the targeting sequence is selected from the group consisting of a secretory protein signal sequence, a membrane protein signal sequence, a nuclear localization sequence, a nucleolar localization signal sequence, an endoplasmic reticulum localization sequence, a peroxisome localization sequence, a mitochondrial localization sequence, and a protein binding motif sequence. [The present invention 1043] The fusion protein of any one of claims 1001 to 1042, wherein the variant NS3 protease is derived from an HCV NS3 protease having the amino acid sequence of SEQ ID NO:2. [The present invention 1044] A polynucleotide encoding any one of the fusion proteins of the present invention Nos. 1001 to 1043. [The present invention 1045] A vector comprising the polynucleotide of the present invention. [The present invention 1046] A cell comprising the fusion protein of any one of the present inventions 1001 to 1043, the polynucleotide of the present invention 1044, or the vector of the present invention 1045. [The present invention 1047] The cell of the present invention 1046, which is an immune cell or a cell line derived from an immune cell. [The present invention 1048] The cell of the present invention 1047, wherein the immune cell is selected from the group consisting of T cells, B cells, NK cells, NKT cells, innate lymphoid cells, mast cells, eosinophils, basophils, macrophages, neutrophils, dendritic cells, and any combination thereof. [The present invention 1049] The cell of the present invention 1046, which is a mesenchymal stromal cell. [The present invention 1050] A pharmaceutical composition comprising any one of the fusion proteins of the present inventions 1001 to 1043 and an excipient. [The present invention 1051] A pharmaceutical composition comprising the cells of any one of the present inventions 1046 to 1049 and an excipient. [The present invention 1052] A method of treating a subject in need of treatment comprising administering a pharmaceutical composition of the present invention 1050 or the present invention 1051. [The present invention 1053] 1. A method for modulating the activity of a protein of interest, comprising: a) providing a population of cells comprising a fusion protein of any one of claims 1001 to 1043, a polynucleotide of claim 1044, or a vector of claim 1045; b) contacting the population of cells with a protease inhibitor; The method comprising: [The present invention 1054] The method of claim 1053, further comprising the step of removing said protease inhibitor from said population of cells. [The present invention 1055] The method of claim 1053 or 1054, further comprising administering said population of cells to a subject in need of a cell-based therapy. [The present invention 1056] A method for treating a subject in need of cell-based therapy, comprising administering to the subject a population of cells comprising any of the fusion proteins of the present inventions 1001 to 1043, the polynucleotide of the present invention 1044, or the vector of the present invention 1045. [The present invention 1057] The method of claim 1056, wherein said population of cells is cultured in the presence of a protease inhibitor capable of inhibiting an inhibitory protease. [The present invention 1058] The method of claim 1056, wherein said population of cells is cultured in the absence of a protease inhibitor capable of inhibiting an inhibitory protease. [The present invention 1059] The method according to any one of claims 1056 to 1058, further comprising the step of administering to said subject said protease inhibitor capable of inhibiting an inhibitory protease. [The present invention 1060] The method of claim 1059, further comprising the step of discontinuing the use of said protease inhibitor capable of inhibiting an inhibitory protease in said subject. These and other embodiments of the present disclosure will readily occur to those of ordinary skill in the art in light of the disclosure herein.

[0045] These and other features, aspects, and advantages of the present disclosure will become better understood with regard to the following description and accompanying drawings. [Brief description of the drawings]

[0046] [Figure 1] Shown is the normalized CAR expression percentage in cells transfected to express one of four different fusion proteins. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] Detailed Description The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of molecular biology, chemistry, biochemistry, virology, and immunology within the skill of the art. Such techniques are explained fully in the literature. See, for example, Hepatitis C Viruses: Genomes and Molecular Biology (SLTan ed., Taylor & Francis, 2006), Fundamental Virology, 3 rd Edition,vol.I&II(BNFields and DMKnipe,eds.), Handbook of Experimental Immunology,Vols.I-IV(DMWeir and CCBlackwell eds.,Blackwell Scientific Publications), ALLehninger,Biochemistry(Worth Publishers,Inc.,current addition), Sambrook,et al.,Molecular Cloning:A Laboratory Manual(3 rd Edition, 2001), and Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.).

[0048] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0049] definition In describing this disclosure, the following terms will be employed and are intended to be defined as follows:

[0050] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a "fusion protein" includes a mixture of two or more fusion proteins, and the like.

[0051] The term "about" is meant to encompass a deviation of plus or minus 5 percent, particularly in relation to a given amount.

[0052] The term "protease" as used herein refers to a protease that can be inactivated by the presence or absence of a specific agent (e.g., that binds to the protease). In some embodiments, the protease is active (cleaves the cognate cleavage site) in the absence of the specific agent and is inactive (does not cleave the cognate cleavage site) in the presence of the specific agent. In some embodiments, the specific agent is a protease inhibitor. In some embodiments, the protease inhibitor specifically inhibits a given protease of the disclosure.

[0053] Non-limiting examples of proteases include Hepatitis C virus proteases (e.g., NS3 and NS2-3), signal peptidases, proprotein convertases of the subtilisin / kexin family (furin, PCI, PC2, PC4, PACE4, PC5, PC), proprotein convertases that cleave at hydrophobic residues (e.g., Leu, Phe, Val, or Met), proprotein convertases that cleave at small amino acid residues such as Ala or Thr, proopiomelanocortin convertase (PCE), chromaffin granule acetylcholinesterase (CHA), and chromaffin granule acetylcholinesterase (CGA). These include glutamic acid proteases (CGAP), prohormone thiol proteases, carboxypeptidases (e.g., carboxypeptidase E / H, carboxypeptidase D, and carboxypeptidase Z), aminopeptidases (e.g., arginine aminopeptidase, lysine aminopeptidase, aminopeptidase B), prolyl endopeptidase, aminopeptidase N, insulin-degrading enzyme, calpain, high molecular weight proteases, and caspases 1, 2, 3, 4, 5, 6, 7, 8, and 9.Other proteases include aminopeptidase N, puromycin-sensitive aminopeptidase, angiotensin-converting enzyme, pyroglutamyl peptidase II, dipeptidyl peptidase IV, N-arginine dibasic convertase, endopeptidase 24.15, endopeptidase 24.16, amyloid precursor protein secretase alpha, beta, and gamma, angiotensin-converting enzyme secretase, TGF alpha secretase, TF alpha secretase, FAS ligand secretase, TNF receptor-I and -II secretase, CD30 secretase, KL1 and KL These include, but are not limited to, 2 secretase, IL6 receptor secretase, CD43, CD44 secretase, CD16-1 and CD16-11 secretase, L-selectin secretase, folate receptor secretase, MMP1, 2, 3, 7, 8, 9, 10, 11, 12, 13, 14, and 15, urokinase plasminogen activator, tissue plasminogen activator, plasmin, thrombin, BMP-1 (procollagen C-peptidase), ADAM1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11, and granzymes A, B, C, D, E, F, G, and H.For a discussion of proteases, see, for example, VYH Hook, Proteolytic and cellular mechanisms in prohormone and proprotein processing, RG Landes Company, Austin, Tex., USA (1998), NM Hooper et al., Biochem. J. 321: 265-279 (1997), Z. Werb, Cell 91: 439-442 (1997), TG Wolfsberg et al., J. Cell Biol. 131: 275-278 (1995), K. Murakami and JD Atlinger, Biochem. Biophys. Res. Comm. 146: 1249-1259 (1987), T. Berg et al., Biochem. J. 307: 313-326 (1995), and MJ Myth and JA Trapani, Immunology Today 16:202-206 (1995), RV Talanian et al., J. Biol. Chem. 272:9677-9682 (1997), and NA Thomberry et al., J. Biol. Chem. 272:17907-17911 (1997), the disclosures of which are incorporated herein.

[0054] "Nonstructural protein 3 (NS3)" nucleic acid, oligonucleotide, protein, polypeptide, or peptide refers to a molecule derived from Hepatitis C virus (HCV), including any isolate of HCV having any genotype (e.g., seven genotypes 1-7) or subtype. The molecule need not be physically derived from HCV, but may be synthetically or recombinantly produced. Many NS3 nucleic acid and protein sequences are known. Representative sequences are listed in the National Center for Biotechnology Information (NCBI) database. For example, NCBI entries: Accession numbers: YP_001491553, YP_001469631, YP_001469632, NP_803144, NP_671491, YP_001469634, YP_001469630, YP_001469633, ADA68311, ADA68307, AFP99000, AFP98987, ADA68322, AFP99033, ADA68330, AFP99056, AFP99041, CBF60982, CBF60817, See AHH29575, AIZ00747, AIZ00744, ABI36969, ABN05226, KF516075, KF516074, KF516056, AB826684, AB826683, JX171009, JX171008, JX171000, EU847455, EF154714, GU085487, JX171065, JX171063, all of which sequences (as entered by the filing date of this application) are incorporated by reference herein. Any of these sequences, or variants thereof, including sequences having at least about 80-100% sequence identity thereto (including any percent identity within this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto), can be used to construct fusion proteins as described herein or recombinant polynucleotides encoding such fusion proteins.

[0055] "Nonstructural protein 4A (NS4A)" nucleic acid, oligonucleotide, protein, polypeptide, or peptide refers to a molecule derived from HCV, including any isolate of HCV having any genotype (e.g., seven genotypes 1-7) or subtype. The molecule need not be physically derived from HCV, but may be synthetically or recombinantly produced. Many NS4A nucleic acid and protein sequences are known. Representative sequences are listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries: Accession Nos. NP_751925, YP_001491554, GU945462, HQ822054, FJ932208, FJ932207, FJ932205, and FJ932199, all of which sequences (as entered by the filing date of this application) are incorporated herein by reference. Any of these sequences, or variants thereof, including sequences having at least about 80-100% sequence identity thereto (including any percent identity within this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto), can be used to construct fusion proteins as described herein or recombinant polynucleotides encoding such fusion proteins.

[0056] A "polyprotein" nucleic acid, oligonucleotide, protein, polypeptide, or peptide refers to a molecule derived from HCV, including any isolate of HCV having any genotype (e.g., the seven genotypes 1-7) or subtype. The molecules need not be physically derived from HCV, but may be synthetically or recombinantly produced. Many polyprotein nucleic acid and protein sequences are known. Representative HCV polyprotein sequences are listed in the National Center for Biotechnology Information (NCBI) database. See, e.g., NCBI entries: Accession Nos. YP_001469631, NP_671491, YP_001469633, YP_001469630, YP_001469634, YP_001469632, NC_009824, NC_004102, NC_009825, NC_009827, NC_009823, NC_009826, and EF108306, all of which sequences (as entered by the filing date of this application) are incorporated by reference herein. Any of these sequences, or variants thereof, including sequences having at least about 80-100% sequence identity thereto (including any percent identity within this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto), can be used to construct fusion proteins as described herein or recombinant polynucleotides encoding such fusion proteins.

[0057] For discussions of genetic diversity and phylogenetic analysis of Hepatitis C virus, see also Smith et al. (2014) Hepatology 59(1):318-327, Simmonds et al. (2005) Hepatology 42(4):962-973, Kuiken et al. (2009) Methods Mol. Biol. 510:33-53, Ho et al. (2015) J. Virol. Methods 219:28-37, Echeverria et al. (2015) World J. Hepatol. 7(6):831-845, and Jackowiak et al. (2014) Infect Genet Evol. 21:67-82, which are incorporated by reference in their entireties.

[0058] The term "fusion protein," "fusion polypeptide," "degron fusion protein," or "degron fusion" as used herein refers to a fusion that includes a degron combined with a protease and a selected polypeptide of interest as part of a single continuous chain of amino acids (which chain does not occur in nature). The degron may be linked to the polypeptide of interest via a cleavable linker that includes a cleavage site that can be recognized by the protease of the fusion to allow for self-removal of the protease and the degron from the polypeptide of interest. The location of the cleavage site in the fusion may be selected to allow for release of the polypeptide of interest from the fusion essentially unmodified or minimally modified (e.g., less than 10 extra amino acids). The fusion polypeptide may be designed for N- or C-terminal attachment of the degron to the polypeptide of interest. The fusion polypeptide may also contain sequences exogenous to the degron, protease, and polypeptide of interest. For example, the fusion may include a targeting or localization sequence, a detectable label, or a tag sequence.

[0059] The term "cell receptor" as used herein refers to a membrane protein that responds specifically to a particular extracellular stimulus and generates an intracellular signal that produces a specific functional response. Non-limiting examples of these stimuli / signals include soluble factors that are generated locally (e.g., synaptic transmission) or remotely (e.g., hormones and growth factors), ligands on the surface of other cells (e.g., antigens such as cancer antigens), or the extracellular matrix itself. Non-limiting examples of cell receptors include G protein-coupled receptors, receptor tyrosine kinases, ligand-gated ion channels, integrins, cytokine receptors, and chimeric antigen receptors (CARs).

[0060] The term "chimeric antigen receptor" or alternatively "CAR" as used herein refers to a polypeptide or set of polypeptides that, when expressed in an immune effector cell, provides the cell with specificity for a target cell, typically a cancer cell, and generates an intracellular signal. In some embodiments, a CAR comprises at least an extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain"), including a functional signaling domain derived from a stimulatory molecule and / or a costimulatory molecule. In some aspects, the set of polypeptides are contiguous with each other. In some embodiments, the CAR further comprises a spacer domain between the extracellular antigen binding domain and the transmembrane domain. In some embodiments, the set of polypeptides comprises a recruitment domain, such as a dimerization or multimerization domain, that can bind the polypeptides to each other. In some embodiments, a CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain including a functional signaling domain derived from a stimulatory molecule. In one aspect, a CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one aspect, a CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In some embodiments, a CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule.

[0061] The term "extracellular protein binding domain" as used herein refers to a molecular binding domain that is typically an ectodomain of a cellular receptor, located outside the cell, and exposed to the extracellular space. An extracellular protein binding domain can include any molecule (e.g., a protein or peptide) that can bind to another protein or peptide. In some embodiments, the extracellular protein binding domain includes an antibody, an antigen-binding fragment thereof, F(ab), F(ab'), a single-chain variable fragment (scFv), or a single-domain antibody (sdAb). In some embodiments, the extracellular protein binding domain binds to a hormone, a growth factor, a cell surface ligand (e.g., an antigen such as a cancer antigen), or the extracellular matrix.

[0062] The term "intracellular signaling domain" as used herein refers to a functional endodomain of a cell receptor located within the cell. For example, the signaling domain transmits a signal (e.g., a growth / survival signal) to the cell after the molecular binding domain binds to an antigen. In some embodiments, the signaling domain is a CD3-zeta protein that contains three immunoreceptor tyrosine-based activation motifs (ITAMs). Other examples of signaling domains include CD28, 4-1BB, and OX40. In some embodiments, a cell receptor contains two or more signaling domains, each of which is referred to as a co-signaling domain.

[0063] The term "transmembrane domain" as used herein refers to a domain that spans a cell membrane. In some embodiments, the transmembrane domain comprises a hydrophobic alpha helix. Different transmembrane domains result in different receptor stabilities. In some embodiments, the transmembrane domain of the cell receptor of the present disclosure comprises a CD3-zeta transmembrane domain or a CD28 transmembrane domain.

[0064] The term "recruitment domain" as used herein refers to an interaction motif found in a variety of proteins, such as helicases, kinases, mitochondrial proteins, caspases, other cytoplasmic factors, etc. Recruitment domains mediate the formation of large protein complexes through direct interactions between recruitment domains. In some embodiments, the recruitment domains of the present disclosure are dimerization or multimerization domains.

[0065] The term "cell-based therapy" as used herein refers to a therapeutic method using cells (e.g., immune cells and / or stem cells) to deliver a gene or polypeptide of interest, such as a therapeutic protein, to a patient (subject). Cell-based therapy, as provided herein, also encompasses prophylactic and diagnostic regimens. Thus, the gene of interest (and the encoded product of interest) used in cell-based therapy can be a prophylactic molecule (e.g., an antigen intended to induce an immune response) or a detectable molecule (e.g., a fluorescent protein or other visible molecule).

[0066] As used herein, the term "cognate cleavage site" refers to the specific sequence or sequence motif that is recognized and cleaved by a protease of the present disclosure. A protease cleavage site comprises a specific amino acid sequence or motif that is recognized by the protease during proteolytic cleavage, typically comprising 1-6 amino acids around either side of the scissile bond that are used to bind to the active site of the protease and recognize it as a substrate.

[0067] The term "cleavable linker" refers to a linker that includes a cleavage site. A cleavable linker may include a cleavage site specific for an enzyme, such as a protease or other cleaving agent. A cleavable linker is typically cleavable under physiological conditions.

[0068] The term "degron" as used herein refers to a protein or a portion thereof that is important in regulating protein degradation rates. In various embodiments of the present disclosure, various degrons known in the art may be used, including, but not limited to, short amino acid sequences, structural motifs, and exposed amino acids. Degrons identified from various organisms may be used. In some embodiments, the degron of the present disclosure comprises a degradation sequence. In some embodiments, the degron is an auto-excising degron. An auto-excising degron is a degron that is fused to a polypeptide of interest such that the protease of the present disclosure can cleave the fusion protein containing the polypeptide of interest to separate the degron from the polypeptide of interest. The protease itself may or may not be removed from the fusion protein containing the polypeptide of interest after cleavage.

[0069] The term "degradation sequence" as used herein refers to a sequence that promotes the degradation of attached proteins through either the proteasome or the autophagy-lysosomal pathway. In a preferred embodiment, the degradation sequence is a polypeptide that destabilizes a protein when fused to the protein, such that the half-life of the protein is reduced by at least two-fold. Many different degradation sequences / signals (e.g., of the ubiquitin-proteasome system) are known in the art, any of which can be used as provided herein. The degradation sequence can be operably linked to a cellular receptor, but need not be adjacent to it, as long as the degradation sequence still functions to direct the degradation of the cellular receptor. In some embodiments, the degradation sequence induces the rapid degradation of the cellular receptor. For a discussion of degradation sequences and their function in protein degradation, see, e.g., Kanemaki et al. (2013) Pflugers Arch. 465(3):419-425; Erales et al. (2014) Biochim Biophys Acta 1843(1):216-221; Schrader et al. (2009) Nat. Chem. Biol. 5(11):815-822; Ravid et al. (2008) Nat. Rev. Mol. Cell. Biol. 9(9):679-690; Tasaki et al. (2007) Trends Biochem Sci. 32(11):520-528; Meinnel et al. (2006) Biol. Chem. 387(7):839-851; Kim et al. (2013) Autophagy 9(7):1100-1103, Varshavsky (2012) Methods Mol. Biol. 832:1-11, and Fayadat et al. (2003) Mol Biol Cell. 14(3):1268-1278.

[0070] The terms "polypeptide" and "protein" refer to a polymer of amino acid residues and are not limited to a minimum length. Thus, peptides, oligopeptides, dimers, multimers, and the like, are included in this definition. Both full-length proteins and fragments thereof are encompassed by this definition. The terms also include post-expression modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, hydroxylation, and the like. Furthermore, for purposes of this disclosure, "polypeptide" refers to a protein that includes modifications such as deletions, additions, and substitutions to the native sequence, so long as the protein maintains a desired activity. These modifications may be deliberate, such as through site-directed mutagenesis, or may be accidental, such as through errors due to, for example, mutations of the host that produces the protein or PCR amplification.

[0071] By "derivative" is intended any suitable modification of the native polypeptide of interest, a fragment of a native polypeptide, or their respective analogs, such as glycosylation, phosphorylation, polymer conjugation (such as with polyethylene glycol), or addition of other exogenous moieties, so long as the desired biological activity of the native polypeptide is retained. Methods for making polypeptide fragments, analogs, and derivatives are generally available in the art.

[0072] By "fragment" is intended a molecule consisting of only a portion of the intact full-length sequence and structure. Fragments can include C-terminal deletions, N-terminal deletions, and / or internal deletions of the polypeptide. An active fragment of a particular protein or polypeptide will generally include at least about 5-10 contiguous amino acid residues of the full-length molecule, preferably at least about 15-25 contiguous amino acid residues of the full-length molecule, and most preferably at least about 20-50 or more contiguous amino acid residues of the full-length molecule, or any integer between 5 amino acids and the full-length sequence, provided that the fragment retains biological activity, such as catalytic activity, ligand binding activity, regulatory activity, degron proteolytic signaling, or fluorescent properties.

[0073] "Substantially purified" generally refers to the isolation of a substance (compound, polynucleotide, protein, polypeptide, polypeptide composition) such that the substance constitutes a majority percentage of the sample in which it is present. Typically, in a sample, the substantially purified component constitutes 50% of the sample, preferably 80%-85%, more preferably 90-95%. Techniques for purifying polynucleotides and polypeptides of interest are well known in the art and include, for example, ion exchange chromatography, affinity chromatography, and sedimentation according to density.

[0074] "Isolated," when referring to a polypeptide, means that the indicated molecule is separated and discrete from the whole organism in which it is found in nature or exists in the substantial absence of other biological macromolecules of the same type. With reference to polynucleotides, the term "isolated" refers to a nucleic acid molecule that lacks all or a portion of sequences that are normally associated with it in nature, or a sequence that is present in nature but has heterologous sequences associated with it, or a molecule that has been separated from the chromosome.

[0075] The terms "label" and "detectable label" refer to detectable molecules, including, but not limited to, radioisotopes, stable (non-radioactive) heavy isotopes, fluorophores, chemiluminescent materials, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin or haptens), and the like. The term "fluorophore" refers to a substance or portion thereof that is capable of exhibiting fluorescence in the detectable range. Specific examples of labels that may be used with the present disclosure include radioactive labels (e.g., H, I, S, C, or P), stable (non-radioactive) heavy isotopes (e.g., 13 C or 15N), phycoerythrin, Alexa dyes, fluorescein, 7-nitrobenzo-2-oxa-1,3-diazole (NBD), YPet, CyPet, cascade blue, allophycocyanin, Cy3, Cy5, Cy7, rhodamine, dansyl, umbelliferone, Texas Red, luminol, acladim esters, biotin or other streptavidin-binding proteins, magnetic beads, electron-dense reagents, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP) Enzyme tags include, but are not limited to, enhanced yellow fluorescent protein (EYFP), blue fluorescent protein (BFP), red fluorescent protein (RFP), Dronpa, Padron, mApple, mCherry, rsCherry, rsCherryRev, firefly luciferase, Renilla luciferase, NADPH, beta-galactosidase, horseradish peroxidase, glucose oxidase, alkaline phosphatase, chloramphenicol acetyltransferase, and urease. Enzyme tags are used with their cognate substrates.The term also includes color-coded microspheres of known fluorescent intensity (see, e.g., microspheres using xMAP technology made by Luminex, Austin, TX), microspheres containing quantum dot nanocrystals, e.g., containing different ratios and combinations of quantum dot colors (see, e.g., Qdot nanocrystals made by Life Technologies, Carlsbad, CA), glass-coated metal nanoparticles (see, e.g., SERS nanotags made by Nanoplex Technologies, Inc., Mountain View, CA), barcode materials (see, e.g., submicron sized striped metal rods such as the Nanobarcodes produced by Nanoplex Technologies, Inc.), coded microparticles with colored barcodes (see, e.g., CellCards made by Vitra Bioscience, vitrabio.com), and glass microparticles with digital holographic code images (see, e.g., CyVera microbeads made by Ulumina, San Diego, CA). As with many standard procedures associated with the practice of this disclosure, those of skill in the art will recognize additional labels that can be used.

[0076] "Homology" refers to the percent identity between two polynucleotides or two polypeptide molecules. Two nucleic acid or two polypeptide sequences are "substantially homologous" to each other when the sequences exhibit at least about 50% sequence identity, preferably at least about 75% sequence identity, more preferably at least about 80%-85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95%-98% sequence identity. As used herein, substantially homologous also refers to sequences that exhibit complete identity to a specified sequence.

[0077] Generally, "identity" refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotide or polypeptide sequences, respectively. Percent identity can be determined by direct comparison of sequence information between two molecules by aligning the sequences, counting the number of exact matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. Readily available computer programs can be used to aid in the analysis, for example, ALIGN, Dayhoff, MOin Atlas of Protein Sequence and Structure MODayhoff ed., 5 Suppl.3:353 358, National biomedical Research Foundation, Washington, DC, adapts the local homology algorithm of Smith and Waterman Advances in Appl.Math.2:482 489,1981 for peptide analysis. Programs for determining nucleotide sequence identity are available in the Wisconsin Sequence Analysis Package, version 8 (available from Genetics Computer Group, Madison, WI), such as the BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waterman algorithm. These programs are easily used with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package mentioned above. For example, the percent identity of a particular nucleotide sequence to a reference sequence can be determined using the Smith and Waterman homology algorithm, with a default scoring table and a gap penalty of six nucleotide positions.

[0078] Another method for establishing percent identity in the context of the present disclosure is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok and distributed by IntelliGenetics, Inc. (Mountain View, CA). From this suite of packages, the Smith Waterman algorithm can be employed, in which default parameters are used in the scoring table (e.g., gap opening penalty 12, gap extension penalty 1, and gap 6). From the data generated, the "match" value reflects "sequence identity". Other suitable programs for calculating percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: Genetic code=standard, filter=none, strand=both, cutoff=60, expect=10, Matrix=BLOSUM62, Descriptions=50 sequences, sort by=HIGH SCORE, Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss protein+Spupdate+PIR. Details of these programs are readily available.

[0079] Alternatively, homology can be determined by polynucleotide hybridization under conditions that form stable double strands between homologous regions, followed by digestion with single-strand specific nuclease(s) and sizing of digested fragments. Substantially homologous DNA sequences can be identified, for example, in Southern hybridization experiments under stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, for example, Sambrook et al., supra, DNA Cloning, supra, Nucleic Acid Hybridization, supra.

[0080] "Recombinant" as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, viral, semisynthetic, or synthetic origin that is not related, by reason of its origin or manipulation, to all or a portion of the polynucleotide with which it is naturally associated. The term "recombinant" as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. Generally, a gene of interest is cloned and then expressed in a transformed organism, as further described below. The host organism expresses the foreign gene to produce the protein under expression conditions.

[0081] The term "transformation" refers to the insertion of an exogenous polynucleotide into a host cell, regardless of the method used for the insertion. For example, it includes direct uptake, transduction, or f-mating. The exogenous polynucleotide may be maintained as a non-integrated vector, such as a plasmid, or alternatively, may be integrated into the host genome.

