Fusion Constructs for Controlling Protein Function

Abstract

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.

Claims

1. A fusion protein, comprising: 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; and wherein the one or more mutations decrease immunogenicity when the fusion protein is expressed in a mammalian cell.

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 amino acid substitutions within SEQ ID NO: 1.

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 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.

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. 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 comprise 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 comprise 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 comprise 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 1-10, further comprising an HCV NS4A co-factor.

12. The fusion protein of any one of claims 1-11, further comprising a degron, wherein the degron is operably linked to the polypeptide of interest.

13. The fusion protein of claim 12, wherein 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 repeat of SP2 and NB (SP2-NB-SP2 of influenza A or influenza B) (SEQ ID NO: 49), RPB (four copies of residues 1688-1702 of yeast RPB) (SEQ ID NO: 50), SPmix (tandem repeat of SP1 and SP2 (SP2-SP1-SP2-SP1-SP2 of 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, mouse ODC (residues 422-461), mouse ODC_DA (residues 422-461 of mODC including D433A and D434A point mutations) (SEQ ID NO: 54), an APC/C degron, a COP1 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 phytohormone-dependent SCF-LRR-binding degron, an SCF ubiquitin ligase binding phosphodegron, a phytohormone-dependent SCF-LRR-binding degron, a DSGxxS (SEQ ID NO: 55) phospho-dependent degron, an Siah binding motif, an SPOP SBC docking motif, and a PCNA binding PIP box.

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

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

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

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

22. The fusion protein of any one of claims 1-21, 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 fusion protein of any one of claims 1-21, wherein the cognate protease cleavage site comprises an amino acid sequence selected from the group consisting of CMSADLEVVTSTWVLVGGVL (SEQ ID NO: 4), YQEFDEMEECSQHLPYIEQG (SEQ ID NO. 5), WISSECTTPCSGSWLRDIWD (SEQ ID NO: 6), and GADTEDVVCCSMSYSWTGAL (SEQ ID NO: 7).

24. The fusion protein of any one of claims 1-21, wherein the cognate protease cleavage site comprises an amino acid sequence selected from the group consisting of ADLEVVTSTWL (SEQ ID NO 8), DEMEECSQHL (SEQ ID NO: 9), ECTTPCSGSWL (SEQ ID NO: 10), and EDVVPCSMG (SEQ ID NO: 11).

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

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

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

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

29. The fusion protein of any one of claims 1-28, wherein the polypeptide of interest is selected from the group consisting of 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.

30. The fusion protein of any one of claims 1-28, 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 1-31, wherein the polypeptide of interest 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-coupled T cell receptor (ACTR), a T cell receptor fusion construct (TRUC), and a chimeric antigen receptor (CAR).

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

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

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

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

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

38. The fusion protein of claim 37, wherein the cognate protease cleavage site is localized between the multiple domains of the polypeptide of interest.

39. The fusion protein of any one of claims 1-38, wherein the variant HCV NS3 protease can be repressed by a protease inhibitor.

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

41. The fusion protein of any one of claims 1-40, 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 1-42, 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 of any one of claims 1-43.

45. A vector comprising the polynucleotide of claim 44.

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

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

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

49. The cell of claim 46, wherein the cell is a mesenchymal stromal cell.

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

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

52. A method of treating a subject in need thereof, comprising administering the pharmaceutical composition of claim 50 or claim 51.

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

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

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

56. 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 1-43, the polynucleotide of claim 44, or the vector of claim 45.

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

58. The method of claim 56, wherein the population of cells was cultured in the absence of a protease inhibitor capable of inhibiting the repressible protease.

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

60. The method of claim 59, further comprising the step of withdrawing the protease inhibitor capable of inhibiting the repressible protease from the subject.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0044] These and other features, aspects, and advantages of the present disclosure will become better understood with regard to the following description, and accompanying drawings.

[0045] FIG. 1 depicts the normalized percentage CAR expression in cells transfected to express one of four different fusion proteins.

DETAILED DESCRIPTION

[0046] 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, e.g., Hepatitis C Viruses: Genomes and Molecular Biology (S. L. Tan ed., Taylor & Francis, 2006), Fundamental Virology, 3′ Edition, vol. I & II (B. N. Fields and D. M. Knipe, eds.); Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell eds., Blackwell Scientific Publications); A. L Lehninger, Biochemistry (Worth Publishers, Inc, current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (3.sup.rd Edition, 2001), Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.).

[0047] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entireties.

Definitions

[0048] In describing the present disclosure, the following terms will be employed, and are intended to be defined as indicated below.