[0082] "Recombinant host cell", "host cell", "cell", "cell line", "cell culture", and other such terms referring to microorganisms or higher eukaryotic cell lines refer to cells that can be or have been used as recipients of recombinant vectors or other transferred DNA, including the progeny of transfected cells. Host cells can be cultured as single cells or multicellular entities (e.g., tissues, organs, or organoids containing recombinant vectors).

[0083] A "coding sequence" or a sequence "encoding" a selected polypeptide is a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences (or "control elements"). The boundaries of the coding sequence may be determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. Coding sequences can include, but are not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic DNA sequences from viral or prokaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3' to the coding sequence.

[0084] Exemplary "control elements" include, but are not limited to, transcriptional promoters, transcriptional enhancer elements, transcription termination signals, polyadenylation sequences (located 3' to a translation stop codon), sequences for optimizing translation initiation (located 5' to the coding sequence), and translation termination sequences.

[0085] "Operably linked" refers to the arrangement of elements, such that the components so described are configured to perform their normal functions. For example, a given promoter operably linked to a coding sequence can cause expression of the coding sequence when the appropriate enzymes are present. The promoter need not be contiguous with the coding sequence, so long as it functions to induce expression of the coding sequence. Thus, for example, an intervening untranslated but transcribed sequence can be present between the promoter sequence and the coding sequence, and the promoter sequence can still be considered to be "operably linked" to the coding sequence. In another example, a degron operably linked to a polypeptide can promote degradation of the polypeptide when the appropriate cellular degradation system (e.g., proteasome or autophagosome degradation) is present. The degron need not be contiguous with the polypeptide, so long as it functions to direct degradation of the polypeptide.

[0086] "Encoded by" refers to a nucleic acid sequence that encodes a polypeptide sequence, where the polypeptide sequence or a portion thereof includes an amino acid sequence of at least 3-5 amino acids, more preferably at least 8-10 amino acids, and even more preferably at least 15-20 amino acids from the polypeptide encoded by the nucleic acid sequence.

[0087] "Expression cassette" or "expression construct" refers to an assembly capable of directing the expression of a sequence(s) or gene(s) of interest. An expression cassette generally includes control elements such as a promoter (directing the transcription of the sequence(s) or gene(s) of interest) operably linked to the sequence(s) or gene(s) of interest, as described above, and often also includes a polyadenylation sequence. In certain embodiments of the present disclosure, the expression cassettes described herein may be contained within a plasmid construct. In addition to the components of the expression cassette, the plasmid construct may also include one or more selectable markers, a signal that allows the plasmid construct to exist as single-stranded DNA (e.g., M13 origin of replication), at least one multiple cloning site, and a "mammalian" origin of replication (e.g., SV40 or adenovirus origin of replication).

[0088] "Purified polynucleotide" refers to a polynucleotide of interest or a fragment thereof that is substantially free of the proteins with which the polynucleotide naturally accompanies, e.g., less than about 50%, preferably less than about 70%, and more preferably less than about 90% of the proteins with which the polynucleotide naturally accompanies. Techniques for purifying a polynucleotide of interest are well known in the art and include, for example, disruption of cells containing the polynucleotide with chaotropic agents, and separation of the polynucleotide(s) and proteins by ion exchange chromatography, affinity chromatography, and sedimentation according to density.

[0089] The term "transfection" is used to refer to the uptake of foreign DNA by a cell. A cell is "transfected" when foreign DNA is introduced inside the cell membrane. Many transfection techniques are commonly known in the art. See, for example, Graham et al. (1973) Virology, 52:456; Sambrook et al. (2001) Molecular Cloning, a laboratory manual, 3 rSee, for example, Davis et al. (1995) Basic Methods in Molecular Biology, 2nd edition, Cold Spring Harbor Laboratories, New York; Davis et al. (1995) Basic Methods in Molecular Biology, 2nd edition, McGraw-Hill; and Chu et al. (1981) Gene 13:197. Using such techniques, one or more exogenous DNA moieties can be introduced into a suitable host cell. The term refers to both stable and transient uptake of genetic material, including uptake of peptide or antibody-bound DNA.

[0090] A "vector" is capable of introducing a nucleic acid sequence into a target cell (e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes). Typically, "vector construct", "expression vector", and "gene transfer vector" refer to any nucleic acid construct capable of directing the expression of a nucleic acid of interest and introducing a nucleic acid sequence into a target cell. Thus, the term includes cloning and expression vehicles, as well as viral vectors.

[0091] The terms "variant", "analog", and "mutein" refer to biologically active derivatives of a reference molecule that retain a desired activity, such as fluorescence or oligomerization properties. In general, the terms "variant" and "analog" refer to compounds having a native polypeptide sequence and structure with one or more amino acid additions, substitutions (generally of a conservative nature) and / or deletions relative to the native molecule, so long as the modifications do not destroy the biological activity and are "substantially homologous" to the reference molecule as defined below. Generally, the amino acid sequence of such an analog has a high degree of sequence homology to the reference sequence when the two sequences are aligned, e.g., greater than 50%, generally greater than 60%-70%, and even more particularly 80%-85% or more, e.g., at least 90%-95% or more amino acid sequence homology. Often, an analog contains the same number of amino acids, but includes substitutions, as described herein. The term "mutein" further includes polypeptides having one or more amino acid-like molecules, including, but not limited to, compounds containing only amino and / or imino molecules, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted bonds, and other modifications known in the art, both naturally occurring and non-naturally occurring (e.g., synthetic), cyclized, branched molecules, etc. The term also includes molecules containing one or more N-substituted glycine residues ("peptoids") and other synthetic amino acids or peptides. (See, e.g., U.S. Patent Nos. 5,831,005, 5,877,278, and 5,977,301, Nguyen et al., Chem. Biol. (2000) 7:463-473, and Simon et al., Proc. Natl. Acad. Sci. USA (1992) 89:9367-9371 for a description of peptoids.) Methods for making polypeptide analogs and muteins are known in the art and are further described below.

[0092] As explained above, analogs generally include substitutions of a conservative nature, i.e., substitutions that occur within a family of amino acids whose side chains are related. Specifically, amino acids are generally classified into four families: (1) acidic-aspartate and glutamate, (2) basic-lysine, arginine, histidine, (3) non-polar-alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and (4) uncharged polar-glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. For example, isolated substitutions of leucine with isoleucine or valine, aspartate with glutamate, threonine with serine, or similar conservative substitutions of amino acids with structurally related amino acids can be reasonably expected not to significantly affect biological activity. For example, a polypeptide of interest can contain up to about 5-10 conservative or non-conservative amino acid substitutions, or even up to about 15-25 conservative or non-conservative amino acid substitutions, or any integer between 5 and 25, so long as the desired function of the molecule remains intact. One of skill in the art can readily determine the regions of a molecule of interest that are tolerant of alteration by reference to Hopp / Woods and Kyte-Doolittle plots, which are well known in the art.

[0093] "Gene transfer" or "gene delivery" refers to a method or system for reliably inserting DNA or RNA of interest into a host cell. Such methods may result in transient expression of non-integrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g., episomes), or integration of the transferred genetic material into the genomic DNA of the host cell. Gene delivery expression vectors include, but are not limited to, bacterial plasmid vectors, viral vectors, non-viral vectors, vectors derived from alphaviruses, poxviruses, and vaccinia viruses.

[0094] The term "derived from" is used herein to identify the original source of a molecule, but is not meant to limit the manner in which the molecule is made, which may be, for example, by chemical synthesis or recombinant means.

[0095] A polynucleotide "derived from" a specified sequence refers to a polynucleotide sequence that contains a contiguous sequence of approximately at least about 6 nucleotides, preferably at least about 8 nucleotides, more preferably at least about 10-12 nucleotides, and even more preferably at least about 15-20 nucleotides corresponding thereto, i.e., identical or complementary to a region of the specified nucleotide sequence. A derived polynucleotide is not necessarily physically derived from a nucleotide sequence of interest, but may be generated in any manner, including, but not limited to, chemical synthesis, replication, reverse transcription, or transcription, which is based on information provided by the sequence of bases within the region(s) from which the polynucleotide is derived. It may therefore represent either the sense or antisense orientation of the original polynucleotide.

[0096] The term "heterologous" refers to sequences that are not normally linked together and / or not normally associated with a particular cell, with respect to nucleic acid sequences, such as coding sequences and control sequences. Thus, a "heterologous" region of a nucleic acid construct or vector is a segment of nucleic acid within or attached to another nucleic acid molecule that is not found in association with the other molecule in nature. For example, a heterologous region of a nucleic acid construct may include a coding sequence adjacent to a sequence that is not found in association with the natural coding sequence. Another example of a heterologous coding sequence is a construct in which the coding sequence itself is not found in nature (e.g., a synthetic sequence with different codons than the natural gene). Similarly, a cell transformed with a construct that is not normally present in the cell would be considered heterologous for the purposes of this disclosure.

[0097] By "recombinant virus" is meant a virus that has been genetically altered, for example, by the addition or insertion of a heterologous nucleic acid construct into the particle.

[0098] As used herein, "recombinant virion" refers to a viral particle that contains a recombinant viral vector (e.g., a conditionally replicating viral vector encoding a degron fusion protein). Generally, a recombinant virion contains one or more structural proteins and a viral vector. A recombinant virion may also contain a nucleocapsid structure and, in some cases, a lipid envelope derived from a host cell membrane.

[0099] The term "subject" refers to any invertebrate or vertebrate subject, including, but not limited to, humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species, farm animals such as cows, sheep, pigs, goats, and horses, domestic mammals such as dogs and cats, laboratory animals including rodents such as mice, rats, and guinea pigs, chickens, turkeys, and other birds, domestic animals such as ducks, geese, and birds including wild and game birds. The term does not denote a particular age. Thus, both adult and newborn individuals are intended to be subjects.

[0100] "Recombinant animal" refers to a non-human subject that is the recipient of a recombinant vector or other transferred DNA, and includes the progeny of the recombinant animal.

[0101] Other Rules of Interpretation Ranges recited herein are understood to be shorthand for all of the values ​​within the range, including the recited endpoints. For example, the range 1 to 50 is understood to include any number, combination of numbers, or subranges from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

[0102] Unless otherwise indicated, reference to a compound having one or more stereocenters contemplates each stereoisomer and all combinations of stereoisomers.

[0103] overview Before describing the present disclosure in detail, it is to be understood that the present disclosure is not limited to particular formulations or process parameters, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing only particular embodiments of the present disclosure, and is not intended to be limiting.

[0104] Although a number of methods and materials similar or equivalent to those described herein can be used in the practice of the present disclosure, the preferred materials and methods are described herein.

[0105] The present disclosure is based on the discovery that certain mutations within the immunodominant epitopes of the disclosed proteases, such as the Hepatitis C Virus (HCV) nonstructural protein 3 (NS3) protease, can affect not only the immunogenicity but also the activity of the protease when fused to a polypeptide of interest. Such mutations can be used to reduce immunogenicity and modulate the activity of the protease when used in therapeutic applications, such as the small molecule assisted shut-off (SMASh) technique, in which a polypeptide of interest is fused and thereby expressed in a minimally modified form. In such applications, the degron can be removed from the protein of interest by a cis-encoded protease (e.g., a viral NS3 protease). Clinically available protease inhibitors can be used to block protease cleavage such that the degron is retained after inhibitor addition to the subsequently synthesized protein copy. The degron when attached causes rapid degradation of the linked protein. Alternatively, the proteases of the present disclosure may be fused to a polypeptide of interest having functional domains, or in the case of a multi-domain polypeptide, may be interdomain, such that the addition of a protease inhibitor can control one or more functions of the polypeptide of interest. As disclosed herein, the use of such inhibitory proteases allows for the reversible and dose-dependent shut-off of a variety of proteins with a high dynamic range in multiple cell types.

[0106] Fusion proteins Certain aspects of the present disclosure relate to fusion proteins, which comprise a variant protease (e.g., a variant HCV NS3 protease) and a cognate protease cleavage site fused to a selected polypeptide of interest in an arrangement designed to control the function and / or production of the polypeptide of interest. The cleavage site can be recognized by the protease of the fusion protein to allow for cleavage of one or more domains within the polypeptide of interest. The location of the cleavage site in the fusion is preferably selected to allow for controlled function and / or expression of the polypeptide of interest. Fusion proteins of the present disclosure can be designed with regard to N- or C-terminal attachment of the protease to the polypeptide of interest. Fusion proteins can also contain sequences exogenous to the protease of interest, the cognate cleavage site, and the polypeptide. For example, the fusion can include a targeting or localization sequence, or a tag sequence. In addition, the fusion protein can include a detectable label (e.g., a fluorescent, bioluminescent, chemiluminescent, colorimetric, or isotopic label) to facilitate monitoring the production and degradation of the polypeptide of interest.

[0107] Variant Proteases Certain aspects of the present disclosure relate to fusion proteins comprising variant proteases, which contain one or more mutations that reduce immunogenicity and / or modulate protease activity when the fusion protein is expressed in a mammalian cell.

[0108] The variant protease of the present disclosure may be derived from any suitable protease known in the art. For example, any of the proteases listed in Table 1 may be used to produce the variant protease of the present disclosure. When a protease is selected, its cognate cleavage site and a protease inhibitor known in the art to bind and inhibit the protease may be used in combination. Exemplary combinations for use are provided in Table 1 below. Representative sequences of proteases are available from public databases, including UniProt, through the uniprot.org website. The UniProt accession numbers of the proteases are also provided in Table 1 below.

[0109] [Table 1] TIFF2025032195000008.tif215169TIFF2025032195000009.tif228169TIFF2025032195000010.tif228169 TIFF2025032195000011.tif228169TIFF2025032195000012.tif228169TIFF2025032195000013.tif228169 TIFF2025032195000014.tif224169TIFF2025032195000015.tif255169TIFF2025032195000016.tif227169 TIFF2025032195000017.tif227169TIFF2025032195000018.tif227169TIFF2025032195000019.tif129169

[0110] Exemplary proteases that can be used in the fusion proteins of the present disclosure include Hepatitis C virus proteases (e.g., NS3 and NS2-3), signal peptidases, proprotein convertases of the subtilisin / kexin family (furin, PCI, PC2, PC4, PACE4, PC5, PC), proprotein convertases that cleave at hydrophobic residues (e.g., Leu, Phe, Val, or Met), proprotein convertases that cleave at small amino acid residues such as Ala or Thr, proopiomelanocortin convertase (PCE), These include chromaffin granule aspartic protease (CGAP), prohormone thiol proteases, carboxypeptidases (e.g., carboxypeptidase E / H, carboxypeptidase D, and carboxypeptidase Z), aminopeptidases (e.g., arginine aminopeptidase, lysine aminopeptidase, aminopeptidase B), prolyl endopeptidase, aminopeptidase N, insulin-degrading enzyme, calpain, high molecular weight proteases, and caspases 1, 2, 3, 4, 5, 6, 7, 8, and 9.Other proteases include aminopeptidase N, puromycin-sensitive aminopeptidase, angiotensin-converting enzyme, pyroglutamyl peptidase II, dipeptidyl peptidase IV, N-arginine dibasic convertase, endopeptidase 24.15, endopeptidase 24.16, amyloid precursor protein secretase alpha, beta, and gamma, angiotensin-converting enzyme secretase, TGF alpha secretase, TF alpha secretase, FAS ligand secretase, TNF receptor-I and -II secretase, CD30 secretase, KL1 and Proteases selected for use in the fusion protein include, but are not limited to, KL2 secretase, IL6 receptor secretase, CD43, CD44 secretase, CD16-1 and CD16-11 secretase, L-selectin secretase, folate receptor secretase, MMP1, 2, 3, 7, 8, 9, 10, 11, 12, 13, 14, and 15, urokinase plasminogen activator, tissue plasminogen activator, plasmin, thrombin, BMP-1 (procollagen C-peptidase), ADAM1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11, granzyme A, B, C, D, E, F, G, and H. It is preferred that the protease selected for use in the fusion protein is highly selective for the cleavage site in the cleavable linker. Additionally, the protease activity is preferably inhibitable with an inhibitor that is cell permeable and not toxic to the cells or subjects being studied.For a discussion of proteases, see, for example, VYH Hook, Proteolytic and cellular mechanisms in prohormone and proprotein processing, RG Landes Company, Austin, Tex., USA (1998), NM Hooper et al., Biochem. J. 321:265-279 (1997), Z. Werb, Cell 91:439-442 (1997), TG Wolfsberg et al., J. Cell Biol. 131:275-278 (1995), K. Murakami and JD Atlinger, Biochem. Biophys. Res. Comm. 146:1249-1259 (1987), T. Berg et al., Biochem. J. 307:313-326 (1995), and MJ Myth and JA Trapani, Immunology Today 16:202-206 (1995), RV Talanian et al., J. Biol. Chem. 272:9677-9682 (1997), and NA Thomberry et al., J. Biol. Chem. 272:17907-17911 (1997), the disclosures of which are incorporated herein.

[0111] In certain embodiments, the protease used in the fusion protein is derived from Hepatitis C Virus (HCV). In some embodiments, the protease is HCV nonstructural protein 3 (NS3) protease. NS3 contains an N-terminal serine protease domain and a C-terminal helicase domain. The protease domain of NS3 forms a heterodimer with HCV nonstructural protein 4A (NS4A cofactor), which activates the proteolytic activity. The NS3 protease may include the entire NS3 protein or a proteolytically active fragment thereof, or may further include the activated NS4A cofactor region. Advantages of using the NS3 protease include that it is highly selective and can be sufficiently inhibited by several non-toxic cell-permeable drugs currently available clinically. NS3 protease inhibitors that may be used in the practice of the present disclosure include, but are not limited to, simeprevir, danoprevir, asunaprevir, cilprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and voxiloprevir.

[0112] When NS3 protease is used in the fusion protein, the cleavable linker of the fusion protein can include an NS3 protease cleavage site (e.g., a homologous cleavage site). Exemplary NS3 protease cleavage sites that can be used in the cleavable linker include the four junctions between the nonstructural (NS) proteins of the HCV polyprotein that are normally cleaved by NS3 protease during HCV infection, including NS3 / NS4A, NS4A / NS4B, NS4B / NS5A, and NS5A / NS5B junction cleavage sites. For a description of the representative sequences of NS3 protease and its cleavage sites for various strains of HCV, see, for example, Hepatitis C Viruses: Genomes and Molecular Biology (SLTan ed., Taylor&Francis, 2006), Chapter 6, pp.163-206 (incorporated herein by reference in its entirety).

[0113] The NS3 nucleic acid and protein sequences can be derived from HCV, including any isolate of HCV having any genotype (e.g., the seven genotypes 1-7) or subtype. Many NS3 nucleic acid and protein sequences are known. Representative NS3 sequences are provided in Table 1. Additional representative sequences are listed in the National Center for Biotechnology Information (NCBI) database. For example, NCBI entries: Accession numbers: YP_001491553, YP_001469631, YP_001469632, NP_803144, NP_671491, YP_001469634, YP_001469630, YP_001469633, ADA68311, ADA68307, AFP99000, AFP98987, ADA68322, AFP99033, ADA68330, AFP99056, AFP99041, CBF60982, CBF60817, See AHH29575, AIZ00747, AIZ00744, ABI36969, ABN05226, KF516075, KF516074, KF516056, AB826684, AB826683, JX171009, JX171008, JX171000, EU847455, EF154714, GU085487, JX171065, JX171063, all of which sequences (as entered by the filing date of this application) are incorporated by reference herein. Any of these sequences, or variants thereof, including sequences having at least about 80-100% sequence identity thereto (including any percent identity within this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto), can be used to construct fusion proteins as described herein or recombinant polynucleotides encoding such fusion proteins.

[0114] The NS4A nucleic acid and protein sequences can be derived from HCV, including any isolate of HCV having any genotype (e.g., the seven genotypes 1-7) or subtype. Many NS4A nucleic acid and protein sequences are known. Representative sequences are listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries: Accession Nos. NP_751925, YP_001491554, GU945462, HQ822054, FJ932208, FJ932207, FJ932205, and FJ932199, all of which sequences (as entered by the filing date of this application) are incorporated herein by reference. Any of these sequences, or variants thereof, including sequences having at least about 80-100% sequence identity thereto (including any percent identity within this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto), can be used to construct fusion proteins as described herein or recombinant polynucleotides encoding such fusion proteins.

[0115] HCV polyprotein nucleic acid and protein sequences can be derived from HCV, including any isolate of HCV having any genotype (e.g., the seven genotypes 1-7) or subtype. Many HCV polyprotein nucleic acid and protein sequences are known. Representative HCV polyprotein sequences are listed in the National Center for Biotechnology Information (NCBI) database. See, e.g., NCBI entries: Accession Nos. YP_001469631, P_671491, YP_001469633, YP_001469630, YP_001469634, YP_001469632, NC_009824, NC_004102, NC_009825, NC_009827, NC_009823, NC_009826, and EF108306, all of which sequences (as entered by the filing date of this application) are incorporated by reference herein. Any of these sequences, or variants thereof, including sequences having at least about 80-100% sequence identity thereto (including any percent identity within this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto), can be used to construct fusion proteins as described herein or recombinant polynucleotides encoding such fusion proteins.

[0116] In some embodiments, the NS3 protease is derived from HCV 1a. In some embodiments, the HCV 1a polyprotein has the following amino acid sequence (SEQ ID NO:1): TIFF2025032195000020.tif96167TIFF2025032195000021.tif227167TIFF2025032195000022.tif205167

[0117] In some embodiments, the fusion protein of the disclosure comprises a variant NS3 protease derived from an HCV 1a polyprotein having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the variant protease comprises one or more mutations, such as amino acid substitutions that reduce immunogenicity. In some embodiments, the variant protease comprises two or more mutations, three or more mutations, four or more mutations, five or more mutations, six or more mutations, seven or more mutations, eight or more mutations, nine or more mutations, ten or more mutations, eleven or more mutations, twelve or more mutations, thirteen or more mutations, fourteen or more mutations, fifteen or more mutations, sixteen or more mutations, seventeen or more mutations, eighteen or more mutations, nineteen or more mutations, or twenty or more mutations. In some embodiments, the variant protease comprises 1 mutation, 2 mutations, 3 mutations, 4 mutations, 5 mutations, 6 mutations, 7 mutations, 8 mutations, 9 mutations, 10 mutations, 11 mutations, 12 mutations, 13 mutations, 14 mutations, 15 mutations, 16 mutations, 17 mutations, 18 mutations, 19 mutations, or 20 mutations. In some embodiments, one or more of the mutations is an amino acid substitution.

[0118] Variant proteases may contain one or more mutations within an immunodominant epitope that results in reduced immunogenicity of the protease and / or within an epitope that results in modulation of the catalytic activity of the protease (see, e.g., Soderholm J, et al. Gut. 2006 Feb;55(2):266-74, Soumana D et al. ACS Chem Biol. 2014 Nov 21;9(11):2485-90, and Wertheimer AM et al. Hepatology. 2003 Mar;37(3):577-89). For example, the one or more mutations can be within a region corresponding to positions 1038-1047 of SEQ ID NO:1, positions 1057-1081 of SEQ ID NO:1, positions 1073-1081 of SEQ ID NO:1, positions 1073-1082 of SEQ ID NO:1, positions 1127-1141 of SEQ ID NO:1, positions 1131-1138 of SEQ ID NO:1, positions 1169-1177 of SEQ ID NO:1, and / or positions 1192-1206 of SEQ ID NO:1. TIFF2025032195000023.tif33162, and any combination thereof.

[0119] In some embodiments, the one or more mutations are one or more amino acid substitutions selected from a position corresponding to 1062 of SEQ ID NO:1, a position corresponding to 1069 of SEQ ID NO:1, a position corresponding to 1070 of SEQ ID NO:1, a position corresponding to 1071 of SEQ ID NO:1, a position corresponding to 1072 of SEQ ID NO:1, a position corresponding to 1074 of SEQ ID NO:1, a position corresponding to 1075 of SEQ ID NO:1, a position corresponding to 1077 of SEQ ID NO:1, a position corresponding to 1078 of SEQ ID NO:1, a position corresponding to 1079 of SEQ ID NO:1, a position corresponding to 1080 of SEQ ID NO:1, a position corresponding to 1031 of SEQ ID NO:1, a position corresponding to 1074 of SEQ ID NO:1, a position corresponding to 1132 of SEQ ID NO:1, a position corresponding to 1133 of SEQ ID NO:1, a position corresponding to 1195 of SEQ ID NO:1, a position corresponding to 1196 of SEQ ID NO:1, a position corresponding to 1201 of SEQ ID NO:1, a position corresponding to 1202 of SEQ ID NO:1, and any combination thereof.

[0120] In some embodiments, the one or more mutations are one or more amino acid substitutions selected from a substitution of Ile to Leu at a position corresponding to position 1074 of SEQ ID NO:1, a substitution of Ile to Met at a position corresponding to position 1074 of SEQ ID NO:1, a substitution of Asn to Ala at a position corresponding to position 1075 of SEQ ID NO:1, a substitution of Val to Ala at a position corresponding to position 1077 of SEQ ID NO:1, a substitution of Cys to Phe at a position corresponding to position 1078 of SEQ ID NO:1, a substitution of Trp to Ala at a position corresponding to position 1079 of SEQ ID NO:1, a substitution of Thr to Ala at a position corresponding to position 1080 of SEQ ID NO:1, a substitution of Val to Ala at a position corresponding to position 1081 of SEQ ID NO:1, a substitution of Val to Asn at a position corresponding to position 1081 of SEQ ID NO:1, and any combination thereof. In some embodiments, the one or more mutations are one or more amino acid substitutions selected from a Thr to Ala substitution at a position corresponding to position 1080 of SEQ ID NO:1, a Val to Ala substitution at a position corresponding to position 1077 of SEQ ID NO:1, a Val to Ala substitution at a position corresponding to position 1081 of SEQ ID NO:1, and any combination thereof. In some embodiments, the one or more mutations include a Thr to Ala substitution at a position corresponding to position 1080 of SEQ ID NO:1. In some embodiments, the one or more mutations include a Thr to Ala substitution at a position corresponding to position 1080 of SEQ ID NO:1, and a Val to Ala substitution at a position corresponding to position 1077 of SEQ ID NO:1. In some embodiments, the one or more mutations include a Thr to Ala substitution at a position corresponding to position 1080 of SEQ ID NO:1, and a Val to Ala substitution at a position corresponding to position 1081 of SEQ ID NO:1.