[0049] It must 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.

[0050] The term “about,” particularly in reference to a given quantity, is meant to encompass deviations of plus or minus five percent.

[0051] 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, a protease is active (cleaves a cognate cleavage site) in the absence of the specific agent and is inactive (does not cleave a 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 present disclosure.

[0052] Non-limiting examples of proteases include hepatitis C virus proteases (e.g., NS3 and NS2-3); signal peptidase; proprotein convertases of the subtilisin/kexin family (furin, PCI, PC2, PC4, PACE4, PC5, PC); proprotein convertases cleaving at hydrophobic residues (e.g., Leu, Phe, Val, or Met); proprotein convertases cleaving at small amino acid residues such as Ala or Thr; proopiomelanocortin converting enzyme (PCE); chromaffin granule aspartic protease (CGAP); prohormone thiol protease, 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 protease; and, caspases 1, 2, 3, 4, 5, 6, 7, 8, and 9 Other proteases include, but are not limited to, 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 secretases alpha, beta and gamma, angiotensin converting enzyme secretase; TGF alpha secretase; T F alpha secretase; FAS ligand secretase; TNF receptor-I and -II secretases; CD30 secretase; KL1 and KL2 secretases; IL6 receptor secretase, CD43, CD44 secretase; CD 16-1 and CD 16-11 secretases; L-selectin secretase; Folate receptor secretase; MMP 1, 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); ADAM 1, 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, e.g., V. Y. H. Hook, Proteolytic and cellular mechanisms in prohormone and proprotein processing, R G Landes Company, Austin, Tex., USA (1998); N. M. Hooper et al., Biochem. J. 321: 265-279 (1997); Z. Werb, Cell 91: 439-442 (1997); T. G. Wolfsberg et al., J. Cell Biol. 131: 275-278 (1995); K. Murakami and J. D. Etlinger, Biochem. Biophys. Res. Comm. 146: 1249-1259 (1987); T. Berg et al., Biochem. J. 307: 313-326 (1995); M. J. Smyth and J. A. Trapani, Immunology Today 16: 202-206 (1995), R V. Talanian et al., J. Biol. Chem. 272: 9677-9682 (1997); and N. A Thomberry et al., J. Biol. Chem. 272: 17907-17911 (1997), the disclosures of which are incorporated herein.

[0053] A “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 A number of NS3 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. 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, 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 date of filing of this application) are herein incorporated by reference. Any of these sequences or a variant thereof comprising a sequence 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 a fusion protein or a recombinant polynucleotide encoding such a fusion protein, as described herein.

[0054] A “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. A number of 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 date of filing of this application) are herein incorporated by reference. Any of these sequences or a variant thereof comprising a sequence 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 a fusion protein or a recombinant polynucleotide encoding such a fusion protein, as described herein.

[0055] 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., seven genotypes 1-7) or subtype. The molecule need not be physically derived from HCV, but may be synthetically or recombinantly produced. A number of polyprotein nucleic acid and protein sequences are known Representative HCV polyprotein sequences are listed in the National Center for Biotechnology Information (NCBI) database. See, for example, 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 EF 108306; all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference. Any of these sequences or a variant thereof comprising a sequence 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 a fusion protein or a recombinant polynucleotide encoding such a fusion protein, as described herein.

[0056] For a discussion 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; herein incorporated by reference in their entireties.

[0057] The terms “fusion protein,” “fusion polypeptide,” “degron fusion protein,” or “degron fusion” as used herein refer to a fusion comprising a degron in combination 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 connected to the polypeptide of interest through a cleavable linker comprising a cleavage site capable of being recognized by the protease of the fusion to allow self-removal of the protease and degron from the polypeptide of interest. The position of the cleavage site in the fusion may be chosen to allow release of the polypeptide of interest from the fusion essentially unmodified or with little modification (e.g., less than 10 extra amino acids). The fusion polypeptides may be designed for N-terminal or C-terminal attachment of the degron to the polypeptide of interest. The fusion polypeptides may also contain sequences exogenous to the degron, protease, and polypeptide of interest. For example, the fusion may include targeting or localization sequences, detectable labels, or tag sequences.

[0058] The term, “cell receptor” as used herein, refers to a membrane protein that responds specifically to individual extracellular stimuli and generates intracellular signals that give rise to a particular functional responses. Non-limiting examples of these stimuli/signals include soluble factors generated locally (for example, synaptic transmission) or distantly (for example, hormones and growth factors), ligands on the surface of other cells (e.g., an antigen, such as a cancer antigen), 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).