[0121] In some embodiments, the variant protease may include one or more additional mutations, such as amino acid substitutions, that modulate or otherwise adjust the enzymatic activity of the protease. In some embodiments, the variant protease includes two or more additional mutations, three or more additional mutations, four or more additional mutations, five or more additional mutations, six or more additional mutations, seven or more additional mutations, eight or more additional mutations, nine or more additional mutations, or ten or more additional mutations. In some embodiments, the variant protease includes one additional mutation, two additional mutations, three additional mutations, four additional mutations, five additional mutations, six additional mutations, seven additional mutations, eight additional mutations, nine additional mutations, or ten additional mutations. In some embodiments, the one or more additional mutations are amino acid substitutions. In some embodiments, the one or more additional mutations are amino acid substitutions at one or more positions corresponding to position 1074 of SEQ ID NO:1, position 1078 of SEQ ID NO:1, and / or position 1079 of SEQ ID NO:1. In some embodiments, the one or more additional mutations reduce the enzymatic activity of the protease. In some embodiments, the one or more additional mutations that reduce the enzymatic activity of the protease are one or more additional amino acid substitutions selected from an Ile to Ala substitution at a position corresponding to position 1074 of SEQ ID NO:1, a Trp to Ala substitution at a position corresponding to position 1079 of SEQ ID NO:1, and any combination thereof. In some embodiments, the one or more additional mutations increase the enzymatic activity of the protease. In some embodiments, the one or more additional mutations that increase the enzymatic activity of the protease are one or more additional amino acid substitutions comprising a Cys to Ala substitution at a position corresponding to position 1078 of SEQ ID NO:1.

[0122] In some embodiments, the fusion proteins of the disclosure comprise a variant NS3 protease derived from the HCV NS3 protease having the following amino acid sequence: TIFF2025032195000024.tif26164.

[0123] In some embodiments, the fusion protein further comprises an HCV NS4A cofactor. In some embodiments, the NS4A cofactor is It has the amino acid sequence of TIFF2025032195000025.tif4150.

[0124] Cognate protease cleavage site Certain aspects of the present disclosure relate to fusion proteins comprising variant proteases and cognate cleavage sites recognized by the proteases. When a protease is selected, its cognate cleavage site and a protease inhibitor known in the art to bind and inhibit the protease may be used in combination. Any suitable protease, cognate cleavage site, and cognate protease inhibitor may be used. Exemplary combinations or proteases, cognate cleavage sites, and cognate protease inhibitors are provided in Table 1 below.

[0125] When NS3 protease is used, the cognate cleavage site comprises the NS3 protease cleavage site. Exemplary NS3 protease cleavage sites include the four junctions between the nonstructural (NS) proteins of HCV polyprotein that are normally cleaved by NS3 protease during HCV infection, including NS3 / NS4A, NS4A / NS4B, NS4B / NS5A, and NS5A / NS5B junction cleavage sites. For a description of the representative sequences of NS3 protease and its cleavage sites for various strains of HCV, see, for example, Hepatitis C Viruses: Genomes and Molecular Biology (SLTan ed., Taylor&Francis, 2006), Chapter 6, pp.163-206 (incorporated herein by reference in its entirety). For example, the sequences of the HCV NS3 / 4A protease cleavage site, the HCV NS4A / 4B protease cleavage site (SEQ ID NO: 9, 44), the HCV NS4B / 5A protease cleavage site, and the HCV NS5A / 5B protease cleavage site (SEQ ID NO: 11, 45) are provided in Table 1.

[0126] In some embodiments, the cognate cleavage site of the NS3 protease includes those listed in Table 1. In some embodiments, the cognate cleavage site of the NS3 protease, such as the variant NS3 protease of the disclosure, is In some embodiments, the cognate cleavage site of the NS3 protease, such as the variant NS3 protease of the disclosure, is selected from: TIFF2025032195000027.tif12128. In some embodiments, the cognate cleavage site comprises one or more mutations, e.g., one or more amino acid substitutions. In some embodiments, the mutations at the cognate cleavage site can modulate or otherwise regulate the enzymatic activity and / or catalytic rate of the protease. For example, in some embodiments, the one or more mutations can increase the enzymatic activity and / or catalytic rate of the protease. Alternatively, in some embodiments, the one or more mutations can decrease the enzymatic activity and / or catalytic rate of the protease.

[0127] Degron Certain aspects of the present disclosure relate to fusion proteins comprising a polypeptide of interest, a protease, a cognate protease cleavage site, and further comprising a degron or an auto-excising degron.

[0128] The degron of the present disclosure may comprise a sequence of amino acids that provides a degradation signal that directs the polypeptide to cellular degradation. The degron may promote the degradation of the attached polypeptide through either the proteasome or the autophagy-lysosome pathway. In the fusion protein of the present disclosure, the degron must be operably linked to the polypeptide of interest, but does not have to be adjacent to it, as long as the degron still functions to direct the degradation of the polypeptide of interest. Preferably, the degron induces rapid degradation of the polypeptide of interest. For a discussion of degrons and their function in protein degradation, see, e.g., Kanemaki et al. (2013) Pflugers Arch. 465(3):419-425; Erales et al. (2014) Biochim Biophys Acta 1843(1):216-221; Schrader et al. (2009) Nat. Chem. Biol. 5(11):815-822; Ravid et al. (2008) Nat. Rev. Mol. Cell. Biol. 9(9):679-690; Tasaki et al. (2007) Trends Biochem Sci. 32(11):520-528; Meinnel et al. (2006) Biol. Chem. 387(7):839-851; Kim et al. (2013) Autophagy, which are incorporated herein by reference. 9(7):1100-1103, Varshavsky (2012) Methods Mol. Biol. 832:1-11, and Fayadat et al. (2003) Mol Biol Cell. 14(3):1268-1278.

[0129] Degrons with degradation sequences known in the art may be used in various embodiments of the present disclosure. In some embodiments, the degrons of the present disclosure may be derived from degrons identified from an organism, or variants thereof. Such degrons include HCV NS4 degron, PEST (two copies of residues 277-307 of IκBα (human) (SEQ ID NO: 46), GRR (residues 352-408 of p105 (human) (SEQ ID NO: 47), DRR (residues 210-295 of Cdc34 (yeast) (SEQ ID NO: 48), SNS (tandem repeats of SP2 and NB (SP2-NB-SP2) (influenza A and influenza B) (SEQ ID NO: 49), RPB (four copies of residues 1688-1702 of RPB1 (yeast) (SEQ ID NO: 50), SPmix (tandem repeats of SP1 and SP2 (SP2-SP1-SP2-SP1- SP2) (influenza A virus M2 protein) (SEQ ID NO: 51), NS2 (three copies of residues 79-93 of influenza A virus NS protein) (SEQ ID NO: 52), ODC (residues 106-142 of ornithine decarboxylase) (SEQ ID NO: 53), Nek2A (human), mODC (amino acids 422-461 (moue)), mODC_DA (amino acids 422-461 of mODC (D433A, D434A point mutations (mouse)) (SEQ ID NO: 54), APC / C degron (e.g., D-box, KEN-box, and ABBA motifs), COP1 These include, but are not limited to, E3 ligase-binding degron motifs, CRL4-Cdt2-binding PIP degrons, actinfilin-binding degrons, KEAP1-binding degrons, KLHL2- and KLHL3-binding degrons, MDM2-binding motifs, N-degrons (e.g., N-boxes, or UBR boxes), hydroxyproline modifications in hypoxia signaling, phytohormone-dependent SCF-LRR-binding degrons, SCF ubiquitin ligase-binding phosphodegrons, phytohormone-dependent SCF-LRR-binding degrons, DSGxxS (SEQ ID NO: 55) phospho-dependent degrons, siah-binding motifs, SPOP SBC docking motifs, and PCNA-binding PIP boxes.

[0130] In some embodiments, the degron comprises a portion of the HCV nonstructural proteins NS3 and NS4A. TIFF2025032195000028.tif4148, or variants thereof comprising a sequence having at least about 80-100% sequence identity (including any percent identity within this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity), wherein the degron is capable of promoting degradation of the polypeptide. It is understood that degrons comprising residues corresponding to the reference sequence of SEQ ID NO:40 in HCV nonstructural proteins NS3 and NS4A from other strains of HCV are also intended to be encompassed by the present disclosure.

[0131] In the fusion protein, the degron can be linked to the N-terminus or C-terminus of the polypeptide of interest. For example, the fusion protein can be represented by the formula NH2-PDLX-COOH or NH2-XLPD-COOH, where P is the amino acid sequence of the protease, D is the amino acid sequence of the degron, L is the amino acid sequence of the linker containing the cleavage site of the protease, and X is the amino acid sequence of the selected polypeptide of interest. The cleavable linker between the polypeptide of interest and the degron is designed for selective cleavage by the specific protease contained in the fusion protein. The cleavage site of the linker contains a specific amino acid sequence or motif recognized by the protease during proteolytic cleavage, typically including 1-6 amino acids around either side of the scissile bond required to bind to the active site of the protease and recognize it as a substrate. The cleavable linker can contain any protease recognition motif known in the art and is typically cleavable under physiological conditions.

[0132] The polypeptides contained in the fusion construct may be directly linked to each other by peptide bonds or may be separated by an intervening amino acid sequence. The fusion polypeptide may also contain a sequence exogenous to the protease of interest or the selected protein. For example, the fusion protein may contain a targeting or localization sequence, a tag sequence, or the sequence of a fluorescent or bioluminescent protein.

[0133] In certain embodiments, the tag sequence is located at the N-terminus or C-terminus of the fusion protein. Exemplary tags that can be used in the practice of the present disclosure include His tag, Strep tag, TAP tag, S tag, SBP tag, Arg tag, calmodulin-binding peptide tag, cellulose-binding domain tag, DsbA tag, c-myc tag, glutathione S-transferase tag, FLAG tag, HAT tag, maltose-binding protein tag, NusA tag, and thioredoxin tag.

[0134] In certain embodiments, the fusion protein comprises a targeting sequence. Exemplary targeting sequences that can be used in the practice of the present disclosure include secretory protein signal sequences, membrane protein signal sequences, nuclear localization sequences, nucleolar localization signal sequences, endoplasmic reticulum localization sequences, peroxisome localization sequences, mitochondrial localization sequences, and protein-protein interaction motif sequences. Examples of targeting sequences include nuclear (e.g., KKKRK, SEQ ID NO: 41), mitochondrial (e.g., TIFF2025032195000029.tif4128), endoplasmic reticulum (e.g., KDEL, SEQ ID NO: 43), peroxisome (e.g., SKL), synapse (e.g., S / TDV or fusion to GAP43, kinesin or tau), plasma membrane (e.g., CaaX), where "a" is an aliphatic amino acid, CC, CXC, CCXX at the C-terminus, or targeting a protein-protein interaction motif (e.g., SH2, SH3, PDZ, WW, RGD, Src homology domain, DNA binding domain, SLiM).

[0135] In certain embodiments, the fusion protein comprises a detectable label. Detectable labels may include any molecule that can be detected. Detectable labels that can be used in the practice of the present disclosure include, but are not limited to, radioisotopes, stable (non-radioactive) heavy isotopes, fluorophores, chemiluminescent materials, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin or haptens), and the like. Specific examples of labels that can be used with the present disclosure include radioactive labels (e.g., H, I, S, C, or P), stable (non-radioactive) heavy isotopes (e.g., 13 C or 15N), phycoerythrin, Alexa dyes, fluorescein, 7-nitrobenzo-2-oxa-1,3-diazole (NBD), YPet, CyPet, cascade blue, allophycocyanin, Cy3, Cy5, Cy7, rhodamine, dansyl, umbelliferone, Texas Red, luminol, acladim esters, biotin or other streptavidin-binding proteins, magnetic beads, electron-dense reagents, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein Examples of suitable enzyme tags include, but are not limited to, fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), blue fluorescent protein (BFP), red fluorescent protein (RFP), Dronpa, Padron, mApple, mCherry, rsCherry, rsCherryRev, firefly luciferase, Renilla luciferase, NADPH, beta-galactosidase, horseradish peroxidase, glucose oxidase, alkaline phosphatase, chloramphenicol acetyltransferase, and urease. Enzyme tags are used with their cognate substrates.These terms also include color-coded microspheres of known fluorescent intensity (see, e.g., microspheres using xMAP technology made by Luminex (Austin, TX)), quantum dot nanocrystals, e.g., microspheres containing different ratios and combinations of quantum dot colors (see, e.g., Qdot nanocrystals made by Life Technologies (Carlsbad, CA)), glass-coated metal nanoparticles (see, e.g., SERS nanotags made by Nanoplex Technologies, Inc. (Mountain View, CA)), barcode materials (see, e.g., submicron sized striped metal rods such as the Nanobarcodes produced by Nanoplex Technologies, Inc.), coded microparticles with colored barcodes (see, e.g., CellCards made by Vitra Bioscience, vitrabio.com), and glass microparticles with digital holographic code images (see, e.g., CyVera microbeads made by Illumina (San Diego, CA)). As with many standard procedures associated with the practice of this disclosure, those of skill in the art will recognize additional labels that can be used.

[0136] Polypeptide of interest In one aspect, the present disclosure provides a fusion protein comprising a polypeptide of interest. The polypeptide of interest selected for inclusion in the fusion protein may be derived from a membrane protein, a receptor, a hormone, a transport protein, a transcription factor, a cytoskeletal protein, an extracellular matrix protein, a signal transduction protein, an enzyme, or any other protein of interest. The polypeptide of interest may comprise an entire protein, or a biologically active domain (e.g., a catalytic domain, a ligand binding domain, or a protein-protein interaction domain), or a polypeptide fragment of the selected protein. In some embodiments, the polypeptide of interest comprises one or more functional and / or structural domains. In some embodiments, the polypeptide of interest comprises multiple functional and / or structural domains.

[0137] In some embodiments, the polypeptide of interest is a therapeutic protein. Examples of suitable therapeutic proteins include, but are not limited to, receptors, antibodies, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytic agents.

[0138] In some embodiments, the polypeptide of interest is a receptor, such as an inducible receptor. Examples of suitable receptors include, but are not limited to, T cell receptors (TCRs), chimeric T cell receptors, artificial T cell receptors, synthetic T cell receptors, chimeric immune receptors, antibody-binding T cell receptors (ACTRs), T cell receptor fusion constructs (TRUCs), and chimeric antigen receptors (CARs).

[0139] In some embodiments, the polypeptide of interest is a cytokine, such as a pro-inflammatory cytokine or an anti-inflammatory cytokine. Examples of suitable cytokines include, but are not limited to, IL-2, IL-7, IL-12, IL-15, IL-18, and IL-21.

[0140] Inducible receptors In one aspect, the polypeptide of interest of the present disclosure is an inducible cellular receptor that comprises an extracellular protein binding domain, a first intracellular signaling domain, and a transmembrane domain located between the extracellular protein binding domain and the first intracellular signaling domain, and is operably linked to a fusion protein. In another aspect, the polypeptide of interest of the present disclosure is an inducible cellular receptor that comprises (a) an extracellular protein binding domain, (b) a first intracellular signaling domain, and (c) a transmembrane domain located between the extracellular protein binding domain and the first intracellular signaling domain.

[0141] ON and OFF switch In some embodiments, the disclosure provides a fusion protein with an "OFF switch", where the polypeptide of interest is an inducible receptor that is selectively inactivated in the presence of a protease inhibitor. An exemplary OFF switch provided herein can be a cellular receptor that includes (a) a molecular binding domain (e.g., an extracellular protein binding domain), (b) an intracellular signaling domain, (c) a transmembrane domain (e.g., located between the molecular binding domain and the signaling domain), and (d) a, where the (a)-(d) components are configured such that the cellular receptor is inactivated (does not transmit an intracellular signal) when the inhibitable protease is inhibited. In some embodiments, the a is located at the C-terminus (carboxy terminus) of the polypeptide of interest, at the N-terminus (amino terminus) of the polypeptide of interest, or within a domain of the polypeptide of interest. The presence of the OFF switch allows cleavage by the protease to remove the a, thereby preserving the structural integrity of the receptor and causing degradation of the receptor when a protease inhibitor is added.

[0142] In some embodiments, the present disclosure provides a fusion protein with an "ON switch," where the polypeptide of interest is an inducible receptor that is selectively activated in the presence of a protease inhibitor. An exemplary ON switch provided herein may be a cellular receptor that includes (a) a molecular binding domain (e.g., an extracellular protein binding domain), (b) a signaling domain, (c) a transmembrane domain (e.g., located between the molecular binding domain and the signaling domain), (d) a protease, and (e) a cognate cleavage site, where the (a)-(e) components are configured such that when the protease is inhibited, the cellular receptor is activated (transmits an intracellular signal). Unlike the OFF switches described above, the ON switch does not include a. Rather, with an ON switch, cleavage by a protease removes a functional element of the cellular receptor (e.g., a signaling domain or a protein binding domain), and addition of a protease inhibitor preserves the structural integrity of the receptor.

[0143] The protease and cognate cleavage site of the ON switch may be located between any two domains of the cellular receptor. For example, the protease and cognate cleavage site may be located between the extracellular protein binding domain and the transmembrane domain. In some embodiments, the protease and cognate cleavage site is located between the transmembrane domain and the intracellular signaling domain. In other embodiments, the protease and cognate cleavage site is located between two co-signaling domains. In some embodiments, the domain of the cellular receptor further comprises a ligand operably linked to the ligand binding domain (e.g., the extracellular protein binding domain). In this case, the protease and cognate cleavage site may be located between the ligand and the ligand binding domain.

[0144] In some embodiments, the inducible cell receptor comprises two polypeptides (e.g., a multi-chain receptor). In such embodiments, a recruitment domain can be used to bring two polypeptides together to activate the receptor. A recruitment domain is a protein domain that binds to each other, thus bringing together two different polypeptides, each of which comprises one of a pair of recruitment domains. A pair of recruitment domains is considered to be assembled together when the two domains bind directly to each other, or when the two domains bind to the same (intermediate) molecule. Non-limiting examples of recruitment domain pairs include (a) FK506 binding protein (FKBP) and FKBP, (b) FKBP and calcineurin catalytic subunit A (CnA), (c) FKBP and cyclophilin, (d) FKBP and FKBP-rapamycin related protein (FRB), (e) gyrase B (GyrB) and GyrB, (f) dihydrofolate reductase (DHFR) and DHFR, (g) DmrB and DmrB, (g) PYL and ABI, (h) Cry2 and CIP, and (i) GAI and GID1.

[0145] In some embodiments of the OFF switch, one polypeptide comprises a protein binding domain, a transmembrane domain, a signaling domain, and a first recruitment domain. In some embodiments, a second polypeptide comprises a second recruitment domain that assembles with the first recruitment domain. In some embodiments, a is located within the first polypeptide or within the second polypeptide. In some embodiments, a protease may be located within one (first) polypeptide, while a cognate cleavage site may be located within the other (second) polypeptide.

[0146] In some embodiments of the ON switch, the first polypeptide can comprise a protein binding domain, a transmembrane domain, a signaling domain, a first recruitment domain, and a cognate cleavage site, hi some embodiments, the second polypeptide comprises a second recruitment domain that assembles with (links directly or indirectly to) the protease and the first recruitment domain.

[0147] Also provided herein is a method of modulating the activity of a cellular receptor (e.g., an OFF switch). In some embodiments of the OFF switch, the method includes providing a cell comprising a cellular receptor comprising (a) an extracellular protein binding domain, (b) an intracellular signaling domain, (c) a transmembrane domain located between the protein binding domain and the signaling domain, (d) a, (e) a protease (e.g., NS3 protease), and (f) a cognate cleavage site, wherein the components (a)-(f) are configured such that the cellular receptor is inactivated when the protease is inhibited, and contacting the cell with a protease inhibitor (e.g., simeprevir, danoprevir, asunaprevir, cilprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, or voxiloprevir) that inhibits the activity of the protease, thereby inactivating the cellular receptor.

[0148] In other embodiments of an ON switch, the method includes providing a cell comprising a cellular receptor comprising (a) an extracellular protein binding domain, (b) an intracellular signaling domain, (c) a transmembrane domain located between the protein binding domain and the signaling domain, (d) a protease (e.g., NS3 protease), and (e) a cognate cleavage site, wherein components (a)-(e) are configured such that the cellular receptor is activated when the inhibitable protease is inhibited, and contacting the cell with a protease inhibitor (e.g., simeprevir, danoprevir, asunaprevir, cilprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, or voxiloprevir) that inhibits activity of the protease, thereby activating the cellular receptor.

[0149] Chimeric antigen receptors (CARs) In one aspect, the polypeptide of interest of the present disclosure is a chimeric antigen receptor (CAR). In general, a CAR is an artificial immune cell receptor engineered to recognize and bind to an antigen expressed by a tumor cell. A CAR may typically include an antibody fragment as an antigen-binding domain, a spacer domain, a hydrophobic alpha-helical transmembrane domain, and one or more intracellular signaling / co-signaling domains, such as (but not limited to) CD3-zeta, CD28, 4-1BB, and / or OX40. A CAR may include a signaling domain or at least two co-signaling domains. In some embodiments, a CAR includes three or four co-signaling domains. In some embodiments, a is located at the C-terminus of the CAR.

[0150] Generally, CARs are engineered for T cells or NK cells and are chimeras of the signaling domain of the T cell receptor (TCR) complex and an antigen recognition domain (e.g., a single chain fragment (scFv) of an antibody) (Enblad et al., Human Gene Therapy. 2015;26(8):498-505). T cells expressing CARs are known in the art as CAR T cells.

[0151] There are at least four generations of CARs, each of which contains different components. First generation CARs link an antibody-derived scFv to the CD3 zeta (ζ or z) intracellular signaling domain of the T cell receptor via a hinge and transmembrane domain. Second generation CARs incorporate additional domains, such as CD28, 4-1BB (41BB), or ICOS, to provide a costimulatory signal. Third generation CARs contain two costimulatory domains fused to the TcR CD3-ζ chain. Third generation costimulatory domains may include, for example, a combination of CD3z, CD27, CD28, 4-1BB, ICOS, or OX40. In some embodiments, a CAR contains an endodomain with an ectodomain (e.g., CD3ζ) typically derived from a single chain variable fragment (scFv), a hinge, a transmembrane domain, and one (first generation), two (second generation), or three (third generation) signaling domains derived from CD3Ζ and / or costimulatory molecules (Maude et al., Blood. 2015; 125(26):4017-4023; Kakarla and Gottschalk, Cancer J. 2014; 20(2):151-155).

[0152] In some embodiments, the chimeric antigen receptor (CAR) is a universal cytokine killing redirected T cell (TRUCK), also known as the fourth generation CAR. TRUCK is a CAR redirected T cell used as a means to produce and release transgenic cytokines that accumulate in target tissue, for example, target tumor tissue. The transgenic cytokines are released upon CAR engagement of the target. TRUCK cells can deposit various therapeutic cytokines in the target. This can provide therapeutic concentrations at the target site and avoid systemic toxicity.

[0153] CARs typically differ in their functional properties. The CD3ζ signaling domain of the T cell receptor, when engaged, induces T cell activation and proliferation, but can result in anergy (lack of response by the body's defense mechanisms, resulting in direct induction of peripheral lymphocyte tolerance). Lymphocytes are considered anergic when they do not respond to a particular antigen. The addition of a costimulatory domain in second-generation CARs improved the replicative capacity and persistence of modified T cells. Although similar antitumor effects are observed in vitro with CD28 or 4-1BB CARs, in vivo preclinical studies suggest that 4-1BB CARs may produce superior proliferation and / or persistence. Clinical trials suggest that both of these second-generation CARs can induce substantial T cell proliferation in vivo, but CARs containing the costimulatory domain of 4-1BB seem to persist longer. Third-generation CARs combine multiple signaling domains (costimulation) to enhance efficacy. Fourth generation CARs are additionally modified with constitutive or inducible expression cassettes for transgenic cytokines that are released by the CAR T cells to regulate T cell responses. See, e.g., Enblad et al., Human Gene Therapy. 2015; 26(8): 498-505; Chmielewski and Hinrich, Expert Opinion on Biological Therapy. 2015; 15(8): 1145-1154.

[0154] In some embodiments, the chimeric antigen receptor of the present disclosure is a first generation CAR. In some embodiments, the chimeric antigen receptor of the present disclosure is a second generation CAR. In some embodiments, the chimeric antigen receptor of the present disclosure is a third generation CAR. In some embodiments, the chimeric antigen receptor of the present disclosure is a fourth generation CAR.

[0155] In some embodiments, a spacer domain or hinge domain is located between the extracellular domain (e.g., including the antigen binding domain) and the transmembrane domain of the CAR, or between the cytoplasmic signaling domain and the transmembrane domain of the CAR. A spacer domain is any oligopeptide or polypeptide that functions to link a transmembrane domain to an extracellular domain and / or a cytoplasmic signaling domain in a polypeptide chain. A hinge domain is any oligopeptide or polypeptide that functions to provide flexibility to the CAR or a domain thereof or to prevent steric hindrance of the CAR or a domain thereof. In some embodiments, a spacer domain or hinge domain can include up to 300 amino acids (e.g., 10-100 amino acids, or 5-20 amino acids). In some embodiments, one or more spacer domain(s) can be included in other regions of the CAR.

[0156] In some embodiments, the CAR is an antigen-specific inhibitory CAR (iCAR), which can be used, for example, to avoid extratumoral toxicity (Fedorov, VD et al. Sci. Transl. Med. 2013, incorporated herein by reference). iCARs contain antigen-specific inhibitory receptors, for example, to block non-specific immune suppression that can result from extratumoral target expression. iCARs can be based, for example, on the inhibitory molecules CTLA-4 or PD-1. In some embodiments, these iCARs block T cell responses from T cells activated by either endogenous T cell receptors or activated CARs. In some embodiments, this inhibitory effect is temporary.

[0157] In some embodiments, CARs may be used in adoptive cell transfer, where immune cells are removed from a subject and modified to express a receptor specific for an antigen, e.g., a tumor-specific antigen. The modified immune cells, which can recognize and kill cancer cells, are then reintroduced into the subject (Pule, et al., Cytotherapy. 2003; 5(3): 211-226; Maude et al., Blood. 2015; 125(26): 4017-4023, each of which is incorporated herein by reference).

[0158] Multi-part CAR In some embodiments, the polypeptide of interest of the present disclosure is a single-chain (polypeptide) cellular receptor or a multi-chain (and therefore multipartite) receptor. Thus, an ON or OFF switch can comprise a single polypeptide, or at least two polypeptides.

[0159] In some embodiments of the OFF switch, the CAR is a multipartite receptor comprising at least two polypeptides. In some embodiments, the CAR comprises a first polypeptide comprising (a) an extracellular protein binding domain (e.g., an antibody fragment), (b) a signaling domain, (c) a transmembrane domain located between the extracellular protein binding domain and the signaling domain, and (d) a first recruitment domain, and a second polypeptide comprising a second recruitment domain that assembles with the signaling domain and the first recruitment domain, where a is located within the first polypeptide and / or the second polypeptide. In some embodiments, a is located at the C-terminus of the first polypeptide and / or the second polypeptide.

[0160] In other embodiments of the OFF switch, the CAR comprises a first polypeptide comprising (a) an extracellular protein binding domain (e.g., an antibody fragment), (b) a signaling domain, (c) a transmembrane domain located between the extracellular protein binding domain and the signaling domain, and (d) a first recruitment domain, and a second polypeptide comprising a second recruitment domain that assembles with the first recruitment domain, wherein the protease is located within the first polypeptide and a cognate cleavage site and a are located within the second polypeptide, or the protease is located within the second polypeptide and a cognate cleavage site and a are located within the first polypeptide. In some embodiments, a is located at the C-terminus of the first polypeptide and / or the second polypeptide.

[0161] In some embodiments of the ON switch, the CAR comprises a first polypeptide comprising: (a) an extracellular protein binding domain (e.g., an antibody fragment); (b) a first intracellular signaling domain; (c) a transmembrane domain located between the antibody fragment and the intracellular signaling domain; (d) a second intracellular signaling domain; and (d) a first recruitment domain, and a second polypeptide comprising a protease and a second recruitment domain that assembles with the first recruitment domain, wherein a cognate cleavage site is located between the antibody fragment and the transmembrane domain, between the transmembrane domain and the first intracellular signaling domain, or between the first intracellular signaling domain and the second intracellular signaling domain.

[0162] In other embodiments of the ON switch, the CAR comprises a first polypeptide comprising: (a) an extracellular protein binding domain (e.g., an antibody fragment); (b) a first intracellular signaling domain; (c) a transmembrane domain located between the antibody fragment and the intracellular signaling domain; (d) a second intracellular signaling domain; and (d) a first recruitment domain, and a second polypeptide comprising a protease and a second recruitment domain that assembles with the first recruitment domain, wherein a cognate cleavage site is located between the antibody fragment and the transmembrane domain, between the transmembrane domain and the first intracellular signaling domain, or between the first intracellular signaling domain and the second intracellular signaling domain.

[0163] Additional CAR adjustment switch In some embodiments, a (e.g., an OFF switch) and / or a protease / cognate cleavage site (e.g., an ON switch) may be combined with an orthogonal CAR regulatory switch to obtain, for example, a logic gate (e.g., AND, OR, NOR, and conditional ON gates) with at least two agent (e.g., drug) inputs that perform a higher order function.

[0164] In some embodiments, a CAR comprises a first polypeptide comprising (a) an extracellular protein binding domain (e.g., an antibody fragment), (b) a signaling domain, (c) a transmembrane domain located between the extracellular protein binding domain and the signaling domain, (d) a first recruitment domain, (e) a, (f) a protease, and (d) a cognate cleavage site, and a second polypeptide comprising a second recruitment domain that assembles with the signaling domain and the first recruitment domain only when the CAR is contacted with an agent required for assembly of the first recruitment domain and the second recruitment domain. In some embodiments, a method of modulating the activity of a CAR comprises contacting a cell comprising the CAR with (a) a protease inhibitor that inhibits activity of the protease, and (b) an agent required for assembly of the first recruitment domain with the second recruitment domain, thereby activating the CAR.

[0165] In other embodiments, a CAR comprises a first polypeptide comprising (a) an extracellular protein binding domain (e.g., an antibody fragment), (b) a signaling domain, (c) a transmembrane domain located between the antibody fragment and the signaling domain, (d) a first recruitment domain, (e) a, (f) a protease, and (g) a cognate cleavage site, and a second polypeptide comprising a second recruitment domain that assembles with the signaling domain and the first recruitment domain unless the CAR is contacted with an agent that prevents assembly of the first recruitment domain with the second recruitment domain. In some embodiments, a method of modulating the activity of a CAR comprises contacting a cell comprising the CAR with (a) a protease inhibitor that inhibits activity of the protease, and (b) an agent that prevents assembly of the first recruitment domain with the second recruitment domain, thereby inactivating the CAR.

[0166] In yet other embodiments, the CAR comprises (a) an antibody fragment, (b) a signaling domain, (c) a transmembrane domain located between the antibody fragment and the signaling domain, (d) a first recruitment domain, and (e) a first polypeptide comprising a protease and a cognate cleavage site, the protease and cognate cleavage site being located between the signaling domain and the first recruitment domain, and a second polypeptide comprising a second recruitment domain that assembles with the signaling domain and the first recruitment domain only when the CAR is contacted with an agent required for assembly of the first recruitment domain with the second recruitment domain. In some embodiments, a method of modulating the activity of a CAR comprises contacting a cell comprising the CAR with (a) a protease inhibitor that inhibits activity of the protease, and (b) an agent required for assembly of the first recruitment domain and the second recruitment domain, thereby activating the CAR.

[0167] In yet other embodiments, the CAR comprises a first polypeptide comprising (a) an antibody fragment, (b) a signaling domain, (c) a transmembrane domain located between the antibody fragment and the signaling domain, and (d) a first recruitment domain, and a second polypeptide comprising a second recruitment domain that assembles with the first recruitment domain only when the CAR is contacted with an agent necessary for assembly of the first recruitment domain with the second recruitment domain, the CAR further comprising a, a protease, a cognate cleavage site, the cognate cleavage site located at the C-terminus of the first polypeptide, and the protease located at the C-terminus of the second polypeptide. In some embodiments, a method of modulating the activity of a CAR comprises contacting a cell comprising the CAR with an agent necessary for assembly of the first recruitment domain with the second recruitment domain, thereby activating the CAR. The method may further comprise contacting the cell with a protease inhibitor that inhibits activity of the protease, thereby inactivating the CAR.

[0168] In some embodiments, the CAR comprises a first polypeptide comprising (a) an antibody fragment, (b) a signaling domain, (c) a transmembrane domain located between the antibody fragment and the signaling domain, (d) a first recruitment domain, (e) an inhibitory domain, and (f) a protease and cognate cleavage site located between the first recruitment domain and the inhibitory domain, and a second polypeptide comprising a second recruitment domain that assembles with the first recruitment domain only when the CAR is contacted with an agent required for assembly of the first recruitment domain with the second recruitment domain. In some embodiments, the method of modulating the activity of a CAR comprises contacting a cell comprising the CAR with an agent required for assembly of the first recruitment domain with the second recruitment domain, thereby activating the CAR. The method may further comprise contacting the cell with a protease that inhibits the activity of the protease, thereby inactivating the CAR.

[0169] The ability of the construct to produce a fusion protein can be determined empirically (eg, detecting EGFP- or AHA-tagged fusion protein by fluorescence microscopy or immunoblotting, respectively).

[0170] Additionally, the production, and in certain embodiments, degradation of the polypeptide of interest can be monitored in the presence and absence of protease inhibitors. The presence of a protease inhibitor prevents the accumulation of new protein copies without affecting the old copies, so the overall level of the polypeptide of interest after adding the protease inhibitor depends on its degradation rate. Thus, the half-life of the polypeptide of interest in the cell can be easily calculated by monitoring its decay. Additionally, the turnover of the polypeptide of interest can be determined by measuring the amount of the polypeptide of interest in the transformed cells before and after contacting the cells with a protease inhibitor, and calculating the turnover of the polypeptide of interest based on the amount of the polypeptide of interest in the cells before and after adding the protease inhibitor. The amount of the polypeptide of interest in the cells can be measured continuously or periodically over a period of time by any suitable method (e.g., immunoblotting or microscopy).

[0171] Production of fusion proteins The fusion proteins of the present disclosure may be produced using recombinant techniques well known in the art. Those skilled in the art can easily determine the nucleotide sequence encoding the desired polypeptide using standard methodology and the teachings herein. Oligonucleotide probes can be devised based on the known sequences and used to probe genomic or cDNA libraries. The sequences can then be further isolated using standard techniques, for example, using restriction enzymes employed to cleave the gene at the desired portion of the full-length sequence. Similarly, known techniques such as phenol extraction can be used to directly isolate the sequence of interest from cells and tissues that contain it, and the sequence can be further manipulated to produce the desired truncation. For a description of techniques used to obtain and isolate DNA, see, for example, Sambrook et al., supra.

[0172] The sequence encoding the polypeptide can also be synthetically generated, for example, based on a known sequence. The nucleotide sequence can be designed with the appropriate codons for the specific amino acid sequence desired. The complete sequence is generally assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, for example, Edge (1981) Nature 292:756; Nambair et al. (1984) Science 223:1299; Jay et al. (1984) J.Biol.Chem.259:6311; Stemmer et al. (1995) Gene 164:49-53.

[0173] Recombinant techniques can be readily used to clone sequences encoding polypeptides useful in the claimed fusion proteins, which can then be mutated in vitro by substitution of the appropriate base pair(s) to provide the codon for the desired amino acid. Such changes can involve as little as one base pair, resulting in a single amino acid change, or can encompass several base pair changes.

[0174] Alternatively, mutations can be effected using mismatched primers that hybridize to the parent nucleotide sequence (generally the cDNA corresponding to the RNA sequence) at a temperature below the melting temperature of the mismatched duplex. Primers can be specifically made by keeping primer length and base composition within relatively narrow limits and centering the mutant base. See, for example, Innis et al., (1990) PCR Applications: Protocols for Functional Genomics; Zoller and Smith, Methods Enzymol. (1983) 100:468. Primer extension is effected using DNA polymerase, and clones containing the cloned products and mutated DNA are derived by separation of the selected primer-extended strand.

[0175] Selection can be achieved using mutant primers as hybridization probes. This technique is also applicable to the generation of multiple point mutations. See, e.g., Dalbie-McFarland et al. Proc. Natl. Acad. Sci USA (1982) 79:6409.

[0176] Once the coding sequences have been isolated and / or synthesized, they can be cloned into any suitable vector or replicon for expression. As is evident from the teachings herein, a variety of vectors encoding modified polypeptides can be produced by creating expression constructs that are operably linked, in various combinations, to polynucleotides encoding polypeptides having deletions or mutations therein.

[0177] Many cloning vectors are known to those skilled in the art, and the selection of a suitable cloning vector is a matter of choice. Examples of recombinant DNA vectors for cloning and the host cells they can transform include bacteriophage λ (E. coli), pBR322 (E. coli), pACYC177 (E. coli), pKT230 (gram-negative bacteria), pGVl106 (gram-negative bacteria), pLAFRl (gram-negative bacteria), pME290 (non-E. coli gram-negative bacteria), pHV14 (E. coli and Bacillus subtilis), pBD9 (Bacillus), pU61 (Streptomyces), pUC6 (Streptomyces), YIp5 (Saccharomyces), YCpl9 (Saccharomyces), and bovine papilloma virus (mammalian cells). See generally, DNA Cloning: Vols. I & II, supra; Sambrook et al., supra; B. Perbal, supra.

[0178] Insect cell expression systems such as the baculovirus system can also be used and are known to those skilled in the art and described, for example, in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Materials and methods for baculovirus / insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego Calif. ("MaxBac" kit).

[0179] Plant expression systems can also be used to produce the fusion proteins described herein. Generally, such systems use virus-based vectors to transfect plant cells with heterologous genes. For a description of such systems, see, for example, Porta et al., Mol. Biotech. (1996) 5:209-221, and Hackland et al., Arch. Virol. (1994) 139:1-22.

[0180] Viral systems such as the vaccinia-based infection / transfection system described in Tomei et al., J. Virol. (1993) 67:4017-4026 and Selby et al., J. Gen. Virol. (1993) 74:1103-1113 will also find use in the present disclosure. In this system, cells are first transfected in vitro with a vaccinia virus recombinant encoding the bacteriophage T7 RNA polymerase. This polymerase exhibits exquisite specificity in that it only transcribes templates that carry a T7 promoter. After infection, cells are transfected with DNA of interest driven by the T7 promoter. The polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA, which is then translated into protein by the host translation machinery. The method provides high-level, transient, cytoplasmic production of large amounts of RNA and its translation product(s).

[0181] The gene can be placed under the control of a promoter, a ribosome binding site (for bacterial expression), and optionally an operator (collectively referred to herein as "control" elements) so that the DNA sequence encoding the desired polypeptide is transcribed into RNA in a host cell transformed by a vector containing this expression construct. The coding sequence may or may not contain a signal peptide or leader sequence. Both naturally occurring signal peptides and heterologous sequences can be used in the present disclosure. Leader sequences can be removed by the host in post-translational processing. See, for example, U.S. Patent Nos. 4,431,739, 4,425,437, and 4,338,397. Such sequences include, but are not limited to, the TPA leader, as well as the honeybee melittin signal sequence.

[0182] Other regulatory sequences may be desirable that allow the expression of protein sequences to be regulated relative to the growth of host cells. Such regulatory sequences are known to those skilled in the art, and examples include those that turn on or off the expression of genes in response to chemical or physical stimuli, including the presence of regulatory compounds. Other types of regulatory elements may also be present in vectors, such as enhancer sequences.

[0183] The control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into the vector. Alternatively, the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site.

[0184] In some cases, it may be necessary to modify the coding sequence so that it can be attached to the control sequences in the proper orientation, i.e., to maintain the proper reading frame. Mutants or analogs can be prepared by deleting portions of the protein-encoding sequence, inserting sequences, and / or substituting one or more nucleotides within the sequence. Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, are well known to those skilled in the art. See, for example, Sambrook et al., supra; DNA Cloning, Vols. I and II, supra; and Nucleic Acid Hybridization, supra.

[0185] The expression vector is then used to transform a suitable host cell. Many mammalian cell lines are known in the art, including, but not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC), such as Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), Vero293 cells, etc. Similarly, bacterial hosts such as E. coli, Bacillus subtilis, and Streptococcus species will find use with the present expression constructs. Yeast hosts useful in the present disclosure include, among others, Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica. Insect cells for use with baculovirus expression vectors include Aedes aegypti, Autographa calif or nica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni, among others.

[0186] Depending on the expression system and host selected, the fusion proteins of the present disclosure are produced by growing host cells transformed with the above-mentioned expression vectors under conditions in which the protein of interest is expressed. The selection of appropriate growth conditions is within the skill of the art.

[0187] In one embodiment, the transformed cells secrete the polypeptide product into the surrounding medium. Certain regulatory sequences can be included in the vector to enhance secretion of the protein product, for example, using the tissue plasminogen activator (TP A) leader sequence, interferon (y or a) signal sequence, or other signal peptide sequences from known secreted proteins. The secreted polypeptide product can then be isolated by various techniques described herein, for example, but not limited to, using standard purification techniques such as hydroxyapatite resin, column chromatography, ion exchange chromatography, size exclusion chromatography, electrophoresis, HPLC, immunoadsorption techniques, affinity chromatography, immunoprecipitation, etc. Alternatively, the transformed cells are disrupted using chemical, physical, or mechanical means, which lyses the cells while keeping the recombinant polypeptide substantially intact. Intracellular proteins can also be obtained by removing components from the cell wall or membrane, for example, by the use of detergents or organic solvents, such that leakage of the polypeptide occurs. Such methods are known to those skilled in the art and are described, for example, in Protein Purification Applications: A Practical Approach, (Simon Roe, Ed., 2001).

[0188] For example, methods of disrupting cells for use with the present disclosure include, but are not limited to, sonication or ultrasonication, agitation, liquid or solid extrusion, heat treatment, freeze-thawing, drying, explosive vacuum, osmotic shock, treatment with lytic enzymes including proteases such as trypsin, neuraminidase and lysozyme, alkaline treatment, and the use of detergents and solvents such as bile salts, sodium dodecyl sulfate, Triton, P40 and CHAPS. The particular technique used to disrupt cells is largely a matter of choice and will depend on the cell type in which the polypeptide is expressed, the culture conditions, and any pretreatments used.

[0189] After cell disruption, cell debris is generally removed by centrifugation, and the polypeptides produced within the cells are further purified using standard purification techniques, including, but not limited to, column chromatography, ion exchange chromatography, size exclusion chromatography, electrophoresis, FIPLC, immunoadsorption techniques, affinity chromatography, and immunoprecipitation.

[0190] For example, one method for obtaining intracellular polypeptides of the present disclosure involves affinity purification, such as by immunoaffinity chromatography using antibodies {e.g., previously generated antibodies) or by lectin affinity chromatography. Particularly preferred lectin resins are those that recognize mannose moieties, such as, but not limited to, resins derived from Galanthus nivalis agglutinin (GNA), Lens culinaris agglutinin (LCA or lentil lectin), Pisum sativum agglutinin (PSA or pea lectin), Narcissus pseudonarcissus agglutinin (PA), and Allium ursinum agglutinin (AUA). Selection of a suitable affinity resin is within the skill of one of ordinary skill in the art. After affinity purification, the polypeptides can be further purified by conventional techniques well known in the art, such as any of the techniques described above.

[0191] Polynucleotides encoding fusion proteins In another aspect, the present disclosure provides polynucleotides encoding the fusion proteins of the present disclosure, and vectors comprising such polynucleotides. In some embodiments, the polynucleotide comprises a sequence encoding an inducible cellular receptor (e.g., a CAR), and the sequence encoding the extracellular protein binding domain is adjacent to and in the same reading frame as the sequence encoding the intracellular signaling domain and the transmembrane domain.

[0192] Polynucleotides can be codon-optimized for expression in mammalian cells. In some embodiments, the entire sequence of the polynucleotide is codon-optimized for expression in mammalian cells. Codon optimization refers to the discovery that the frequency of synonymous codons (i.e., codons that code for the same amino acid) coding DNA is biased in different species. Such codon degeneracy allows the same polypeptide to be coded by a variety of nucleotide sequences. Various codon optimization methods are known in the art, including, for example, the methods disclosed in at least U.S. Patent Nos. 5,786,464 and 6,114,148.

[0193] A polynucleotide encoding a fusion protein can be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the polynucleotide, by deriving it from a vector known to contain it, or by isolating it directly from cells and tissues containing it using standard techniques. Alternatively, the polynucleotide can be produced synthetically rather than cloned.

[0194] The polynucleotide may be cloned into a vector. In some embodiments, expression vectors known in the art are used. For example, the polynucleotides described herein may be inserted into an expression vector to generate an expression cassette that can produce a degron fusion protein in a suitable host cell (e.g., tissue, organ, organoid, or subject). The expression cassette typically includes a control element operably linked to the coding sequence, which allows the expression of the gene in vivo in the target species. For example, typical promoters for mammalian cell expression include SV40 early promoter, CMV promoter such as CMV immediate early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP), and herpes simplex virus promoter. Other non-viral promoters, such as the promoter from mouse metallothionein gene, are also found to be used for mammalian expression. Typically, there is also a transcription termination and polyadenylation sequence located 3' of the translation stop codon. There is also a sequence for optimizing the initiation of translation, preferably located 5' of the coding sequence. Examples of transcription terminator / polyadenylation signals include those derived from SV40, as described in Sambrook et al., supra, as well as the bovine growth hormone terminator sequence.

[0195] Enhancer elements can also be used herein to increase the expression level of mammalian constructs. Examples include the SV40 early gene enhancer described in Dijkema et al., EMPO J. (1985) 4:761, the enhancer / promoter derived from the Rous sarcoma virus long terminal repeat (LTR) described in Gorman et al., Proc. Natl. Acad. Sci. USA (1982b) 79:6777, and elements derived from human CMV, such as those contained in the CMV intron A sequence described in Boshart et al., Cell (1985) 41:521.

[0196] The construct encoding the fusion protein can be administered to the subject or introduced into cell, tissue, organ or organoid using standard gene delivery protocol.The method for gene delivery is known in the art.See, for example, U.S. Patent No. 5,399,346, U.S. Patent No. 5,580,859, U.S. Patent No. 5,589,466.The gene can be delivered directly to the subject, or alternatively, can be delivered ex vivo to the cells derived from the subject and reimplanted into the subject.

[0197] Many virus-based systems have been developed for gene transfer into mammalian cells, including adenoviruses, retroviruses (γ-retroviruses and lentiviruses), poxviruses, adeno-associated viruses, baculoviruses, and herpes simplex viruses (see, e.g., Warnock et al. (2011) Methods Mol. Biol. 737:1-25, Walther et al. (2000) Drugs 60(2):249-271, and Lundstrom (2003) Trends Biotechnol. 21(3):117-122, which are incorporated herein by reference).

[0198] For example, retroviruses provide a convenient platform for gene delivery systems. Selected sequences can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of a subject either in vivo or ex vivo. Several retroviral systems have been described (see U.S. Pat. No. 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, AD (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109; and Ferry et al. (2011) Curr Pharm Des. 17(24):2516-2527). Lentiviruses are a class of retroviruses that are particularly useful for delivering polynucleotides to mammalian cells because they are capable of infecting both dividing and non-dividing cells (see, e.g., Lois et al. (2002) Science 295:868-872; Durand et al. (2011) Viruses 3(2):132-159, incorporated herein by reference).

[0199] A number of adenoviral vectors have also been described. Unlike retroviruses, which integrate into the host genome, adenoviruses exist extrachromosomally, thus minimizing the risks associated with insertional mutagenesis (Haj-Ahmad and Graham, J. Virol. (1986) 57:267-274; Bett et al., J. Virol. (1993) 67:5911-5921; Mittereder et al., Human Gene Therapy (1994) 5:717-729; Seth et al., J. Virol. (1994) 68:933-940; Barr et al., Gene Therapy (1994) 1:51-58; Berkner, KL BioTechniques (1988) 6:616-629; and Rich et al., Human Gene Therapy (1993) 4:461-476).

[0200] Additionally, various adeno-associated virus (AAV) vector systems have been developed for gene delivery. AAV vectors can be readily constructed using techniques well known in the art. See, for example, U.S. Patent Nos. 5,173,414 and 5,139,941, International Publication Nos. WO 92 / 01070 (published January 23, 1992) and WO 93 / 03769 (published March 4, 1993), Lebkowski et al., Molec. Cell. Biol. (1988) 8:3988-3996, Vincent et al., Vaccines 90 (1990) (Cold Spring Harbor Laboratory Press), Carter, B. J. Current Opinion in Biotechnology (1992) 3:533-539, Muzyczka, N. Current Topics in Microbiol, and Immunol. (1992) 158:97-129, Kotin, R. M. Human Gene Therapy (1994) 5:793-801, Shelling and See Smith, Gene Therapy (1994) 1:165-169, and Zhou et al., J. Exp. Med. (1994) 179:1867-1875.

[0201] Another vector system that is useful for delivering the polynucleotides of the present disclosure is the enterally administered recombinant poxvirus vaccine described by Small, Jr., PA, et al. (U.S. Patent No. 5,676,950, issued October 14, 1997, incorporated herein by reference).

[0202] Additional viral vectors used to deliver nucleic acid molecules encoding the fusion proteins of the present disclosure include those derived from the pox family of viruses, including vaccinia virus and avian poxvirus. By way of example, vaccinia virus recombinants expressing fusion proteins can be constructed as follows: DNA encoding a particular fusion protein coding sequence is first inserted into an appropriate vector so that it is adjacent to a vaccinia promoter and adjacent vaccinia DNA sequences (e.g., sequences encoding thymidine kinase (TK)). This vector is then used to transfect cells that are simultaneously infected with vaccinia. Homologous recombination serves to insert the gene encoding the coding sequence of interest into the viral genome in addition to the vaccinia promoter. The resulting TK-recombinants can be selected by culturing cells in the presence of 5-bromodeoxyuridine and picking viral plaques that are resistant to it.

[0203] Alternatively, avian pox viruses, such as fowl pox and canary pox viruses, can also be used to deliver genes. Recombinant avian pox viruses expressing immunogens from mammalian pathogens are known to confer protective immunity when administered to non-avian species. The use of avian pox vectors is particularly desirable in humans and other mammalian species, because members of the genus Avian pox can only productively replicate in susceptible avian species and are therefore not infectious in mammalian cells. Methods for producing recombinant avian pox viruses are known in the art and use genetic recombination, as described above for the production of vaccinia viruses. See, for example, WO91 / 12882, WO89 / 03429, and WO92 / 03545.

[0204] Molecular conjugate vectors, such as the adenovirus chimeric vectors described in Michael et al., J. Biol. Chem. (1993) 268:6866-6869 and Wagner et al., Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can also be used for gene delivery.

[0205] Members of the alphavirus genus, such as, but not limited to, vectors derived from Sindbis virus (SIN), Semliki Forest virus (SFV), and Venezuelan equine encephalitis virus (VEE), may also be used as viral vectors to deliver the polynucleotides of the present disclosure. For a description of Sindbis virus-derived vectors useful in the practice of the present methods, see Dubensky et al. (1996) J. Virol. 70:508-519 and International Publication Nos. WO95 / 07995, WO96 / 17072, as well as Dubensky, Jr., TW, et al., U.S. Patent No. 5,843,723, issued December 1, 1998, and Dubensky, Jr., TW, et al., U.S. Patent No. 5,789,245, issued August 4, 1998, both of which are incorporated herein by reference. Particularly preferred are chimeric alphavirus vectors comprised of sequences from Sindbis virus and Venezuelan equine encephalitis virus (see, e.g., Perri et al. (2003) J. Virol. 77:10394-10403 and International Publication Nos. WO 02 / 099035, WO 02 / 080982, WO 01 / 81609, and WO 00 / 61772, which are incorporated by reference in their entireties.

[0206] A vaccinia-based infection / transfection system can be conveniently used to provide inducible, transient expression of a coding sequence of interest (e.g., a fusion protein expression cassette) in a host cell. In this system, cells are first infected in vitro with a vaccinia virus recombinant encoding the bacteriophage T7 RNA polymerase. This polymerase exhibits exquisite specificity in that it only transcribes templates that carry the T7 promoter. After infection, cells are transfected with a polynucleotide of interest, driven by the T7 promoter. The polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA, which is then translated into protein by the host translation machinery. This method provides high-level, transient, cytoplasmic production of large amounts of RNA and its translation products. See, e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990) 87:6743-6747; Fuerst et al., Proc. Natl. Acad. Sci. USA (1986) 83:8122-8126.