[0059] The term, “chimeric antigen receptor” or alternatively a “CAR” as used herein refers to a polypeptide or a set of polypeptides, which when expressed in an immune effector cell, provides the cell with specificity for a target cell, typically a cancer cell, and with intracellular signal generation. 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”) comprising a functional signaling domain derived from a stimulatory molecule and/or 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 include recruitment domains, such as dimerization or multimerization domains, that can couple the polypeptides to one another. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising 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, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule.

[0060] The term, “extracellular protein binding domain” as used herein, refers to a molecular binding domain which is typically an ectodomain of a cell receptor and is located outside the cell, exposed to the extracellular space. Am extracellular protein binding domain can include any molecule (e.g., protein or peptide) capable of binding to another protein or peptide. In some embodiments, an extracellular protein binding domain comprises 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, an 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.

[0061] The term, “intracellular signaling domain” as used herein, refers to a functional endodomain of a cell receptor located inside the cell. Following binding of the molecular binding domain to an antigen, for example, the signaling domain transmits a signal (e.g., proliferative/survival signal) to the cell. In some embodiments, the signaling domain is a CD3-zeta protein, which includes three immunoreceptor tyrosine-based activation motifs (ITAMs) Other examples of signaling domains include CD28, 4-1BB, and OX40. In some embodiments, a cell receptor comprises more than one signaling domain, each referred to as a co-signaling domain.

[0062] The term, “transmembrane domain” as used herein, refers to a domain that spans a cellular membrane. In some embodiments, a transmembrane domain comprises a hydrophobic alpha helix. Different transmembrane domains result in different receptor stability. In some embodiments, a transmembrane domain of a cell receptor of the present disclosure comprises a CD3-zeta transmembrane domain or a CD28 transmembrane domain.

[0063] The term, “recruitment domain” as used herein, refers to an interaction motif found in various proteins, such as helicases, kinases, mitochondrial proteins, caspases, other cytoplasmic factors, etc. The recruitment domains mediate formation of a large protein complex via direct interactions between recruitment domains. In some embodiments, recruitment domains of the present disclosure are dimerization or multimerization domains.

[0064] 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 to a patient (a subject) a gene or polypeptide of interest, such as a therapeutic protein Cell based-therapies, as provided herein, also encompass preventative and diagnostic regimes. Thus, a gene of interest (and encoded product of interest) used in a cell-based therapy may 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).

[0065] The term, “cognate cleavage site” as used herein, refers to a specific sequence or sequence motif recognized by and cleaved by a protease of the present disclosure. A cleavage site for a protease includes the specific amino acid sequence or motif recognized by the protease during proteolytic cleavage and typically includes the surrounding one to six amino acids on either side of the scissile bond, which bind to the active site of the protease and are used for recognition as a substrate.

[0066] The term “cleavable linker” refers to a linker comprising a cleavage site. The cleavable linker may include a cleavage site specific for an enzyme, such as a protease or other cleavage agent A cleavable linker is typically cleavable under physiological conditions.

[0067] The term, “degron” as used herein, refers to a protein or a part thereof that is important in regulation of protein degradation rates. Various degrons known in the art, including but not limited to short amino acid sequences, structural motifs, and exposed amino acids, can be used in various embodiments of the present disclosure. Degrons identified from a variety of organisms can be used. In some embodiments, degrons of the present disclosure comprise a degradation sequence. In some embodiments, the degron is a self-excising degron. A self-excising degron is a degron that is fused to a polypeptide of interest such that a protease of the present disclosure is capable of cleaving 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 following cleavage.

[0068] The term, “degradation sequence” as used herein, refers to a sequence that promotes degradation of an attached protein through either the proteasome or autophagy-lysosome pathways. In preferred embodiments, a degradation sequence is a polypeptide that destabilize a protein such that half-life of the protein is reduced at least two-fold, when fused to the protein Many different degradation sequences/signals (e.g., of the ubiquitin-proteasome system) are known in the art, any of which may be used as provided herein A degradation sequence may be operably linked to a cell receptor, but need not be contiguous with it as long as the degradation sequence still functions to direct degradation of the cell receptor. In some embodiments, the degradation sequence induces rapid degradation of the cell 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(1l1):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; herein incorporated by reference.

[0069] 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 within the definition. Both full length proteins and fragments thereof are encompassed by the definition. The terms also include postexpression modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation, hydroxylation, and the like. Furthermore, for purposes of the present disclosure, a “polypeptide” refers to a protein which includes modifications, such as deletions, additions and substitutions to the native sequence, so long as the protein maintains the desired activity. These modifications may be deliberate, as through site directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.