[0207] As an alternative approach to infection with vaccinia or avian pox virus recombinants, or to delivery of genes using other viral vectors, an amplification system can be used that results in high levels of expression after introduction into a host cell. Specifically, a T7 RNA polymerase promoter can be engineered that precedes the coding region of the T7 RNA polymerase. Translation of RNA derived from this template produces T7 RNA polymerase, which then transcribes more templates. At the same time, there will be cDNAs whose expression is controlled under the control of the T7 promoter. Thus, some of the T7 RNA polymerase produced from translation of the amplified template RNA will lead to transcription of the desired gene. Since some T7 RNA polymerase is required to initiate amplification, T7 RNA polymerase can be introduced into the cell along with the template(s) to prime the transcription reaction. The polymerase can be introduced as a protein or on a plasmid that encodes the RNA polymerase. For further discussion of T7 systems and their use to transform cells, see, e.g., International Publication No. WO 94 / 26911, Studier and Moffatt, J. Mol. Biol. (1986) 189:113-130, Deng and Wolff, Gene (1994) 143:245-249, Gao et al., Biochem. Biophys. Res. Commun. (1994) 200:1201-1206, Gao and Huang, Nuc. Acids Res. (1993) 21:2867-2872, Chen et al., Nuc. Acids Res. (1994) 22:2114-2120, and U.S. Patent No. 5,135,855.

[0208] The synthetic expression cassette of interest can also be delivered without a viral vector. For example, the synthetic expression cassette can be packaged as DNA or RNA in liposomes before delivery to the subject or the cells derived therefrom. Lipid encapsulation is generally achieved using liposomes that can stably bind or encapsulate and retain nucleic acid. The ratio of concentrated DNA to lipid preparations can vary, but is generally 1:1 (mg DNA:micromoles lipid), or more lipid. For a review of the use of liposomes as carriers for the delivery of nucleic acid, see, for example, Hug and Sleight, Biochim.Biophys.Acta (1991) 1097:1-17; Straubinger et al., in Methods of Enzymology (1983), Vol.101, pp.512-527.

[0209] The liposome preparations used in the present disclosure include cationic (positively charged), anionic (negatively charged) and neutral preparations, with cationic liposomes being particularly preferred.Cationic liposomes have been shown to mediate the intracellular delivery of plasmid DNA (Feigner et al., Proc. Natl. Acad. Sci. USA (1987) 84: 7413-7416), mRNA (Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86: 6077-6081), and purified transcription factors (Debs et al., J. Biol. Chem. (1990) 265; 10189-10192) in a functional form.

[0210] Cationic liposomes are readily available. For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are available under the trade name Lipofectin from GIBCO BRL, Grand Island, NY. (See also Feigner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416). Other commercially available lipids include (DDAB / DOPE) and DOTAP / DOPE (Boerhinger). Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, for example, Szoka et al., Proc. Natl. Acad. Sci. USA (1978) 75:4194-4198, PCT Publication No. WO 90 / 11092, for a description of the synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.

[0211] Similarly, anionic and neutral liposomes are readily available, for example from Avanti Polar Lipids (Birmingham, AL), or can be easily prepared using readily available materials. Such materials include phosphatidylcholine, cholesterol, phosphatidylethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylethanolamine (DOPE), and the like. These materials can also be mixed with DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.

[0212] Liposomes can comprise multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs).The various liposome-nucleic acid complexes are prepared using methods known in the art. For example, Straubinger et al., in METHODS OF IMMUNOLOGY (1983), Vol. 101, pp. 512-527, Szoka et al., Proc. Natl. Acad. Sci. USA (1978) 75:4194-4198, Papahadjopoulos et al. al., Biochim. Biophys. Acta (1975) 394:483, Wilson et al., Cell (1979) 17:77), Deamer and Bangham, Biochim. Biophys. Acta (1976) 443:629, Ostro et al. al.,Biochem.Biophys.Res.Commun.(1977)76:836, Fraley et al. See, e.g., Enoch and Strittmatter, Proc. Natl. Acad. Sci. USA (1979) 76:3348), Enoch and Strittmatter, Proc. Natl. Acad. Sci. USA (1979) 76:145), Fraley et al., J. Biol. Chem. (1980) 255:10431, Szoka and Papahadjopoulos, Proc. Natl. Acad. Sci. USA (1978) 75:145, and Schaefer-Ridder et al., Science (1982) 215:166.

[0213] The DNA and / or peptide(s) can also be delivered in cochleate lipid compositions similar to those described by Papahadjopoulos et al., Biochem. Biophys. Acta (1975) 394:483-491. See also U.S. Patent Nos. 4,663,161 and 4,871,488.

[0214] The expression cassette of interest may also be encapsulated, adsorbed or associated with particulate carriers. Examples of particulate carriers include those derived from polymethylmethacrylate polymers, and microparticles derived from poly(lactide) and poly(lactide-co-glycolide), known as PLG. See, for example, Jeffery et al., Pharm.Res.(1993)10:362-368; McGee JP,et al., J Microencapsul.14(2):197-210,1997; O'Hagan DT,et al., Vaccine 11(2):149-54,1993.

[0215] In addition, other microparticle systems and polymers can be used for in vivo or ex vivo delivery of the nucleic acid of interest. For example, polymers such as polylysine, polyarginine, polyornithine, spermine, spermidine, and conjugates of these molecules are useful for introducing the nucleic acid of interest. Similarly, DEAE-dextran-mediated transfection, calcium phosphate precipitation, or precipitation using other insoluble inorganic salts such as strontium phosphate, aluminum silicate including bentonite and kaolin, chromium oxide, magnesium silicate, talc, etc., may be used with the present method. For a review of delivery systems useful for gene transfer, see, for example, Feigner, PL, Advanced Drug Delivery Reviews (1990) 5:163-187. Peptoids (Zuckerman, RN, et al. U.S. Patent No. 5,831,005, issued November 3, 1998, incorporated herein by reference) can also be used for delivery of the constructs of the present disclosure.

[0216] Additionally, biolistic delivery systems employing particulate carriers such as gold and tungsten are particularly useful for delivering the synthetic expression cassettes of the present disclosure. The particles are coated with the synthetic expression cassette(s) to be delivered using gun powder discharge from a "gene gun" and accelerated to high velocities, typically under a reducing atmosphere. See, for example, U.S. Patent Nos. 4,945,050, 5,036,006, 5,100,792, 5,179,022, 5,371,015, and 5,478,744 for descriptions of such techniques and devices that are therefore useful. Also, needleless injection systems can be used (Davis, HL, et al, Vaccine 12:1503-1509, 1994; Bioject, Inc., Portland, Oreg.).

[0217] Recombinant vectors can be formulated into compositions for delivery to vertebrate subjects. Compositions generally contain one or more "pharmaceutically acceptable excipients or vehicles", such as, for example, water, saline, glycerol, polyethylene glycol, hyaluronic acid, ethanol, etc. In addition, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, surfactants, etc., may be present in such vehicles. Certain facilitators of nucleic acid uptake and / or expression may be included in the composition or may be co-administered.

[0218] Once formulated, the compositions of the present disclosure can be administered directly to a subject (e.g., as described above) using methods such as those described above, or alternatively, can be delivered ex vivo to cells derived from a subject.For example, the methods for ex vivo delivery and reimplantation of transformed cells into a subject are known in the art, and include, for example, dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, lipofectamine and LT-1-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotide(s) in liposome, and direct microinjection of DNA into nucleus.

[0219] Direct delivery of the synthetic expression cassette composition in vivo is generally accomplished with or without a viral vector by injection using either a conventional syringe, a needle-free device such as Bioject™, or a gene gun such as the Accell gene delivery system (PowderMed Ltd, Oxford, England), as described above.

[0220] The present disclosure also includes RNA constructs that can be directly transfected into cells. Methods for generating mRNA for use in transfection involve in vitro transcription (IVT) of a template with specifically designed primers, followed by polyA addition, to produce a construct containing 3' and 5' untranslated sequences ("UTRs") (e.g., 3' and / or 5' UTRs as described herein), a 5' cap (e.g., 5' cap as described herein), and / or an internal ribosome entry site (IRES) (e.g., an IRES as described herein), the nucleic acid to be expressed, and a polyA tail. RNA produced in this manner can efficiently transfect different types of cells.

[0221] cell In one aspect, the present disclosure provides a cell expressing a fusion protein of the present disclosure or comprising a polynucleotide or vector encoding the fusion protein. The cell can be a stem cell, progenitor cell, and / or immune cell modified to express the fusion protein described herein. In some embodiments, a cell line derived from an immune cell is used. As provided herein, non-limiting examples of cells include mesenchymal stem cells (MSCs), natural killer (NK) cells, NKT cells, innate lymphoid cells, mast cells, eosinophils, basophils, macrophages, neutrophils, mesenchymal stem cells, dendritic cells, T cells (e.g., CD8+ T cells, CD4+ T cells, gamma delta T cells, and regulatory T cells (CD4+, FOXP3+, CD25+)), and B cells. In some embodiments, the cell is a stem cell, such as a pluripotent stem cell, an embryonic stem cell, an adult stem cell, a bone marrow stem cell, an umbilical cord stem cell, or other stem cell.

[0222] The cells may be modified to express the fusion proteins provided herein. In some embodiments, the fusion protein comprises an inducible receptor. The inducible receptor may comprise a single chain receptor (i.e., a single fusion protein) or a multi-chain receptor (i.e., multiple fusion proteins). When the inducible cell receptor is a multi-chain receptor, the cell comprises multiple fusion proteins. Thus, the present disclosure provides cells (e.g., cell populations) engineered to express an inducible receptor, such as a chimeric antigen receptor (CAR), which comprises an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

[0223] Pharmaceutical Compositions Pharmaceutical compositions of the present disclosure can include a fusion protein, or cells expressing a fusion protein (e.g., a plurality of fusion protein-expressing cells), as described herein, in combination with one or more pharma- ceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions can include buffers, such as neutral buffered saline, phosphate buffered saline, carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol, proteins, polypeptides, or amino acids, such as glycine, antioxidants, chelating agents, such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), and preservatives.

[0224] The pharmaceutical compositions of the present disclosure may be administered in a manner appropriate to the disease to be treated (or prevented). Appropriate dosages may be determined by clinical trials, but the amount and frequency of administration may be determined by factors such as the condition of the patient and the type and severity of the patient's disease.

[0225] In a preferred embodiment, the pharmaceutical composition is substantially free of contaminants such as endotoxins, mycoplasma, replication-competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD3 / anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, vector packaging cells or plasmid components, bacteria, and fungi. The pharmaceutical composition can be free of bacteria such as Alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilus influenza, Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumonia, and Group A Streptococcus pyogenes.

[0226] Methods for preparing therapeutic cells In one aspect, the disclosure provides methods for preparing modified cells containing fusion proteins for experimental or therapeutic use.

[0227] Ex vivo procedures for generating therapeutic fusion protein modified cells are well known in the art. For example, cells are isolated from a mammal (e.g., human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a fusion protein disclosed herein. The fusion protein modified cells can be administered to a mammalian recipient to provide therapeutic benefits. The mammalian recipient can be a human, and the fusion protein modified cells can be autologous with respect to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic with respect to the recipient. Procedures for ex vivo expansion of hematopoietic stem cells and hematopoietic progenitor cells are described in U.S. Patent No. 5,199,942, which is incorporated herein by reference, and can be applied to the cells of the present disclosure. Other suitable methods are known in the art, and thus the present disclosure is not limited to any particular method of ex vivo expansion of cells.

[0228] How to use In one aspect, the present disclosure provides a type of cell therapy in which a cell population is genetically modified to express a fusion protein provided herein, and the modified cells are administered to a subject in need thereof. In some embodiments, the method includes culturing a cell population (e.g., in a cell culture medium) to a desired cell density (e.g., a cell density sufficient for a particular cell-based therapy). In some embodiments, the cell population is cultured in the absence of a protease inhibitor that inhibits the activity of a protease, or in the presence of a protease inhibitor that inhibits the activity of a protease.

[0229] In another aspect, the present disclosure provides a type of therapy in which a pharmaceutical composition comprising a fusion protein provided herein is administered to a subject in need thereof.

[0230] In some embodiments, the methods include administering a protease inhibitor that inhibits activity of the protease after administration of the modified cells or pharmaceutical composition, hi some embodiments, the methods further include withdrawing the use of the protease inhibitor after administration of the modified cells or pharmaceutical composition.

[0231] In some embodiments, administration of a protease inhibitor to a subject induces degradation of a polypeptide of interest. In some embodiments, administration of a protease inhibitor protects a polypeptide of interest from degradation. In some embodiments, withdrawal of the use of a protease inhibitor in a subject induces degradation of a polypeptide of interest. In some embodiments, withdrawal of the use of a protease inhibitor in a subject protects a polypeptide of interest from degradation.

[0232] In some embodiments, administration of a protease inhibitor to a subject induces activation of the polypeptide of interest. In some embodiments, administration of a protease inhibitor induces inhibition of the polypeptide of interest. In some embodiments, withdrawal of the protease inhibitor in a subject induces activation of the polypeptide of interest. In some embodiments, withdrawal of the protease inhibitor in a subject induces inhibition of the polypeptide of interest.

[0233] In some embodiments, the population of cells is cultured in the presence of a protease inhibitor that inhibits the activity of the protease to degrade the polypeptide of interest and produce an expanded cell population. For example, in some embodiments, the fusion protein comprises a located at the C-terminus of the polypeptide of interest such that when the cells are cultured in the presence of the protease inhibitor, the protease is inactivated, e.g., unable to cleave the cognate cleavage site that separates the C-terminus of the polypeptide of interest from the degron. Thus, the degron remains fused to the polypeptide of interest and promotes degradation of the polypeptide via either the proteasome or the autophagic-lysosomal pathway. This is particularly advantageous, for example, when the polypeptide of interest is a product that is toxic to the cell or inhibits cell survival and / or cell proliferation / expansion.

[0234] In some embodiments, the cell population is cultured for a period of time that results in the production of an expanded cell population that contains at least twice the number of cells of the starting population. In some embodiments, the cell population is cultured for a period of time that results in the production of an expanded cell population that contains at least four times the number of cells of the starting population. In some embodiments, the cell population is cultured for a period of time that results in the production of an expanded cell population that contains at least 16 times the number of cells of the starting population.

[0235] In some embodiments, the method further comprises withdrawing the use of protease inhibitors in the expanded cell population. The protease inhibitors can be removed, for example, by simply washing the cells with fresh culture medium. In the absence of the protease inhibitors, the cells are capable of producing the polypeptide of interest in vivo, for example, after administration of the cells to a subject in need.

[0236] Thus, in some embodiments, the method includes delivering cells of the expanded cell population to a subject in need of a cell-based therapy. In some embodiments, the subject is a human subject. In some embodiments, the subject in need has cancer (e.g., primary or metastatic cancer).

[0237] Thus, in some embodiments, the polypeptide of interest encodes a therapeutic protein. Examples of therapeutic proteins include, but are not limited to, T cell receptors (TCRs), chimeric T cell receptors, artificial T cell receptors, synthetic T cell receptors, chimeric immunoreceptors, antibody-binding T cell receptors (ACTRs), T cell receptor fusion constructs (TRUCs), chimeric antigen receptors (CARs), antibodies, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytic agents.

[0238] In some embodiments, the method may include administering to the subject a protease inhibitor that inhibits the activity of a protease to degrade the polypeptide of interest. The protease inhibitor may be administered at any time after administration of the cell-based therapy (the expanded cells containing the polypeptide of interest). In some embodiments, the protease inhibitor is administered 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 9 months, 1 year, 2 years, 3 years, 4 years, or 5 years after the subject has received the cell-based therapy. In some embodiments, the protease inhibitor is administered according to the health status of the subject.

[0239] Also provided herein are methods of modulating the activity of a protein of interest either in vivo or ex vivo. In some embodiments, the activity of a protein of interest is modulated in vivo by delivering to a subject in need of cell-based therapy a cell population comprising a polynucleotide encoding a fusion protein of the present disclosure comprising a protein of interest fused to a sequence encoding a degron, and administering to the subject a protease inhibitor that inhibits the activity of a protease to degrade the protein of interest. In some embodiments, the protein of interest is a therapeutic protein. In some embodiments, the method can include ceasing the use of a protease inhibitor that inhibits the activity of a protease in the subject. The use of the protease inhibitor can be ceasing at any time after administration of the cell-based therapy (the expanded cells containing the gene of interest). In some embodiments, the use of the protease inhibitor is ceasing 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 9 months, 1 year, 2 years, 3 years, 4 years, or 5 years after the subject has received the cell-based therapy. In some embodiments, the use of the protease inhibitor is discontinued for 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 9 months, 1 year, 2 years, 3 years, 4 years, or 5 years. In some embodiments, the use of the protease inhibitor is discontinued depending on the subject's health condition.

[0240] In some embodiments, the activity of the protein of interest is modulated by providing a population of cells comprising a fusion protein or a polynucleotide encoding the fusion protein of the present disclosure and contacting the population of cells with a protease inhibitor that inhibits the activity of the protease. In some embodiments, the method further comprises removing the protease inhibitor from the cell population. The protease inhibitor can be removed any time after contacting the cell population with the protease inhibitor. In some embodiments, the protease inhibitor is removed 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 9 months, 1 year, 2 years, 3 years, 4 years, or 5 years after contacting the cell population with the protease inhibitor. In some embodiments, the protease inhibitor is removed 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 9 months, 1 year, 2 years, 3 years, 4 years, or 5 years. In some embodiments, the cell population is administered to a subject in need of cell-based therapy.

[0241] kit The fusion proteins or nucleic acids encoding them, as well as the conditionally replicating viral vectors, can be provided in a kit with appropriate instructions and other necessary reagents for preparing or using them, as described above. The kit may contain the fusion proteins, and / or recombinant constructs for producing the fusion proteins, and / or the conditionally replicating viral vectors, and / or cells (either already transfected or isolated) in separate containers. Additionally, the kit may contain instructions (e.g., written, tape, VCR, CD-ROM, DVD, Blu-ray, flash drive, etc.) for using the fusion proteins or viral vectors. The kit may further include a protease inhibitor, such as an HCV NS3 protease inhibitor, including, for example, simeprevir, danoprevir, asunaprevir, cilprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and voxiloprevir. The kit may also contain other packaged reagents and materials (e.g., transfection reagents, buffers, media, etc.). EXAMPLES

[0242] Example 1: Single and double deimmunized variants of the NS3 protease / NS4 degron fusion protein Four different fusion proteins were generated that contain: 1) a chimeric antigen receptor (CAR) polypeptide of interest, 2) a variant HCV NS3 protease, 3) a cognate protease cleavage site, and 4) an HCV NS4 degron operably linked to the CAR polypeptide of interest. The four different fusion proteins differ from each other based on one or more mutations in the variant HCV NS3 protease. The mutations of the different fusion proteins were tested to determine whether they could reduce immunogenicity while maintaining protease activity, thereby ensuring control over the CAR. Specifically, the four fusion proteins have the following mutations in the variant HCV NS3 protease:

[0243] Fusion protein 1 (T1080A): Contains a Thr to Ala substitution at the position corresponding to position 1080 of the sequence shown in SEQ ID NO:1.

[0244] Fusion protein 2 (T1080A, V1077A): Contains a Thr to Ala substitution at the position corresponding to position 1080 of the sequence shown in SEQ ID NO:1 and a Val to Ala substitution at the position corresponding to position 1077 of the sequence shown in SEQ ID NO:1.

[0245] Fusion protein 3 (T1080A, W1079A): Contains a Thr to Ala substitution at the position corresponding to position 1080 of the sequence shown in SEQ ID NO:1 and a Trp to Ala substitution at the position corresponding to position 1079 of the sequence shown in SEQ ID NO:1.

[0246] Fusion protein 4 (T1080A, V1081A): Contains a Thr to Ala substitution at the position corresponding to position 1080 of the sequence shown in SEQ ID NO:1 and a Val to Ala substitution at the position corresponding to position 1081 of the sequence shown in SEQ ID NO:1.

[0247] On day 0, all total T cell populations were isolated from peripheral blood mononuclear cells (PBMCs) and stimulated using Dynabeads®. On day 1, T cell populations underwent lentiviral transduction. On day 2, cell medium was changed to remove lentivirus and LentiBlast medium. On day 4, Dynabeads® were removed. On day 7, cell medium was changed and T cells were treated. To test the control of the CAR polypeptide of the fusion protein, a first population was treated with 2 μM asunaprevir, a small molecule inhibitor of hepatitis C, while a second population was left untreated (no ASV). On day 9, flow cytometry using YFP and myc tag (Alexa647) fluorescent tags was performed on ASV-treated and non-ASV-treated T cells to determine the CAR expression levels in the cells.

[0248] Figure 1 shows the normalized % CAR expression in cells transfected to express one of four different fusion proteins. Cells were treated with asunaprevir (+ASV) or left untreated (no ASV). Each value was normalized to CAR expression in T1080A variant expressing untreated cells. Notably, fusion protein 2 (T1080A, V1077A), fusion protein 3 (T1080A, W1079A), and fusion protein 4 (T1080A, V1081A) showed near or even higher (e.g., 100%-150%) levels of CAR expression compared to fusion protein 1 (T1080A), thereby indicating that the additional mutations do not impair degron function. For three of the fusion proteins, namely, fusion protein 1 (T1080A), fusion protein 2 (T1080A, V1077A), and fusion protein 4 (T1080A, V1081A), asunaprevir treatment significantly reduced the relative rate of CAR expression, indicating that inhibition of HCV NS3 protease by asunaprevir directly led to a reduction in CAR expression levels. Collectively, these results demonstrate the controllability of expression of a polypeptide of interest, such as CAR, on deimmunized fusion proteins by using small molecule knockdown (e.g., using asunaprevir).

[0249] array TIFF2025032195000030.tif213166TIFF2025032195000031.tif229166TIFF2025032195000032.tif221166TIFF20250321950 00033.tif229166TIFF2025032195000034.tif221166TIFF2025032195000035.tif223166TIFF2025032195000036.tif164166

[0250] Sequence information SEQUENCE LISTING <110> SENTI BIOSCIENCES, INC. <120> FUSION CONSTRUCTS FOR CONTROLLING PROTEIN FUNCTION <150> US 62 / 797,043 <151> 2019-01-25 <160> 56 <170> PatentIn version 3.5 <210> 1 <211> 3011 <212> PRT <213> Hepacivirus C <400> 1 Met Ser Thr Asn Pro Lys Pro Gln Lys Lys Asn Lys Arg Asn Thr Asn 1 5 10 15 Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly 20 25 30 Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala 35 40 45 Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro 50 55 60 Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Thr Trp Ala Gln Pro Gly 65 70 75 80 Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Cys Gly Trp Ala Gly Trp 85 90 95 Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro 100 105 110 Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys 115 120 125 Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu 130 135 140 Gly Gly Ala Ala Arg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp 145 150 155 160 Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile 165 170 175 Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Val Pro Ala Ser Ala Tyr 180 185 190 Gln Val Arg Asn Ser Thr Gly Leu Tyr His Val Thr Asn Asp Cys Pro 195 200 205 Asn Ser Ser Ile Val Tyr Glu Ala Ala Asp Ala Ile Leu His Thr Pro 210 215 220 Gly Cys Val Pro Cys Val Arg Glu Gly Asn Ala Ser Arg Cys Trp Val 225 230 235 240 Ala Met Thr Pro Thr Val Ala Thr Arg Asp Gly Lys Leu Pro Ala Thr 245 250 255 Gln Leu Arg Arg His Ile Asp Leu Leu Val Gly Ser Ala Thr Leu Cys 260 265 270 Ser Ala Leu Tyr Val Gly Asp Leu Cys Gly Ser Val Phe Leu Val Gly 275 280 285 Gln Leu Phe Thr Phe Ser Pro Arg Arg His Trp Thr Thr Gln Gly Cys 290 295 300 Asn Cys Ser Ile Tyr Pro Gly His Ile Thr Gly His Arg Met Ala Trp 305 310 315 320 Asp Met Met Met Asn Trp Ser Pro Thr Thr Ala Leu Val Met Ala Gln 325 330 335 Leu Leu Arg Ile Pro Gln Ala Ile Leu Asp Met Ile Ala Gly Ala His 340 345 350 Trp Gly Val Leu Ala Gly Ile Ala Tyr Phe Ser Met Val Gly Asn Trp 355 360 365 Ala Lys Val Leu Val Val Leu Leu Leu Phe Ala Gly Val Asp Ala Glu 370 375 380 Thr His Val Thr Gly Gly Ser Ala Gly His Thr Val Ser Gly Phe Val 385 390 395 400 Ser Leu Leu Ala Pro Gly Ala Lys Gln Asn Val Gln Leu Ile Asn Thr 405 410 415 Asn Gly Ser Trp His Leu Asn Ser Thr Ala Leu Asn Cys Asn Asp Ser 420 425 430 Leu Asn Thr Gly Trp Leu Ala Gly Leu Phe Tyr His His Lys Phe Asn 435 440 445 Ser Ser Gly Cys Pro Glu Arg Leu Ala Ser Cys Arg Pro Leu Thr Asp 450 455 460 Phe Asp Gln Gly Trp Gly Pro Ile Ser Tyr Ala Asn Gly Ser Gly Pro 465 470 475 480 Asp Gln Arg Pro Tyr Cys Trp His Tyr Pro Pro Lys Pro Cys Gly Ile 485 490 495 Val Pro Ala Lys Ser Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser 500 505 510 Pro Val Val Val Gly Thr Thr Asp Arg Ser Gly Ala Pro Thr Tyr Ser 515 520 525 Trp Gly Glu Asn Asp Thr Asp Val Phe Val Leu Asn Asn Thr Arg Pro 530 535 540 Pro Leu Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Ser Thr Gly Phe 545 550 555 560 Thr Lys Val Cys Gly Ala Pro Pro Cys Val Ile Gly Gly Ala Gly Asn 565 570 575 Asn Thr Leu His Cys Pro Thr Asp Cys Phe Arg Lys His Pro Asp Ala 580 585 590 Thr Tyr Ser Arg Cys Gly Ser Gly Pro Trp Ile Thr Pro Arg Cys Leu 595 600 605 Val Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Ile Asn Tyr 610 615 620 Thr Ile Phe Lys Ile Arg Met Tyr Val Gly Gly Val Glu His Arg Leu 625 630 635 640 Glu Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asp Leu Glu Asp 645 650 655 Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Thr Thr Thr Gln Trp 660 665 670 Gln Val Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser Thr Gly 675 680 685 Leu Ile His Leu His Gln Asn Ile Val Asp Val Gln Tyr Leu Tyr Gly 690 695 700 Val Gly Ser Ser Ile Ala Ser Trp Ala Ile Lys Trp Glu Tyr Val Val 705 710 715 720 Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg Val Cys Ser Cys Leu Trp 725 730 735 Met Met Leu Leu Ile Ser Gln Ala Glu Ala Ala Leu Glu Asn Leu Val 740 745 750 Ile Leu Asn Ala Ala Ser Leu Ala Gly Thr His Gly Leu Val Ser Phe 755 760 765 Leu Val Phe Phe Cys Phe Ala Trp Tyr Leu Lys Gly Lys Trp Val Pro 770 775 780 Gly Ala Val Tyr Thr Phe Tyr Gly Met Trp Pro Leu Leu Leu Leu Leu 785 790 795 800 Leu Ala Leu Pro Gln Arg Ala Tyr Ala Leu Asp Thr Glu Val Ala Ala Ala 805 810 815 Ser Cys Gly Gly Val Val Leu Val Gly Leu Met Ala Leu Thr Leu Ser 820 825 830 Pro Tyr Tyr Lys Arg Tyr Ile Ser Trp Cys Leu Trp Trp Leu Gln Tyr 835 840 845 Phe Leu Thr Arg Val Glu Ala Gln Leu His Val Trp Ile Pro Pro Leu 850 855 860 Asn Val Arg Gly Gly Arg Asp Ala Val Ile Leu Leu Met Cys Ala Val 865 870 875 880 His Pro Thr Leu Val Phe Asp Ile Thr Lys Leu Leu Leu Ala Val Phe 885 890 895 Gly Pro Leu Trp Ile Leu Gln Ala Ser Leu Leu Lys Val Pro Tyr Phe 900 905 910 Val Arg Val Gln Gly Leu Leu Arg Phe Cys Ala Leu Ala Arg Lys Met 915 920 925 Ile Gly Gly His Tyr Val Gln Met Val Ile Ile Lys Leu Gly Ala Leu 930 935 940 Thr Gly Thr Tyr Val Tyr Asn His Leu Thr Pro Leu Arg Asp Trp Ala 945 950 955 960 His Asn Gly Leu Arg Asp Leu Ala Val Ala Val Glu Pro Val Val Phe 965 970 975 Ser Gln Met Glu Thr Lys Leu Ile Thr Trp Gly Ala Asp Thr Ala Ala 980 985 990 Cys Gly Asp Ile Ile Asn Gly Leu Pro Val Ser Ala Arg Arg Gly Arg 995 1000 1005 Glu Ile Leu Leu Gly Pro Ala Asp Gly Met Val Ser Lys Gly Trp 1010 1015 1020 Arg Leu Leu Ala Pro Ile Thr Ala Tyr Ala Gln Gln Thr Arg Gly 1025 1030 1035 Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn 1040 1045 1050 Gln Val Glu Gly Glu Val Gln Ile Val Ser Thr Ala Ala Gln Thr 1055 1060 1065 Phe Leu Ala Thr Cys Ile Asn Gly Val Cys Trp Thr Val Tyr His 1070 1075 1080 Gly Ala Gly Thr Arg Thr Ile Ala Ser Pro Lys Gly Pro Val Ile 1085 1090 1095 Gln Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Pro Ala 1100 1105 1110 Pro Gln Gly Ser Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser 1115 1120 1125 Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val Ile Pro Val Arg 1130 1135 1140 Arg Arg Gly Asp Ser Arg Gly Ser Leu Leu Ser Pro Arg Pro Ile 1145 1150 1155 Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu Leu Cys Pro Ala 1160 1165 1170 Gly His Ala Val Gly Ile Phe Arg Ala Ala Val Cys Thr Arg Gly 1175 1180 1185 Val Ala Lys Ala Val Asp Phe Ile Pro Val Glu Asn Leu Glu Thr 1190 1195 1200 Thr Met Arg Ser Pro Val Phe Thr Asp Asn Ser Ser Pro Pro Val 1205 1210 1215 Val Pro Gln Ser Phe Gln Val Ala His Leu His Ala Pro Thr Gly 1220 1225 1230 Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly 1235 1240 1245 Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly 1250 1255 1260 Phe Gly Ala Tyr Met Ser Lys Ala His Gly Ile Asp Pro Asn Ile 1265 1270 1275 Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ser Pro Ile Thr Tyr 1280 1285 1290 Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly 1295 1300 1305 Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ala 1310 1315 1320 Thr Ser Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr 1325 1330 1335 Ala Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly 1340 1345 1350 Ser Val Thr Val Pro His Pro Asn Ile Glu Glu Val Ala Leu Ser 1355 1360 1365 Thr Thr Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Leu Glu 1370 1375 1380 Val Ile Lys Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys 1385 1390 1395 Lys Cys Asp Glu Leu Ala Ala Lys Leu Val Ala Leu Gly Ile Asn 1400 1405 1410 Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr 1415 1420 1425 Ser Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly 1430 1435 1440 Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val 1445 1450 1455 Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu 1460 1465 1470 Thr Ile Thr Leu Pro Gln Asp Ala Val Ser Arg Thr Gln Arg Arg 1475 1480 1485 Gly Arg Thr Gly Arg Gly Lys Pro Gly Ile Tyr Arg Phe Val Ala 1490 1495 1500 Pro Gly Glu Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys 1505 1510 1515 Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala 1520 1525 1530 Glu Thr Thr Val Arg Leu Arg Ala Tyr Met Asn Thr Pro Gly Leu 1535 1540 1545 Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Gly Val Phe Thr 1550 1555 1560 Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln 1565 1570 1575 Ser Gly Glu Asn Leu Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val 1580 1585 1590 Cys Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp Gln Met Trp 1595 1600 1605 Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro 1610 1615 1620 Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu Ile Thr Leu Thr 1625 1630 1635 His Pro Val Thr Lys Tyr Ile Met Thr Cys Met Ser Ala Asp Leu 1640 1645 1650 Glu Val Val Thr Ser Thr Trp Val Leu Val Gly Gly Val Leu Ala 1655 1660 1665 Ala Leu Ala Ala Tyr Cys Leu Ser Thr Gly Cys Val Val Ile Val 1670 1675 1680 Gly Arg Val Val Leu Ser Gly Lys Pro Ala Ile Ile Pro Asp Arg 1685 1690 1695 Glu Val Leu Tyr Arg Glu Phe Asp Glu Met Glu Glu Cys Ser Gln 1700 1705 1710 His Leu Pro Tyr Ile Glu Gln Gly Met Met Leu Ala Glu Gln Phe 1715 1720 1725 Lys Gln Lys Ala Leu Gly Leu Leu Gln Thr Ala Ser Arg Gln Ala 1730 1735 1740 Glu Val Ile Ala Pro Ala Val Gln Thr Asn Trp Gln Lys Leu Glu 1745 1750 1755 Thr Phe Trp Ala Lys His Met Trp Asn Phe Ile Ser Gly Ile Gln 1760 1765 1770 Tyr Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Ile Ala 1775 1780 1785 Ser Leu Met Ala Phe Thr Ala Ala Val Thr Ser Pro Leu Thr Thr 1790 1795 1800 Ser Gln Thr Leu Leu Phe Asn Ile Leu Gly Gly Trp Val Ala Ala 1805 1810 1815 Gln Leu Ala Ala Pro Gly Ala Ala Thr Ala Phe Val Gly Ala Gly 1820 1825 1830 Leu Ala Gly Ala Ala Ile Gly Ser Val Gly Leu Gly Lys Val Leu 1835 1840 1845 Ile Asp Ile Leu Ala Gly Tyr Gly Ala Gly Val Ala Gly Ala Leu 1850 1855 1860 Val Ala Phe Lys Ile Met Ser Gly Glu Val Pro Ser Thr Glu Asp 1865 1870 1875 Leu Val Asn Leu Leu Pro Ala Ile Leu Ser Pro Gly Ala Leu Val 1880 1885 1890 Val Gly Val Val Cys Ala Ala Ile Leu Arg Arg His Val Gly Pro 1895 1900 1905 Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu Ile Ala Phe Ala 1910 1915 1920 Ser Arg Gly Asn His Val Ser Pro Thr His Tyr Val Pro Glu Ser 1925 1930 1935 Asp Ala Ala Ala Arg Val Thr Ala Ile Leu Ser Ser Leu Thr Val 1940 1945 1950 Thr Gln Leu Leu Arg Arg Leu His Gln Trp Ile Ser Ser Glu Cys 1955 1960 1965 Thr Thr Pro Cys Ser Gly Ser Trp Leu Arg Asp Ile Trp Asp Trp 1970 1975 1980 Ile Cys Glu Val Leu Ser Asp Phe Lys Thr Trp Leu Lys Ala Lys 1985 1990 1995 Leu Met Pro Gln Leu Pro Gly Ile Pro Phe Val Ser Cys Gln Arg 2000 2005 2010 Gly Tyr Lys Gly Val Trp Arg Val Asp Gly Ile Met His Thr Arg 2015 2020 2025 Cys His Cys Gly Ala Glu Ile Thr Gly His Val Lys Asn Gly Thr 2030 2035 2040 Met Arg Ile Val Gly Pro Arg Thr Cys Arg Asn Met Trp Ser Gly 2045 2050 2055 Thr Phe Pro Ile Asn Ala Tyr Thr Thr Gly Pro Cys Thr Pro Leu 2060 2065 2070 Pro Ala Pro Asn Tyr Thr Phe Ala Leu Trp Arg Val Ser Ala Glu 2075 2080 2085 Glu Tyr Val Glu Ile Arg Gln Val Gly Asp Phe His Tyr Val Thr 2090 2095 2100 Gly Met Thr Thr Asp Asn Leu Lys Cys Pro Cys Gln Val Pro Ser 2105 2110 2115 Pro Glu Phe Phe Thr Glu Leu Asp Gly Val Arg Leu His Arg Phe 2120 2125 2130 Ala Pro Pro Cys Lys Pro Leu Leu Arg Glu Glu Val Ser Phe Arg 2135 2140 2145 Val Gly Leu His Glu Tyr Pro Val Gly Ser Gln Leu Pro Cys Glu 2150 2155 2160 Pro Glu Pro Asp Val Ala Val Leu Thr Ser Met Leu Thr Asp Pro 2165 2170 2175 Ser His Ile Thr Ala Glu Ala Ala Gly Arg Arg Leu Ala Arg Gly 2180 2185 2190 Ser Pro Pro Ser Val Ala Ser Ser Ser Ala Ser Gln Leo Ser Ala 2195 2200 2205 Pro Ser Leu Lys Ala Thr Cys Thr Ala Asn His Asp Ser Pro Asp 2210 2215 2220 Ala Glu Leu Ile Glu Ala Asn Leu Leu Trp Arg Gln Glu Met Gly 2225 2230 2235 Gly Asn Ile Thr Arg Val Glu Ser Glu Asn Lys Val Val Ile Leu 2240 2245 2250 Asp Ser Phe Asp Pro Leu Val Ala Glu Glu Asp Glu Arg Glu Ile 2255 2260 2265 Ser Val Pro Ala Glu Ile Leu Arg Lys Ser Arg Arg Phe Ala Gln 2270 2275 2280 Ala Leu Pro Val Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu Val 2285 2290 2295 Glu Thr Trp Lys Lys Pro Asp Tyr Glu Pro Pro Val Val His Gly 2300 2305 2310 Cys Pro Leu Pro Pro Pro Lys Ser Pro Pro Val Pro Pro Pro Arg 2315 2320 2325 Lys Lys Arg Thr Val Val Leu Thr Glu Ser Thr Leu Ser Thr Ala 2330 2335 2340 Leu Ala Glu Leu Ala Thr Arg Ser Phe Gly Ser Ser Ser Thr Ser 2345 2350 2355 Gly Ile Thr Gly Asp Asn Thr Thr Thr Ser Ser Glu Pro Ala Pro 2360 2365 2370 Ser Gly Cys Pro Pro Asp Ser Asp Ala Glu Ser Tyr Ser Ser Met 2375 2380 2385 Pro Pro Leu Glu Gly Glu Pro Gly Asp Pro Asp Leu Ser Asp Gly 2390 2395 2400 Ser Trp Ser Thr Val Ser Ser Glu Ala Asn Ala Glu Asp Val Val 2405 2410 2415 Cys Cys Ser Met Ser Tyr Ser Trp Thr Gly Ala Leu Val Thr Pro 2420 2425 2430 Cys Ala Ala Glu Glu Gln Lys Leu Pro Ile Asn Ala Leu Ser Asn 2435 2440 2445 Ser Leu Leu Arg His His Asn Leu Val Tyr Ser Thr Thr Ser Arg 2450 2455 2460 Ser Ala Cys Gln Arg Gln Lys Lys Val Thr Phe Asp Arg Leu Gln 2465 2470 2475 Val Leu Asp Ser His Tyr Gln Asp Val Leu Lys Glu Val Lys Ala 2480 2485 2490 Ala Ala Ser Lys Val Lys Ala Asn Leu Leu Ser Val Glu Glu Ala 2495 2500 2505 Cys Ser Leu Thr Pro Pro His Ser Ala Lys Ser Lys Phe Gly Tyr 2510 2515 2520 Gly Ala Lys Asp Val Arg Cys His Ala Arg Lys Ala Val Thr His 2525 2530 2535 Ile Asn Ser Val Trp Lys Asp Leu Leu Glu Asp Asn Val Thr Pro 2540 2545 2550 Ile Asp Thr Thr Ile Met Ala Lys Asn Glu Val Phe Cys Val Gln 2555 2560 2565 Pro Glu Lys Gly Gly Arg Lys Pro Ala Arg Leu Ile Val Phe Pro 2570 2575 2580 Asp Leu Gly Val Arg Val Cys Glu Lys Met Ala Leu Tyr Asp Val 2585 2590 2595 Val Thr Lys Leu Pro Leu Ala Val Met Gly Ser Ser Tyr Gly Phe 2600 2605 2610 Gln Tyr Ser Pro Gly Gln Arg Val Glu Phe Leu Val Gln Ala Trp 2615 2620 2625 Lys Ser Lys Lys Thr Pro Met Gly Phe Ser Tyr Asp Thr Arg Cys 2630 2635 2640 Phe Asp Ser Thr Val Thr Glu Ser Asp Ile Arg Thr Glu Glu Ala 2645 2650 2655 Ile Tyr Gln Cys Cys Asp Leu Asp Pro Gln Ala Arg Val Ala Ile 2660 2665 2670 Lys Ser Leu Thr Glu Arg Leu Tyr Val Gly Gly Pro Leu Thr Asn 2675 2680 2685 Ser Arg Gly Glu Asn Cys Gly Tyr Arg Arg Cys Arg Ala Ser Gly 2690 2695 2700 Val Leu Thr Thr Ser Cys Gly Asn Thr Leu Thr Cys Tyr Ile Lys 2705 2710 2715 Ala Arg Ala Ala Cys Arg Ala Ala Gly Leu Gln Asp Cys Thr Met 2720 2725 2730 Leu Val Cys Gly Asp Asp Leu Val Val Ile Cys Glu Ser Ala Gly 2735 2740 2745 Val Gln Glu Asp Ala Ala Ser Leu Arg Ala Phe Thr Glu Ala Met 2750 2755 2760 Thr Arg Tyr Ser Ala Pro Pro Gly Asp Pro Pro Gln Pro Glu Tyr 2765 2770 2775 Asp Leu Glu Leu Ile Thr Ser Cys Ser Ser Asn Val Ser Val Ala 2780 2785 2790 His Asp Gly Ala Gly Lys Arg Val Tyr Tyr Leu Thr Arg Asp Pro 2795 2800 2805 Thr Thr Pro Leu Ala Arg Ala Ala Trp Glu Thr Ala Arg His Thr 2810 2815 2820 Pro Val Asn Ser Trp Leu Gly Asn Ile Ile Met Phe Ala Pro Thr 2825 2830 2835 Leu Trp Ala Arg Met Ile Leu Met Thr His Phe Phe Ser Val Leu 2840 2845 2850 Ile Ala Arg Asp Gln Leu Glu Gln Ala Leu Asp Cys Glu Ile Tyr 2855 2860 2865 Gly Ala Cys Tyr Ser Ile Glu Pro Leu Asp Leu Pro Pro Ile Ile 2870 2875 2880 Gln Arg Leu His Gly Leu Ser Ala Phe Ser Leu His Ser Tyr Ser 2885 2890 2895 Pro Gly Glu Ile Asn Arg Val Ala Ala Cys Leu Arg Lys Leu Gly 2900 2905 2910 Val Pro Pro Leu Arg Ala Trp Arg His Arg Ala Arg Ser Val Arg 2915 2920 2925 Ala Arg Leu Leu Ala Arg Gly Gly Arg Ala Ala Ile Cys Gly Lys 2930 2935 2940 Tyr Leu Phe Asn Trp Ala Val Arg Thr Lys Leu Lys Leu Thr Pro 2945 2950 2955 Ile Ala Ala Ala Gly Gln Leu Asp Leu Ser Gly Trp Phe Thr Ala 2960 2965 2970 Gly Tyr Ser Gly Gly Asp Ile Tyr His Ser Val Ser His Ala Arg 2975 2980 2985 Pro Arg Trp Ile Trp Phe Cys Leu Leu Leu Leu Ala Ala Gly Val 2990 2995 3000 Gly Ile Tyr Leu Leu Pro Asn Arg 3005 3010 <210> 2 <211> 186 <212> PRT <213> Hepacivirus C <400> 2 Ala Pro Ile Thr Ala Tyr Ala Gln Gln Thr Arg Gly Leu Leu Gly Cys 1 5 10 15 Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln Val Glu Gly Glu 20 25 30 Val Gln Ile Val Ser Thr Ala Thr Gln Thr Phe Leu Ala Thr Cys Ile 35 40 45 Asn Gly Val Cys Trp Ala Val Tyr His Gly Ala Gly Thr Arg Thr Ile 50 55 60 Ala Ser Pro Lys Gly Pro Val Ile Gln Met Tyr Thr Asn Val Asp Gln 65 70 75 80 Asp Leu Val Gly Trp Pro Ala Pro Gln Gly Ser Arg Ser Leu Thr Pro 85 90 95 Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp 100 105 110 Val Ile Pro Val Arg Arg Arg Gly Asp Ser Arg Gly Ser Leu Leu Ser 115 120 125 Pro Arg Pro Ile Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu Leu 130 135 140 Cys Pro Ala Gly His Ala Val Gly Leu Phe Arg Ala Ala Val Cys Thr 145 150 155 160 Arg Gly Val Ala Lys Ala Val Asp Phe Ile Pro Val Glu Asn Leu Glu 165 170 175 Thr Thr Met Arg Ser Pro Val Phe Thr Asp 180 185 <210> 3 <211> 44 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 3 Thr Trp Val Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys 1 5 10 15 Leu Ser Thr Gly Cys Val Val Ile Val Gly Arg Ile Val Leu Ser Gly 20 25 30 Lys Pro Ala Ile Ile Pro Asp Arg Glu Val Leu Tyr 35 40 <210> 4 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 4 Cys Met Ser Ala Asp Leu Glu Val Val Thr Ser Thr Trp Val Leu Val 1 5 10 15 Gly Gly Val Leu 20 <210> 5 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 5 Tyr Gln Glu Phe Asp Glu Met Glu Glu Cys Ser Gln His Leu Pro Tyr 1 5 10 15 Ile Glu Gln Gly 20 <210> 6 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 6 Trp Ile Ser Ser Glu Cys Thr Thr Pro Cys Ser Gly Ser Trp Leu Arg 1 5 10 15 Asp Ile Trp Asp 20 <210> 7 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 7 Gly Ala Asp Thr Glu Asp Val Val Cys Cys Ser Met Ser Tyr Ser Trp 1 5 10 15 Thr Gly Ala Leu 20 <210> 8 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 8 Ala Asp Leu Glu Val Val Thr Ser Thr Trp Leu 1 5 10 <210> 9 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 9 Asp Glu Met Glu Glu Cys Ser Gln His Leu 1 5 10 <210> 10 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 10 Glu Cys Thr Thr Pro Cys Ser Gly Ser Trp Leu 1 5 10 <210> 11 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 11 Glu Asp Val Val Pro Cys Ser Met Gly 1 5 <210> 12 <211> 186 <212> PRT <213> Hepacivirus C <400> 12 Ala Pro Ile Thr Ala Tyr Ala Gln Gln Thr Arg Gly Leu Leu Gly Cys 1 5 10 15 Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln Val Glu Gly Glu 20 25 30 Val Gln Ile Val Ser Thr Ala Ala Gln Thr Phe Leu Ala Thr Cys Ile 35 40 45 Asn Gly Val Cys Trp Thr Val Tyr His Gly Ala Gly Thr Arg Thr Ile 50 55 60 Ala Ser Ser Lys Gly Pro Val Ile Gln Met Tyr Thr Asn Val Asp Gln 65 70 75 80 Asp Leu Val Gly Trp Pro Ala Pro Gln Gly Ala Arg Ser Leu Thr Pro 85 90 95 Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp 100 105 110 Val Ile Pro Val Arg Arg Arg Gly Asp Gly Arg Gly Ser Leu Leu Ser 115 120 125 Pro Arg Pro Ile Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu Leu 130 135 140 Cys Pro Ala Gly His Ala Val Gly Ile Phe Arg Ala Ala Val Cys Thr 145 150 155 160 Arg Gly Val Ala Lys Ala Val Asp Phe Ile Pro Val Glu Gly Leu Glu 165 170 175 Thr Thr Met Arg Ser Pro Val Phe Ser Asp 180 185 <210> 13 <211> 99 <212> PRT <213> Human immunodeficiency virus 1 <400> 13 Pro Gln Val Thr Leu Trp Gln Arg Pro Leu Val Thr Ile Lys Ile Gly 1 5 10 15 Gly Gln Leu Lys Glu Ala Leu Leu Asp Thr Gly Ala Asp Asp Thr Val 20 25 30 Leu Glu Glu Met Ser Leu Pro Gly Arg Trp Lys Pro Lys Met Ile Gly 35 40 45 Gly Ile Gly Gly Phe Ile Lys Val Arg Gln Tyr Asp Gln Ile Leu Ile 50 55 60 Glu Ile Cys Gly His Lys Ala Ile Gly Thr Val Leu Val Gly Pro Thr 65 70 75 80 Pro Val Asn Ile Ile Gly Arg Asn Leu Leu Thr Gln Ile Gly Cys Thr 85 90 95 Leu Asn Phe <210> 14 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 14 Glu Pro Leu Phe Ala Glu Arg Lys 1 5 <210> 15 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 15 Tyr Val Ala Asp 1 <210> 16 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 16 Val Asp Val Ala Asp 1 5 <210> 17 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 17 Asp Glu Val Asp 1 <210> 18 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 18 Val Glu His Asp 1 <210> 19 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 19 Leu Gly His Asp 1 <210> 20 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 20 Leu Gln Thr Asp Gly 1 5 <210> 21 <211> 1306 <212> PRT <213> Homo sapiens <400> 21 Met Gly Ala Ala Ser Gly Arg Arg Gly Pro Gly Leu Leu Leu Pro Leu 1 5 10 15 Pro Leu Leu Leu Leu Leu Pro Pro Gln Pro Ala Leu Ala Leu Asp Pro 20 25 30 Gly Leu Gln Pro Gly Asn Phe Ser Ala Asp Glu Ala Gly Ala Gln Leu 35 40 45 Phe Ala Gln Ser Tyr Asn Ser Ser Ala Glu Gln Val Leu Phe Gln Ser 50 55 60 Val Ala Ala Ser Trp Ala His Asp Thr Asn Ile Thr Ala Glu Asn Ala 65 70 75 80 Arg Arg Gln Glu Glu Ala Ala Leu Leu Ser Gln Glu Phe Ala Glu Ala 85 90 95 Trp Gly Gln Lys Ala Lys Glu Leu Tyr Glu Pro Ile Trp Gln Asn Phe 100 105 110 Thr Asp Pro Gln Leu Arg Arg Ile Ile Gly Ala Val Arg Thr Leu Gly 115 120 125 Ser Ala Asn Leu Pro Leu Ala Lys Arg Gln Gln Tyr Asn Ala Leu Leu 130 135 140 Ser Asn Met Ser Arg Ile Tyr Ser Thr Ala Lys Val Cys Leu Pro Asn 145 150 155 160 Lys Thr Ala Thr Cys Trp Ser Leu Asp Pro Asp Leu Thr Asn Ile Leu 165 170 175 Ala Ser Ser Arg Ser Tyr Ala Met Leu Leu Phe Ala Trp Glu Gly Trp 180 185 190 His Asn Ala Ala Gly Ile Pro Leu Lys Pro Leu Tyr Glu Asp Phe Thr 195 200 205 Ala Leu Ser Asn Glu Ala Tyr Lys Gln Asp Gly Phe Thr Asp Thr Gly 210 215 220 Ala Tyr Trp Arg Ser Trp Tyr Asn Ser Pro Thr Phe Glu Asp Asp Leu 225 230 235 240 Glu His Leu Tyr Gln Gln Leu Glu Pro Leu Tyr Leu Asn Leu His Ala 245 250 255 Phe Val Arg Arg Ala Leu His Arg Arg Tyr Gly Asp Arg Tyr Ile Asn 260 265 270 Leu Arg Gly Pro Ile Pro Ala His Leu Leu Gly Asp Met Trp Ala Gln 275 280 285 Ser Trp Glu Asn Ile Tyr Asp Met Val Val Pro Phe Pro Asp Lys Pro 290 295 300 Asn Leu Asp Val Thr Ser Thr Met Leu Gln Gln Gly Trp Asn Ala Thr 305 310 315 320 His Met Phe Arg Val Ala Glu Glu Phe Phe Thr Ser Leu Glu Leu Ser 325 330 335 Pro Met Pro Pro Glu Phe Trp Glu Gly Ser Met Leu Glu Lys Pro Ala 340 345 350 Asp Gly Arg Glu Val Val Cys His Ala Ser Ala Trp Asp Phe Tyr Asn 355 360 365 Arg Lys Asp Phe Arg Ile Lys Gln Cys Thr Arg Val Thr Met Asp Gln 370 375 380 Leu Ser Thr Val His His Glu Met Gly His Ile Gln Tyr Tyr Leu Gln 385 390 395 400 Tyr Lys Asp Leu Pro Val Ser Leu Arg Arg Gly Ala Asn Pro Gly Phe 405 410 415 His Glu Ala Ile Gly Asp Val Leu Ala Leu Ser Val Ser Thr Pro Glu 420 425 430 His Leu His Lys Ile Gly Leu Leu Asp Arg Val Thr Asn Asp Thr Glu 435 440 445 Ser Asp Ile Asn Tyr Leu Leu Lys Met Ala Leu Glu Lys Ile Ala Phe 450 455 460 Leu Pro Phe Gly Tyr Leu Val Asp Gln Trp Arg Trp Gly Val Phe Ser 465 470 475 480 Gly Arg Thr Pro Pro Ser Arg Tyr Asn Phe Asp Trp Trp Tyr Leu Arg 485 490 495 Thr Lys Tyr Gln Gly Ile Cys Pro Pro Val Thr Arg Asn Glu Thr His 500 505 510 Phe Asp Ala Gly Ala Lys Phe His Val Pro Asn Val Thr Pro Tyr Ile 515 520 525 Arg Tyr Phe Val Ser Phe Val Leu Gln Phe Gln Phe His Glu Ala Leu 530 535 540 Cys Lys Glu Ala Gly Tyr Glu Gly Pro Leu His Gln Cys Asp Ile Tyr 545 550 555 560 Arg Ser Thr Lys Ala Gly Ala Lys Leu Arg Lys Val Leu Gln Ala Gly 565 570 575 Ser Ser Arg Pro Trp Gln Glu Val Leu Lys Asp Met Val Gly Leu Asp 580 585 590 Ala Leu Asp Ala Gln Pro Leu Leu Lys Tyr Phe Gln Pro Val Thr Gln 595 600 605 Trp Leu Gln Glu Gln Asn Gln Gln Asn Gly Glu Val Leu Gly Trp Pro 610 615 620 Glu Tyr Gln Trp His Pro Pro Leu Pro Asp Asn Tyr Pro Glu Gly Ile 625 630 635 640 Asp Leu Val Thr Asp Glu Ala Glu Ala Ser Lys Phe Val Glu Glu Tyr 645 650 655 Asp Arg Thr Ser Gln Val Val Trp Asn Glu Tyr Ala Glu Ala Asn Trp 660 665 670 Asn Tyr Asn Thr Asn Ile Thr Thr Glu Thr Ser Lys Ile Leu Leu Gln 675 680 685 Lys Asn Met Gln Ile Ala Asn His Thr Leu Lys Tyr Gly Thr Gln Ala 690 695 700 Arg Lys Phe Asp Val Asn Gln Leu Gln Asn Thr Thr Ile Lys Arg Ile 705 710 715 720 Ile Lys Lys Val Gln Asp Leu Glu Arg Ala Ala Leu Pro Ala Gln Glu 725 730 735 Leu Glu Glu Tyr Asn Lys Ile Leu Leu Asp Met Glu Thr Thr Tyr Ser 740 745 750 Val Ala Thr Val Cys His Pro Asn Gly Ser Cys Leu Gln Leu Glu Pro 755 760 765 Asp Leu Thr Asn Val Met Ala Thr Ser Arg Lys Tyr Glu Asp Leu Leu 770 775 780 Trp Ala Trp Glu Gly Trp Arg Asp Lys Ala Gly Arg Ala Ile Leu Gln 785 790 795 800 Phe Tyr Pro Lys Tyr Val Glu Leu Ile Asn Gln Ala Ala Arg Leu Asn 805 810 815 Gly Tyr Val Asp Ala Gly Asp Ser Trp Arg Ser Met Tyr Glu Thr Pro 820 825 830 Ser Leu Glu Gln Asp Leu Glu Arg Leu Phe Gln Glu Leu Gln Pro Leu 835 840 845 Tyr Leu Asn Leu His Ala Tyr Val Arg Arg Ala Leu His Arg His Tyr 850 855 860 Gly Ala Gln His Ile Asn Leu Glu Gly Pro Ile Pro Ala His Leu Leu 865 870 875 880 Gly Asn Met Trp Ala Gln Thr Trp Ser Asn Ile Tyr Asp Leu Val Val 885 890 895 Pro Phe Pro Ser Ala Pro Ser Met Asp Thr Thr Glu Ala Met Leu Lys 900 905 910 Gln Gly Trp Thr Pro Arg Arg Met Phe Lys Glu Ala Asp Asp Phe Phe 915 920 925 Thr Ser Leu Gly Leu Leu Pro Val Pro Pro Glu Phe Trp Asn Lys Ser 930 935 940 Met Leu Glu Lys Pro Thr Asp Gly Arg Glu Val Val Cys His Ala Ser 945 950 955 960 Ala Trp Asp Phe Tyr Asn Gly Lys Asp Phe Arg Ile Lys Gln Cys Thr 965 970 975 Thr Val Asn Leu Glu Asp Leu Val Val Ala His His Glu Met Gly His 980 985 990 Ile Gln Tyr Phe Met Gln Tyr Lys Asp Leu Pro Val Ala Leu Arg Glu 995 1000 1005 Gly Ala Asn Pro Gly Phe His Glu Ala Ile Gly Asp Val Leu Ala 1010 1015 1020 Leu Ser Val Ser Thr Pro Lys His Leu His Ser Leu Asn Leu Leu 1025 1030 1035 Ser Ser Glu Gly Gly Ser Asp Glu His Asp Ile Asn Phe Leu Met 1040 1045 1050 Lys Met Ala Leu Asp Lys Ile Ala Phe Ile Pro Phe Ser Tyr Leu 1055 1060 1065 Val Asp Gln Trp Arg Trp Arg Val Phe Asp Gly Ser Ile Thr Lys 1070 1075 1080 Glu Asn Tyr Asn Gln Glu Trp Trp Ser Leu Arg Leu Lys Tyr Gln 1085 1090 1095 Gly Leu Cys Pro Pro Val Pro Arg Thr Gln Gly Asp Phe Asp Pro 1100 1105 1110 Gly Ala Lys Phe His Ile Pro Ser Ser Val Pro Tyr Ile Arg Tyr 1115 1120 1125 Phe Val Ser Phe Ile Ile Gln Phe Gln Phe His Glu Ala Leu Cys 1130 1135 1140 Gln Ala Ala Gly His Thr Gly Pro Leu His Lys Cys Asp Ile Tyr 1145 1150 1155 Gln Ser Lys Glu Ala Gly Gln Arg Leu Ala Thr Ala Met Lys Leu 1160 1165 1170 Gly Phe Ser Arg Pro Trp Pro Glu Ala Met Gln Leu Ile Thr Gly 1175 1180 1185 Gln Pro Asn Met Ser Ala Ser Ala Met Leu Ser Tyr Phe Lys Pro 1190 1195 1200 Leu Leu Asp Trp Leu Arg Thr Glu Asn Glu Leu His Gly Glu Lys 1205 1210 1215 Leu Gly Trp Pro Gln Tyr Asn Trp Thr Pro Asn Ser Ala Arg Ser 1220 1225 1230 Glu Gly Pro Leu Pro Asp Ser Gly Arg Val Ser Phe Leu Gly Leu 1235 1240 1245 Asp Leu Asp Ala Gln Gln Ala Arg Val Gly Gln Trp Leu Leu Leu 1250 1255 1260 Phe Leu Gly Ile Ala Leu Leu Val Ala Thr Leu Gly Leu Ser Gln 1265 1270 1275 Arg Leu Phe Ser Ile Arg His Arg Ser Leu His Arg His Ser His 1280 1285 1290 Gly Pro Gln Phe Gly Ser Glu Val Glu Leu Arg His Ser 1295 1300 1305 <210> 22 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 22 Glu Val Asn Leu Asp Ala Glu Phe 1 5 <210> 23 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 23 Pro Gln Gly Ile Ala Gly Gln 1 5 <210> 24 <211> 7 <212> PRT <213> Tobacco etch virus <400> 24 Glu Asn Leu Tyr Phe Gln Ser 1 5 <210> 25 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 25 His Pro Phe His Leu 1 5 <210> 26 <211> 619 <212> PRT <213> Dengue virus <400> 26 Ser Gly Val Leu Trp Asp Thr Pro Ser Pro Pro Glu Val Glu Arg Ala 1 5 10 15 Val Leu Asp Asp Gly Ile Tyr Arg Ile Met Gln Arg Gly Leu Leu Gly 20 25 30 Arg Ser Gln Val Gly Val Gly Val Phe Gln Asp Gly Val Phe His Thr 35 40 45 Met Trp His Val Thr Arg Gly Ala Val Leu Met Tyr Gln Gly Lys Arg 50 55 60 Leu Glu Pro Ser Trp Ala Ser Val Lys Lys Asp Leu Ile Ser Tyr Gly 65 70 75 80 Gly Gly Trp Arg Phe Gln Gly Ser Trp Asn Thr Gly Glu Glu Val Gln 85 90 95 Val Ile Ala Val Glu Pro Gly Lys Asn Pro Lys Asn Val Gln Thr Ala 100 105 110 Pro Gly Thr Phe Lys Thr Pro Glu Gly Glu Val Gly Ala Ile Ala Leu 115 120 125 Asp Phe Lys Pro Gly Thr Ser Gly Ser Pro Ile Val Asn Arg Glu Gly 130 135 140 Lys Ile Val Gly Leu Tyr Gly Asn Gly Val Val Thr Thr Ser Gly Thr 145 150 155 160 Tyr Val Ser Ala Ile Ala Gln Ala Lys Ala Ser Gln Glu Gly Pro Leu 165 170 175 Pro Glu Ile Glu Asp Glu Val Phe Arg Lys Arg Asn Leu Thr Ile Met 180 185 190 Asp Leu His Pro Gly Ser Gly Lys Thr Arg Arg Tyr Leu Pro Ala Ile 195 200 205 Val Arg Glu Ala Ile Arg Arg Asn Val Arg Thr Leu Ile Leu Ala Pro 210 215 220 Thr Arg Val Val Ala Ser Glu Met Ala Glu Ala Leu Lys Gly Met Pro 225 230 235 240 Ile Arg Tyr Gln Thr Thr Ala Val Lys Ser Glu His Thr Gly Lys Glu 245 250 255 Ile Val Asp Leu Met Cys His Ala Thr Phe Thr Met Arg Leu Leu Ser 260 265 270 Pro Val Arg Val Pro Asn Tyr Asn Met Ile Ile Met Asp Glu Ala His 275 280 285 Phe Thr Asp Pro Ala Ser Ile Ala Arg Arg Gly Tyr Ile Ser Thr Arg 290 295 300 Val Gly Met Gly Glu Ala Ala Ala Ile Phe Met Thr Ala Thr Pro Pro 305 310 315 320 Gly Ser Val Glu Ala Phe Pro Gln Ser Asn Ala Val Ile Gln Asp Glu 325 330 335 Glu Arg Asp Ile Pro Glu Arg Ser Trp Asn Ser Gly Tyr Glu Trp Ile 340 345 350 Thr Asp Phe Pro Gly Lys Thr Val Trp Phe Val Pro Ser Ile Lys Ser 355 360 365 Gly Asn Asp Ile Ala Asn Cys Leu Arg Lys Asn Gly Lys Arg Val Ile 370 375 380 Gln Leu Ser Arg Lys Thr Phe Asp Thr Glu Tyr Gln Lys Thr Lys Asn 385 390 395 400 Asn Asp Trp Asp Tyr Val Val Thr Thr Asp Ile Ser Glu Met Gly Ala 405 410 415 Asn Phe Arg Ala Asp Arg Val Ile Asp Pro Arg Arg Cys Leu Lys Pro 420 425 430 Val Ile Leu Lys Asp Gly Pro Glu Arg Val Ile Leu Ala Gly Pro Met 435 440 445 Pro Val Thr Val Ala Ser Ala Ala Gln Arg Arg Gly Arg Ile Gly Arg 450 455 460 Asn Gln Asn Lys Glu Gly Asp Gln Tyr Val Tyr Met Gly Gln Pro Leu 465 470 475 480 Asn Asn Asp Glu Asp His Ala His Trp Thr Glu Ala Lys Met Leu Leu 485 490 495 Asp Asn Ile Asn Thr Pro Glu Gly Ile Ile Pro Ala Leu Phe Glu Pro 500 505 510 Glu Arg Glu Lys Ser Ala Ala Ile Asp Gly Glu Tyr Arg Leu Arg Gly 515 520 525 Glu Ala Arg Lys Thr Phe Val Glu Leu Met Arg Arg Gly Asp Leu Pro 530 535 540 Val Trp Leu Ser Tyr Lys Val Ala Ser Glu Gly Phe Gln Tyr Ser Asp 545 550 555 560 Arg Arg Trp Cys Phe Asp Gly Glu Arg Asn Asn Gln Val Leu Glu Glu 565 570 575 Asn Met Asp Val Glu Met Trp Thr Lys Glu Gly Glu Arg Lys Lys Leu 580 585 590 Arg Pro Arg Trp Leu Asp Ala Arg Thr Tyr Ser Asp Pro Leu Ala Leu 595 600 605 Arg Glu Phe Lys Glu Phe Ala Ala Gly Arg Arg 610 615 <210> 27 <211> 618 <212> PRT <213> Dengue virus <400> 27 Ala Gly Val Leu Trp Asp Val Pro Ser Pro Pro Pro Val Gly Lys Ala 1 5 10 15 Glu Leu Glu Asp Gly Ala Tyr Arg Ile Lys Gln Lys Gly Ile Leu Gly 20 25 30 Tyr Ser Gln Ile Gly Ala Gly Val Tyr Lys Glu Gly Thr Phe His Thr 35 40 45 Met Trp His Val Thr Arg Gly Ala Val Leu Met His Lys Gly Lys Arg 50 55 60 Ile Glu Pro Ser Trp Ala Asp Val Lys Lys Asp Leu Ile Ser Tyr Gly 65 70 75 80 Gly Gly Trp Lys Leu Glu Gly Glu Trp Lys Glu Gly Glu Glu Val Gln 85 90 95 Val Leu Ala Leu Glu Pro Gly Lys Asn Pro Arg Ala Val Gln Thr Lys 100 105 110 Pro Gly Leu Phe Lys Thr Asn Ala Gly Thr Ile Gly Ala Val Ser Leu 115 120 125 Asp Phe Ser Pro Gly Thr Ser Gly Ser Pro Ile Ile Asp Lys Lys Gly 130 135 140 Lys Val Val Gly Leu Tyr Gly Asn Gly Val Val Thr Arg Ser Gly Ala 145 150 155 160 Tyr Val Ser Ala Ile Ala Gln Thr Glu Lys Ser Ile Glu Asp Asn Pro 165 170 175 Glu Ile Glu Asp Asp Ile Phe Arg Lys Arg Lys Leu Thr Ile Met Asp 180 185 190 Leu His Pro Gly Ala Gly Lys Thr Lys Arg Tyr Leu Pro Ala Ile Val 195 200 205 Arg Glu Ala Ile Lys Arg Gly Leu Arg Thr Leu Ile Leu Ala Pro Thr 210 215 220 Arg Val Val Ala Ala Glu Met Glu Glu Ala Leu Arg Gly Leu Pro Ile 225 230 235 240 Arg Tyr Gln Thr Pro Ala Ile Arg Ala Glu His Thr Gly Arg Glu Ile 245 250 255 Val Asp Leu Met Cys His Ala Thr Phe Thr Met Arg Leu Leu Ser Pro 260 265 270 Val Arg Val Pro Asn Tyr Asn Leu Ile Ile Met Asp Glu Ala His Phe 275 280 285 Thr Asp Pro Ala Ser Ile Ala Ala Arg Gly Tyr Ile Ser Thr Arg Val 290 295 300 Glu Met Gly Glu Ala Ala Gly Ile Phe Met Thr Ala Thr Pro Pro Gly 305 310 315 320 Ser Arg Asp Pro Phe Pro Gln Ser Asn Ala Pro Ile Met Asp Glu Glu 325 330 335 Arg Glu Ile Pro Glu Arg Ser Trp Ser Ser Gly His Glu Trp Val Thr 340 345 350 Asp Phe Lys Gly Lys Thr Val Trp Phe Val Pro Ser Ile Lys Ala Gly 355 360 365 Asn Asp Ile Ala Ala Cys Leu Arg Lys Asn Gly Lys Lys Val Ile Gln 370 375 380 Leu Ser Arg Lys Thr Phe Asp Ser Glu Tyr Val Lys Thr Arg Thr Asn 385 390 395 400 Asp Trp Asp Phe Val Val Thr Thr Asp Ile Ser Glu Met Gly Ala Asn 405 410 415 Phe Lys Ala Glu Arg Val Ile Asp Pro Arg Arg Cys Met Lys Pro Val 420 425 430 Ile Leu Thr Asp Gly Glu Glu Arg Val Ile Leu Ala Gly Pro Met Pro 435 440 445 Val Thr His Ser Ser Ala Ala Gln Arg Arg Gly Arg Ile Gly Arg Asn 450 455 460 Pro Lys Asn Glu Asn Asp Gln Tyr Ile Tyr Met Gly Glu Pro Leu Glu 465 470 475 480 Asn Asp Glu Asp Cys Ala His Trp Lys Glu Ala Lys Met Leu Leu Asp 485 490 495 Asn Ile Asn Thr Pro Glu Gly Ile Ile Pro Ser Met Phe Glu Pro Glu 500 505 510 Arg Glu Lys Val Asp Ala Ile Asp Gly Glu Tyr Arg Leu Arg Gly Glu 515 520 525 Ala Arg Lys Thr Phe Val Asp Leu Met Arg Arg Gly Asp Leu Pro Val 530 535 540 Trp Leu Ala Tyr Arg Val Ala Ala Glu Gly Ile Asn Tyr Ala Asp Arg 545 550 555 560 Arg Trp Cys Phe Asp Gly Ile Lys Asn Asn Gln Ile Leu Glu Glu Asn 565 570 575 Val Glu Val Glu Ile Trp Thr Lys Glu Gly Glu Arg Lys Lys Leu Lys 580 585 590 Pro Arg Trp Leu Asp Ala Lys Ile Tyr Ser Asp Pro Leu Ala Leu Lys 595 600 605 Glu Phe Lys Glu Phe Ala Ala Gly Arg Lys 610 615 <210> 28 <211> 619 <212> PRT <213> Dengue virus <400> 28 Ser Gly Val Leu Trp Asp Val Pro Ser Pro Pro Glu Thr Gln Lys Ala 1 5 10 15 Glu Leu Glu Glu Gly Val Tyr Arg Ile Lys Gln Gln Gly Ile Phe Gly 20 25 30 Lys Thr Gln Val Gly Val Gly Val Gln Lys Glu Gly Val Phe His Thr 35 40 45 Met Trp His Val Thr Arg Gly Ala Val Leu Thr His Asn Gly Lys Arg 50 55 60 Leu Glu Pro Asn Trp Ala Ser Val Lys Lys Asp Leu Ile Ser Tyr Gly 65 70 75 80 Gly Gly Trp Arg Leu Ser Ala Gln Trp Gln Lys Gly Glu Glu Val Gln 85 90 95 Val Ile Ala Val Glu Pro Gly Lys Asn Pro Lys Asn Phe Gln Thr Met 100 105 110 Pro Gly Ile Phe Gln Thr Thr Thr Gly Glu Ile Gly Ala Ile Ala Leu 115 120 125 Asp Phe Lys Pro Gly Thr Ser Gly Ser Pro Ile Ile Asn Arg Glu Gly 130 135 140 Lys Val Val Gly Leu Tyr Gly Asn Gly Val Val Thr Lys Asn Gly Gly 145 150 155 160 Tyr Val Ser Gly Ile Ala Gln Thr Asn Ala Glu Pro Asp Gly Pro Thr 165 170 175 Pro Glu Leu Glu Glu Glu Met Phe Lys Lys Arg Asn Leu Thr Ile Met 180 185 190 Asp Leu His Pro Gly Ser Gly Lys Thr Arg Lys Tyr Leu Pro Ala Ile 195 200 205 Val Arg Glu Ala Ile Lys Arg Arg Leu Arg Thr Leu Ile Leu Ala Pro 210 215 220 Thr Arg Val Val Ala Ala Glu Met Glu Glu Ala Leu Lys Gly Leu Pro 225 230 235 240 Ile Arg Tyr Gln Thr Thr Ala Thr Lys Ser Glu His Thr Gly Arg Glu 245 250 255 Ile Val Asp Leu Met Cys His Ala Thr Phe Thr Met Arg Leu Leu Ser 260 265 270 Pro Val Arg Val Pro Asn Tyr Asn Leu Ile Ile Met Asp Glu Ala His 275 280 285 Phe Thr Asp Pro Ala Ser Ile Ala Ala Arg Gly Tyr Ile Ser Thr Arg 290 295 300 Val Gly Met Gly Glu Ala Ala Ala Ile Phe Met Thr Ala Thr Pro Pro 305 310 315 320 Gly Thr Ala Asp Ala Phe Pro Gln Ser Asn Ala Pro Ile Gln Asp Glu 325 330 335 Glu Arg Asp Ile Pro Glu Arg Ser Trp Asn Ser Gly Asn Glu Trp Ile 340 345 350 Thr Asp Phe Val Gly Lys Thr Val Trp Phe Val Pro Ser Ile Lys Ala 355 360 365 Gly Asn Asp Ile Ala Asn Cys Leu Arg Lys Asn Gly Lys Lys Val Ile 370 375 380 Gln Leu Ser Arg Lys Thr Phe Asp Thr Glu Tyr Gln Lys Thr Lys Leu 385 390 395 400 Asn Asp Trp Asp Phe Val Val Thr Thr Asp Ile Ser Glu Met Gly Ala 405 410 415 Asn Phe Lys Ala Asp Arg Val Ile Asp Pro Arg Arg Cys Leu Lys Pro 420 425 430 Val Ile Leu Thr Asp Gly Pro Glu Arg Val Ile Leu Ala Gly Pro Met 435 440 445 Pro Val Thr Val Ala Ser Ala Ala Gln Arg Arg Gly Arg Val Gly Arg 450 455 460 Asn Pro Gln Lys Glu Asn Asp Gln Tyr Ile Phe Met Gly Gln Pro Leu 465 470 475 480 Asn Lys Asp Glu Asp His Ala His Trp Thr Glu Ala Lys Met Leu Leu 485 490 495 Asp Asn Ile Asn Thr Pro Glu Gly Ile Ile Pro Ala Leu Phe Glu Pro 500 505 510 Glu Arg Glu Lys Ser Ala Ala Ile Asp Gly Glu Tyr Arg Leu Lys Gly 515 520 525 Glu Ser Arg Lys Thr Phe Val Glu Leu Met Arg Arg Gly Asp Leu Pro 530 535 540 Val Trp Leu Ala His Lys Val Ala Ser Glu Gly Ile Lys Tyr Thr Asp 545 550 555 560 Arg Lys Trp Cys Phe Asp Gly Glu Arg Asn Asn Gln Ile Leu Glu Glu 565 570 575 Asn Met Asp Val Glu Ile Trp Thr Lys Glu Gly Glu Lys Lys Lys Leu 580 585 590 Arg Pro Arg Trp Leu Asp Ala Arg Thr Tyr Ser Asp Pro Leu Ala Leu 595 600 605 Lys Glu Phe Lys Asp Phe Ala Ala Gly Arg Lys 610 615 <210> 29 <211> 618 <212> PRT <213> Dengue virus <400> 29 Ser Gly Ala Leu Trp Asp Val Pro Ser Pro Ala Ala Thr Gln Lys Ala 1 5 10 15 Ala Leu Ser Glu Gly Val Tyr Arg Ile Met Gln Arg Gly Leu Phe Gly 20 25 30 Lys Thr Gln Val Gly Val Gly Ile His Ile Glu Gly Val Phe His Thr 35 40 45 Met Trp His Val Thr Arg Gly Ser Val Ile Cys His Glu Thr Gly Arg 50 55 60 Leu Glu Pro Ser Trp Ala Asp Val Arg Asn Asp Met Ile Ser Tyr Gly 65 70 75 80 Gly Gly Trp Arg Leu Gly Asp Lys Trp Asp Lys Glu Glu Asp Val Gln 85 90 95 Val Leu Ala Ile Glu Pro Gly Lys Asn Pro Lys His Val Gln Thr Lys 100 105 110 Pro Gly Leu Phe Lys Thr Leu Thr Gly Glu Ile Gly Ala Val Thr Leu 115 120 125 Asp Phe Lys Pro Gly Thr Ser Gly Ser Pro Ile Ile Asn Arg Lys Gly 130 135 140 Lys Val Ile Gly Leu Tyr Gly Asn Gly Val Val Thr Lys Ser Gly Asp 145 150 155 160 Tyr Val Ser Ala Ile Thr Gln Ala Glu Arg Ile Gly Glu Pro Asp Tyr 165 170 175 Glu Val Asp Glu Asp Ile Phe Arg Lys Lys Arg Leu Thr Ile Met Asp 180 185 190 Leu His Pro Gly Ala Gly Lys Thr Lys Arg Ile Leu Pro Ser Ile Val 195 200 205 Arg Glu Ala Leu Lys Arg Arg Leu Arg Thr Leu Ile Leu Ala Pro Thr 210 215 220 Arg Val Val Ala Ala Glu Met Glu Glu Ala Leu Arg Gly Leu Pro Ile 225 230 235 240 Arg Tyr Gln Thr Pro Ala Val Lys Ser Glu His Thr Gly Arg Glu Ile 245 250 255 Val Asp Leu Met Cys His Ala Thr Phe Thr Thr Arg Leu Leu Ser Ser 260 265 270 Thr Arg Val Pro Asn Tyr Asn Leu Ile Val Met Asp Glu Ala His Phe 275 280 285 Thr Asp Pro Ser Ser Val Ala Ala Arg Gly Tyr Ile Ser Thr Arg Val 290 295 300 Glu Met Gly Glu Ala Ala Ala Ile Phe Met Thr Ala Thr Pro Pro Gly 305 310 315 320 Thr Thr Asp Pro Phe Pro Gln Ser Asn Ser Pro Ile Glu Asp Ile Glu 325 330 335 Arg Glu Ile Pro Glu Arg Ser Trp Asn Thr Gly Phe Asp Trp Ile Thr 340 345 350 Asp Tyr Gln Gly Lys Thr Val Trp Phe Val Pro Ser Ile Lys Ala Gly 355 360 365 Asn Asp Ile Ala Asn Cys Leu Arg Lys Ser Gly Lys Lys Val Ile Gln 370 375 380 Leu Ser Arg Lys Thr Phe Asp Thr Glu Tyr Pro Lys Thr Lys Leu Thr 385 390 395 400 Asp Trp Asp Phe Val Val Thr Thr Asp Ile Ser Glu Met Gly Ala Asn 405 410 415 Phe Arg Ala Gly Arg Val Ile Asp Pro Arg Arg Cys Leu Lys Pro Val 420 425 430 Ile Leu Pro Asp Gly Pro Glu Arg Val Ile Leu Ala Gly Pro Ile Pro 435 440 445 Val Thr Pro Ala Ser Ala Ala Gln Arg Arg Gly Arg Ile Gly Arg Asn 450 455 460 Pro Ala Gln Glu Asp Asp Gln Tyr Val Phe Ser Gly Asp Pro Leu Lys 465 470 475 480 Asn Asp Glu Asp His Ala His Trp Thr Glu Ala Lys Met Leu Leu Asp 485 490 495 Asn Ile Tyr Thr Pro Glu Gly Ile Ile Pro Thr Leu Phe Gly Pro Glu 500 505 510 Arg Glu Lys Thr Gln Ala Ile Asp Gly Glu Phe Arg Leu Arg Gly Glu 515 520 525 Gln Arg Lys Thr Phe Val Glu Leu Met Arg Arg Gly Asp Leu Pro Val 530 535 540 Trp Leu Ser Tyr Lys Val Ala Ser Ala Gly Ile Ser Tyr Lys Asp Arg 545 550 555 560 Glu Trp Cys Phe Thr Gly Glu Arg Asn Asn Gln Ile Leu Glu Glu Asn 565 570 575 Met Glu Val Glu Ile Trp Thr Arg Glu Gly Glu Lys Lys Lys Leu Arg 580 585 590 Pro Lys Trp Leu Asp Ala Arg Val Tyr Ala Asp Pro Met Ala Leu Lys 595 600 605 Asp Phe Lys Glu Phe Ala Ser Gly Arg Lys 610 615 <210> 30 <211> 10 <212> PRT <213> Hepacivirus C <400> 30 Gly Leu Leu Gly Cys Ile Ile Thr Ser Leu 1 5 10 <210> 31 <211> 26 <212> PRT <213> Hepacivirus C <400> 31 Gly Glu Val Gln Ile Val Ser Thr Ala Ala Gln Thr Phe Leu Ala Thr 1 5 10 15 Cys Ile Asn Gly Val Cys Trp Thr Val Tyr 20 25 <210> 32 <211> 15 <212> PRT <213> Hepacivirus C <400> 32 Gly Glu Val Gln Ile Val Ser Thr Ala Ala Gln Thr Phe Leu Ala 1 5 10 15 <210> 33 <211> 15 <212> PRT <213> Hepacivirus C <400> 33 Gln Thr Phe Leu Ala Thr Cys Ile Asn Gly Val Cys Trp Thr Val 1 5 10 15 <210> 34 <211> 10 <212> PRT <213> Hepacivirus C <400> 34 Cys Ile Asn Gly Val Cys Trp Thr Val Tyr 1 5 10 <210> 35 <211> 15 <212> PRT <213> Hepacivirus C <400> 35 Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val Ile Pro 1 5 10 15 <210> 36 <211> 8 <212> PRT <213> Hepacivirus C <400> 36 Tyr Leu Val Thr Arg His Ala Asp 1 5 <210> 37 <211> 9 <212> PRT <213> Hepacivirus C <400> 37 Leu Leu Cys Pro Ala Gly His Ala Val 1 5 <210> 38 <211> 15 <212> PRT <213> Hepacivirus C <400> 38 Ala Val Asp Phe Ile Pro Val Glu Gly Leu Glu Thr Thr Met Arg 1 5 10 15 <210> 39 <211> 15 <212> PRT <213> Hepacivirus C <400> 39 Lys Ile Asp Thr Lys Tyr Ile Met Thr Cys Met Ser Ala Asp Leu 1 5 10 15 <210> 40 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 40 Pro Ile Thr Lys Ile Asp Thr Lys Tyr Ile Met Thr Cys Met Ser Ala 1 5 10 15 Asp Leu Glu Val Val Thr Ser Thr Trp Val Leu Val Gly Gly Val Leu 20 25 30 Ala Ala Leu Ala Ala Tyr Cys Leu Ser Thr 35 40 <210> 41 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 41 Lys Lys Lys Arg Lys 1 5 <210> 42 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 42 Met Leu Arg Thr Ser Ser Leu Phe Thr Arg Arg Val Gln Pro Ser Leu 1 5 10 15 Phe Arg Asn Ile Leu Arg Leu Gln Ser Thr 20 25 <210> 43 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 43 Lys Asp Glu Leu 1 <210> 44 <211> 304 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 44 Asp Glu Met Glu Glu Cys Ser Gln His Leu Pro Gly Ala Gly Ser Ser 1 5 10 15 Gly Asp Ile Met Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ser Ser Gly 20 25 30 Thr Gly Ser Gly Ser Gly Thr Ser Ala Pro Ile Thr Ala Tyr Ala Gln 35 40 45 Gln Thr Arg Gly Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg 50 55 60 Asp Lys Asn Gln Val Glu Gly Glu Val Gln Ile Val Ser Thr Ala Thr 65 70 75 80 Gln Thr Phe Leu Ala Thr Cys Ile Asn Gly Val Cys Trp Ala Val Tyr 85 90 95 His Gly Ala Gly Thr Arg Thr Ile Ala Ser Pro Lys Gly Pro Val Ile 100 105 110 Gln Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Pro Ala Pro 115 120 125 Gln Gly Ser Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu 130 135 140 Tyr Leu Val Thr Arg His Ala Asp Val Ile Pro Val Arg Arg Arg Gly 145 150 155 160 Asp Ser Arg Gly Ser Leu Leu Ser Pro Arg Pro Ile Ser Tyr Leu Lys 165 170 175 Gly Ser Ser Gly Gly Pro Leu Leu Cys Pro Ala Gly His Ala Val Gly 180 185 190 Leu Phe Arg Ala Ala Val Cys Thr Arg Gly Val Ala Lys Ala Val Asp 195 200 205 Phe Ile Pro Val Glu Asn Leu Glu Thr Thr Met Arg Ser Pro Val Phe 210 215 220 Thr Asp Asn Ser Ser Pro Pro Ala Val Thr Leu Thr His Pro Ile Thr 225 230 235 240 Lys Ile Asp Thr Lys Tyr Ile Met Thr Cys Met Ser Ala Asp Leu Glu 245 250 255 Val Val Thr Ser Thr Trp Val Leu Val Gly Gly Val Leu Ala Ala Leu 260 265 270 Ala Ala Tyr Cys Leu Ser Thr Gly Cys Val Val Ile Val Gly Arg Ile 275 280 285 Val Leu Ser Gly Lys Pro Ala Ile Ile Pro Asp Arg Glu Val Leu Tyr 290 295 300 <210> 45 <211> 303 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 45 Met Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ser Ser Gly Thr Gly Ser 1 5 10 15 Gly Ser Gly Thr Ser Ala Pro Ile Thr Ala Tyr Ala Gln Gln Thr Arg 20 25 30 Gly Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn 35 40 45 Gln Val Glu Gly Glu Val Gln Ile Val Ser Thr Ala Thr Gln Thr Phe 50 55 60 Leu Ala Thr Cys Ile Asn Gly Val Cys Trp Ala Val Tyr His Gly Ala 65 70 75 80 Gly Thr Arg Thr Ile Ala Ser Pro Lys Gly Pro Val Ile Gln Met Tyr 85 90 95 Thr Asn Val Asp Gln Asp Leu Val Gly Trp Pro Ala Pro Gln Gly Ser 100 105 110 Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val 115 120 125 Thr Arg His Ala Asp Val Ile Pro Val Arg Arg Arg Gly Asp Ser Arg 130 135 140 Gly Ser Leu Leu Ser Pro Arg Pro Ile Ser Tyr Leu Lys Gly Ser Ser 145 150 155 160 Gly Gly Pro Leu Leu Cys Pro Ala Gly His Ala Val Gly Leu Phe Arg 165 170 175 Ala Ala Val Cys Thr Arg Gly Val Ala Lys Ala Val Asp Phe Ile Pro 180 185 190 Val Glu Asn Leu Glu Thr Thr Met Arg Ser Pro Val Phe Thr Asp Asn 195 200 205 Ser Ser Pro Pro Ala Val Thr Leu Thr His Pro Ile Thr Lys Ile Asp 210 215 220 Thr Lys Tyr Ile Met Thr Cys Met Ser Ala Asp Leu Glu Val Val Thr 225 230 235 240 Ser Thr Trp Val Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr 245 250 255 Cys Leu Ser Thr Gly Cys Val Val Ile Val Gly Arg Ile Val Leu Ser 260 265 270 Gly Lys Pro Ala Gly Ser Ser Gly Ser Ser Ile Ile Pro Asp Arg Glu 275 280 285 Val Leu Tyr Gln Glu Phe Glu Asp Val Val Pro Cys Ser Met Gly 290 295 300 <210> 46 <211> 64 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 46 Leu Gln Met Leu Pro Glu Ser Glu Asp Glu Glu Ser Tyr Asp Thr Glu 1 5 10 15 Ser Glu Phe Thr Glu Phe Thr Glu Asp Glu Leu Pro Tyr Asp Asp Gly 20 25 30 Ser Leu Gln Met Leu Pro Glu Ser Glu Asp Glu Glu Ser Tyr Asp Thr 35 40 45 Glu Ser Glu Phe Thr Glu Phe Thr Glu Asp Glu Leu Pro Tyr Asp Asp 50 55 60 <210> 47 <211> 57 <212> PRT <213> Homo sapiens <400> 47 Glu Ile Lys Asp Lys Glu Glu Val Gln Arg Lys Arg Gln Lys Leu Met 1 5 10 15 Pro Asn Phe Ser Asp Ser Phe Gly Gly Gly Ser Gly Ala Gly Ala Gly 20 25 30 Gly Gly Gly Met Phe Gly Ser Gly Gly Gly Gly Gly Gly Thr Gly Ser 35 40 45 Thr Gly Pro Gly Tyr Ser Phe Pro His 50 55 <210> 48 <211> 86 <212> PRT <213> Unknown <220> <223> Description of Unknown: yeast sequence <400> 48 Ile Asp Asp Glu Asn Gly Ser Val Ile Leu Gln Asp Asp Asp Tyr Asp 1 5 10 15 Asp Gly Asn Asn His Ile Pro Phe Glu Asp Asp Asp Val Tyr Asn Tyr 20 25 30 Asn Asp Asn Asp Asp Asp Asp Glu Arg Ile Glu Phe Glu Asp Asp Asp 35 40 45 Asp Asp Asp Asp Asp Ser Ile Asp Asn Asp Ser Val Met Asp Arg Lys 50 55 60 Gln Pro His Lys Ala Glu Asp Glu Ser Glu Asp Val Glu Asp Val Glu 65 70 75 80 Arg Val Ser Lys Lys Asp 85 <210> 49 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 49 Pro Glu Ser Met Arg Glu Glu Tyr Arg Lys Glu Gly Ser Lys Arg Ile 1 5 10 15 Lys Cys Pro Asp Cys Glu Pro Phe Cys Asn Lys Arg Gly Ser Pro Glu 20 25 30 Ser Met Arg Glu Glu Tyr Arg Lys Glu 35 40 <210> 50 <211> 68 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 50 Arg Ser Tyr Ser Pro Thr Ser Pro Asn Tyr Ser Pro Thr Ser Pro Ser 1 5 10 15 Gly Ser Tyr Ser Pro Thr Ser Pro Asn Tyr Ser Pro Thr Ser Pro Ser 20 25 30 Gly Gly Ser Arg Ser Tyr Ser Pro Thr Ser Pro Asn Tyr Ser Pro Thr 35 40 45 Ser Pro Ser Gly Ser Tyr Ser Pro Thr Ser Pro Asn Tyr Ser Pro Thr 50 55 60 Ser Pro Ser Gly 65 <210> 51 <211> 59 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 51 Pro Glu Ser Met Arg Glu Glu Tyr Arg Lys Glu Gly Ser Ser Leu Leu 1 5 10 15 Thr Glu Val Glu Thr Pro Gly Ser Pro Glu Ser Met Arg Glu Glu Tyr 20 25 30 Arg Lys Glu Gly Ser Ser Leu Leu Thr Glu Val Glu Thr Pro Gly Ser 35 40 45 Pro Glu Ser Met Arg Glu Glu Tyr Arg Lys Glu 50 55 <210> 52 <211> 51 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 52 Leu Ile Glu Glu Val Arg His Arg Leu Lys Thr Thr Glu Asn Ser Gly 1 5 10 15 Ser Leu Ile Glu Glu Val Arg His Arg Leu Lys Thr Thr Glu Asn Ser 20 25 30 Gly Ser Leu Ile Glu Glu Val Arg His Arg Leu Lys Thr Thr Glu Asn 35 40 45 Ser Gly Ser 50 <210> 53 <211> 37 <212> PRT <213> Unknown <220> <223> Description of Unknown: ornithine decarboxylase sequence <400> 53 Phe Pro Pro Glu Val Glu Glu Gln Asp Asp Gly Thr Leu Pro Met Ser 1 5 10 15 Cys Ala Gln Glu Ser Gly Met Asp Arg His Pro Ala Ala Cys Ala Ser 20 25 30 Ala Arg Ile Asn Val 35 <210> 54 <211> 40 <212> PRT <213> Unknown <220> <223> Description of Unknown: mODC DA sequence <400> 54 Ser His Gly Phe Pro Pro Glu Val Glu Glu Gln Ala Ala Gly Thr Leu 1 5 10 15 Pro Met Ser Cys Ala Gln Glu Ser Gly Met Asp Arg His Pro Ala Ala 20 25 30 Cys Ala Ser Ala Arg Ile Asn Val 35 40 <210> 55 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (4)..(5) <223> Any amino acid <400> 55 Asp Ser Gly Xaa Xaa Ser 1 5 <210> 56 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (1)..(1) <223> Abz <220> <221> MOD_RES <222> (7)..(7) <223> Lys(Dnp) <400> 56 Xaa His Pro Phe His Leu Lys 1 5

Claims

1. A fusion protein comprising: A polypeptide of interest; a variant Hepatitis C virus (HCV) nonstructural protein 3 (NS3) protease; Cognate protease cleavage sites and Including, the variant HCV NS3 protease comprises one or more mutations, the one or more mutations reduce immunogenicity of the fusion protein when expressed in a mammalian cell. The fusion protein.

2. The fusion protein of claim 1 , wherein the variant HCV NS3 protease is derived from an HCV polyprotein comprising the amino acid sequence of SEQ ID NO:

1.

3. The fusion protein of claim 1 or claim 2, wherein the one or more mutations comprise one or more amino acid substitutions.

4. The fusion protein of claim 3, wherein the one or more amino acid substitutions correspond to an amino acid substitution in SEQ ID NO:

1.

5. 5. The fusion protein of claim 4, wherein the one or more amino acid substitutions are at one or more positions corresponding to positions 1038-1047 of SEQ ID NO:1, positions 1057-1081 of SEQ ID NO:1, positions 1073-1081 of SEQ ID NO:1, positions 1073-1082 of SEQ ID NO:1, positions 1127-1141 of SEQ ID NO:1, positions 1131-1138 of SEQ ID NO:1, positions 1169-1177 of SEQ ID NO:1, and / or positions 1192-1206 of SEQ ID NO:

1.

6. The fusion protein of claim 5, wherein the one or more amino acid substitutions are selected from the group consisting of a position corresponding to position 1062 of SEQ ID NO:1, a position corresponding to position 1069 of SEQ ID NO:1, a position corresponding to position 1070 of SEQ ID NO:1, a position corresponding to position 1071 of SEQ ID NO:1, a position corresponding to position 1072 of SEQ ID NO:1, a position corresponding to position 1074 of SEQ ID NO:1, a position corresponding to position 1075 of SEQ ID NO:1, a position corresponding to position 1077 of SEQ ID NO:1, a position corresponding to position 1078 of SEQ ID NO:1, a position corresponding to position 1079 of SEQ ID NO:1, a position corresponding to position 1080 of SEQ ID NO:1, a position corresponding to position 1031 of SEQ ID NO:1, a position corresponding to position 1132 of SEQ ID NO:1, a position corresponding to position 1133 of SEQ ID NO:1, a position corresponding to position 1195 of SEQ ID NO:1, a position corresponding to position 1196 of SEQ ID NO:1, a position corresponding to position 1201 of SEQ ID NO:1, a position corresponding to position 1202 of SEQ ID NO:1, and any combination thereof.

7. 6. The fusion protein of claim 5, wherein the one or more amino acid substitutions are selected from the group consisting of an Ile to Leu substitution at a position corresponding to position 1074 of SEQ ID NO:1, an Ile to Met substitution at a position corresponding to position 1074 of SEQ ID NO:1, an Asn to Ala substitution at a position corresponding to position 1075 of SEQ ID NO:1, a Val to Ala substitution at a position corresponding to position 1077 of SEQ ID NO:1, a Cys to Phe substitution at a position corresponding to position 1078 of SEQ ID NO:1, a Trp to Ala substitution at a position corresponding to position 1079 of SEQ ID NO:1, a Thr to Ala substitution at a position corresponding to position 1080 of SEQ ID NO:1, a Val to Ala substitution at a position corresponding to position 1081 of SEQ ID NO:1, a Val to Asn substitution at a position corresponding to position 1081 of SEQ ID NO:1, and any combination thereof.

8. The fusion protein of claim 5 , wherein the one or more amino acid substitutions include a Thr to Ala substitution at a position corresponding to position 1080 of SEQ ID NO:

1.

9. The fusion protein of claim 5, wherein the one or more amino acid substitutions include a Thr to Ala substitution at a position corresponding to position 1080 of SEQ ID NO:1, and a Val to Ala substitution at a position corresponding to position 1077 of SEQ ID NO:

1.

10. The fusion protein of claim 5, wherein the one or more amino acid substitutions include a Thr to Ala substitution at a position corresponding to position 1080 of SEQ ID NO:1, and a Val to Ala substitution at a position corresponding to position 1081 of SEQ ID NO:

1.

11. The fusion protein of any one of claims 1 to 10, further comprising an HCV NS4A cofactor.

12. The fusion protein of any one of claims 1 to 11, further comprising a degron, said degron being operably linked to said polypeptide of interest.

13. The degron may be selected from the group consisting of HCV NS4 degron, PEST (two copies of residues 277-307 of human IκBα) (SEQ ID NO:46), GRR (residues 352-408 of human p105) (SEQ ID NO:47), DRR (residues 210-295 of yeast Cdc34) (SEQ ID NO:48), SNS (tandem repeats of SP2 and NB of influenza A or influenza B (SP2-NB-SP2) (SEQ ID NO:49), RPB (four copies of residues 1688-1702 of yeast RPB) (SEQ ID NO:50), SPmix (influenza A virus M2 tandem repeats of proteins SP1 and SP2 (SP2-SP1-SP2-SP1-SP2) (SEQ ID NO:51), NS2 (three copies of residues 79-93 of influenza A virus NS protein) (SEQ ID NO:52), ODC (residues 106-142 of ornithine decarboxylase) (SEQ ID NO:53), Nek2A, mouse ODC (residues 422-461), mouse ODC_DA (residues 422-461 of mODC containing D433A and D434A point mutations) (SEQ ID NO:54), APC / C degron, COP1 13. The fusion protein of claim 12, wherein the fusion protein is selected from the group consisting of an E3 ligase binding degron motif, a CRL4-Cdt2 binding PIP degron, an actinfilin binding degron, a KEAP1 binding degron, a KLHL2 and KLHL3 binding degron, an MDM2 binding motif, an N-degron, a hydroxyproline modification in hypoxia signaling, a plant hormone dependent SCF-LRR binding degron, an SCF ubiquitin ligase binding phosphodegron, a plant hormone dependent SCF-LRR binding degron, a DSGxxS (SEQ ID NO:55) phospho-dependent degron, a Siah binding motif, a SPOP SBC docking motif, and a PCNA binding PIP box.

14. The fusion protein of any one of claims 1 to 13, wherein the variant HCV NS3 protease comprises one or more additional mutations.

15. 15. The fusion protein of claim 14, wherein the one or more additional mutations modulate an enzymatic activity of the variant HCV NS3 protease.

16. 16. The fusion protein of claim 14 or claim 15, wherein the one or more additional mutations are one or more additional amino acid substitutions.

17. The fusion protein of claim 16, wherein the one or more additional amino acid substitutions are at one or more positions corresponding to position 1074 of SEQ ID NO:1, position 1078 of SEQ ID NO:1, and / or position 1079 of SEQ ID NO:

1.

18. The fusion protein of claim 17, wherein the one or more additional amino acid substitutions are selected from the group consisting of an Ile to Ala substitution at a position corresponding to position 1074 of SEQ ID NO:1, a Trp to Ala substitution at a position corresponding to position 1079 of SEQ ID NO:1, and any combination thereof.

19. 20. The fusion protein of claim 18, wherein the one or more additional amino acid substitutions reduce an enzymatic activity of the variant HCV NS3 protease.

20. 18. The fusion protein of claim 17, wherein the one or more additional amino acid substitutions comprise a Cys to Ala substitution at a position corresponding to position 1078 of SEQ ID NO:

1.

21. 21. The fusion protein of claim 20, wherein the one or more additional amino acid substitutions increase an enzymatic activity of the variant HCV NS3 protease.

22. 22. The fusion protein of any one of claims 1 to 21, wherein the cognate protease cleavage site comprises an amino acid sequence selected from the group consisting of any of the amino acid sequences listed in Table 1.

23. The cognate protease cleavage site is The fusion protein according to any one of claims 1 to 21, comprising an amino acid sequence selected from the group consisting of:

24. The cognate protease cleavage site is The fusion protein according to any one of claims 1 to 21, comprising an amino acid sequence selected from the group consisting of:

25. The fusion protein of any one of claims 22 to 24, wherein the cognate protease cleavage site comprises one or more mutations.

26. 26. The fusion protein of claim 25, wherein the one or more mutations comprise one or more amino acid substitutions.

27. 27. The fusion protein of claim 25 or claim 26, wherein the one or more mutations increase the catalytic rate of cleavage.

28. 27. The fusion protein of claim 25 or claim 26, wherein the one or more mutations reduce the catalytic rate of cleavage.

29. 29. The fusion protein of any one of claims 1 to 28, wherein the polypeptide of interest is selected from the group consisting of membrane proteins, receptors, hormones, cytokines, transport proteins, transcription factors, cytoskeletal proteins, extracellular matrix proteins, signal transduction proteins, and enzymes.

30. The fusion protein of any one of claims 1 to 28, wherein the polypeptide of interest comprises a biologically active domain of a protein.

31. The fusion protein of claim 30, wherein the biologically active domain is a catalytic domain, a ligand binding domain, or a protein-protein interaction domain.

32. The fusion protein of any one of claims 1 to 31, wherein the polypeptide of interest is a receptor selected from the group consisting of T cell receptors (TCR), chimeric T cell receptors, artificial T cell receptors, synthetic T cell receptors, chimeric immunoreceptors, antibody-binding T cell receptors (ACTR), T cell receptor fusion constructs (TRUC), and chimeric antigen receptors (CAR).

33. The fusion protein of any one of claims 1 to 31, wherein the polypeptide of interest is a chimeric antigen receptor (CAR).

34. The fusion protein of any one of claims 1 to 28, wherein the polypeptide of interest is a cytokine.

35. 35. The fusion protein of claim 34, wherein the cytokine is a proinflammatory cytokine.

36. The fusion protein of any one of claims 1 to 35, wherein the cognate protease cleavage site is located within a domain of the polypeptide of interest.

37. The fusion protein of any one of claims 1 to 35, wherein the polypeptide of interest comprises multiple domains.

38. 38. The fusion protein of claim 37, wherein the cognate protease cleavage sites are located between the multiple domains of the polypeptide of interest.

39. The fusion protein of any one of claims 1 to 38, wherein the variant HCV NS3 protease is capable of being inhibited by a protease inhibitor.

40. 40. The fusion protein of claim 39, wherein the protease inhibitor is selected from the group consisting of simeprevir, danoprevir, asunaprevir, cilprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and voxiloprevir.

41. The fusion protein of any one of claims 1 to 40, further comprising a targeting sequence.

42. The fusion protein of claim 41, wherein the targeting sequence is selected from the group consisting of a secretory protein signal sequence, a membrane protein signal sequence, a nuclear localization sequence, a nucleolar localization signal sequence, an endoplasmic reticulum localization sequence, a peroxisome localization sequence, a mitochondrial localization sequence, and a protein binding motif sequence.

43. The fusion protein of any one of claims 1 to 42, wherein the variant NS3 protease is derived from an HCV NS3 protease having the amino acid sequence of SEQ ID NO:

2.

44. A polynucleotide encoding the fusion protein according to any one of claims 1 to 43.

45. A vector comprising the polynucleotide of claim 44.

46. A cell comprising the fusion protein of any one of claims 1 to 43, the polynucleotide of claim 44, or the vector of claim 45.

47. 47. The cell of claim 46, which is an immune cell or a cell line derived from an immune cell.

48. 48. The cell of claim 47, wherein the immune cell is selected from the group consisting of a T cell, a B cell, a NK cell, a NKT cell, an innate lymphoid cell, a mast cell, an eosinophil, a basophil, a macrophage, a neutrophil, a dendritic cell, and any combination thereof.

49. 47. The cell of claim 46, which is a mesenchymal stromal cell.

50. A pharmaceutical composition comprising the fusion protein of any one of claims 1 to 43 and an excipient.

51. A pharmaceutical composition comprising the cells of any one of claims 46 to 49 and an excipient.

52. 52. A method of treating a subject in need of treatment comprising administering a pharmaceutical composition according to claim 50 or claim 51.

53. 1. A method for modulating the activity of a protein of interest, comprising: a) providing a population of cells comprising the fusion protein of any one of claims 1 to 43, the polynucleotide of claim 44, or the vector of claim 45; b) contacting the population of cells with a protease inhibitor; The method comprising:

54. 54. The method of claim 53, further comprising the step of removing said protease inhibitor from said population of cells.

55. 55. The method of claim 53 or claim 54, further comprising administering the population of cells to a subject in need of a cell-based therapy.

56. 46. ​​A method of treating a subject in need of a cell-based therapy comprising administering to the subject a population of cells comprising the fusion protein of any one of claims 1 to 43, the polynucleotide of claim 44, or the vector of claim 45.

57. 57. The method of claim 56, wherein the population of cells is cultured in the presence of a protease inhibitor capable of inhibiting an inhibitory protease.

58. 57. The method of claim 56, wherein the population of cells is cultured in the absence of a protease inhibitor capable of inhibiting an inhibitory protease.

59. 59. The method of any one of claims 56 to 58, further comprising the step of administering to the subject a protease inhibitor capable of inhibiting an inhibitory protease.

60. 60. The method of claim 59, further comprising the step of discontinuing the use of said protease inhibitor capable of inhibiting an inhibitory protease in said subject.

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