[0070] By “derivative” is intended any suitable modification of the native polypeptide of interest, of a fragment of the native polypeptide, or of their respective analogs, such as glycosylation, phosphorylation, polymer conjugation (such as with polyethylene glycol), or other addition of foreign moieties, as 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.

[0071] By “fragment” is intended a molecule consisting of only a part of the intact full length sequence and structure. The fragment can include a C-terminal deletion an N-terminal deletion, and/or an internal deletion of the polypeptide. Active fragments 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 in question retains biological activity, such as catalytic activity, ligand binding activity, regulatory activity, degron protein degradation signaling, or fluorescence characteristics.

[0072] “Substantially purified” generally refers to isolation of a substance (compound, polynucleotide, protein, polypeptide, polypeptide composition) such that the substance comprises the majority percent of the sample in which it resides. Typically, in a sample, a substantially purified component comprises 50%, preferably 80%-85, more preferably 90-95% of the sample. 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.

[0073] By “isolated” is meant, when referring to a polypeptide, that the indicated molecule is separate and discrete from the whole organism with which the molecule is found in nature or is present in the substantial absence of other biological macro molecules of the same type. The term “isolated” with respect to a polynucleotide is a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences in association therewith; or a molecule disassociated from the chromosome.

[0074] The terms “label” and “detectable label” refer to a molecule capable of detection, including, but not limited to, radioactive isotopes, stable (non-radioactive) heavy isotopes, fluorescers, chemiluminescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin or haptens) and the like. The term “fluorescer” refers to a substance or a portion thereof that is capable of exhibiting fluorescence in the detectable range. Particular examples of labels that may be used with the present disclosure include, but are not limited to radiolabels (e.g., H, I, S, C, or P), stable (non-radioactive) heavy isotopes (e.g., .sup.13C or .sup.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, acradimum 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), 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, chloramphenical acetyl transferase, and urease Enzyme tags are used with their cognate substrate. The terms also include color-coded microspheres of known fluorescent light intensities (see e.g., microspheres with xMAP technology produced by Luminex (Austin, Tex.); microspheres containing quantum dot nanocrystals, for example, containing different ratios and combinations of quantum dot colors (e.g., Qdot nanocrystals produced by Life Technologies (Carlsbad, Calif.); glass coated metal nanoparticles (see e.g., SERS nanotags produced by Nanoplex Technologies, Inc. (Mountain View, Calif.); barcode materials (see e.g., sub-micron sized striped metallic rods such as Nanobarcodes produced by Nanoplex Technologies, Inc.), encoded microparticles with colored bar codes (see e.g., CellCard produced by Vitra Bioscience, vitrabio.com), and glass microparticles with digital holographic code images (see e.g., CyVera microbeads produced by Ulumina (San Diego, Calif.). As with many of the standard procedures associated with the practice of the present disclosure, skilled artisans will be aware of additional labels that can be used.

[0075] “Homology” refers to the percent identity between two polynucleotide 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 over a defined length of the molecules. As used herein, substantially homologous also refers to sequences showing complete identity to the specified sequence.

[0076] In general, “identity” refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of two polynucleotides or polypeptide sequences, respectively Percent identity can be determined by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of 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, such as ALIGN, Dayhoff, M. O. in Atlas of Protein Sequence and Structure M.O. Dayhoff ed., 5 Suppl. 3:353 358, National biomedical Research Foundation, Washington, D.C., which 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, Wis.) for example, the BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waterman algorithm. These programs are readily utilized with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referred to above. For example, percent identity of a particular nucleotide sequence to a reference sequence can be determined using the homology algorithm of Smith and Waterman with a default scoring table and a gap penalty of six nucleotide positions.

[0077] Another method of 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, Calif.). From this suite of packages, the Smith Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the “Match” value reflects “sequence identity” Other suitable programs for calculating the 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.

[0078] Alternatively, homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single stranded specific nuclease(s), and size determination of the digested fragments. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization, supra.

[0079] “Recombinant” as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, viral, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide with which it is associated in nature. The term “recombinant” as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. In general, the gene of interest is cloned and then expressed in transformed organisms, as described further below. The host organism expresses the foreign gene to produce the protein under expression conditions.

[0080] The term “transformation” refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion. For example, direct uptake, transduction or f-mating are included. The exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host genome.

[0081] “Recombinant host cells,” “host cells,” “cells,” “cell lines,” “cell cultures,” and other such terms denoting microorganisms or higher eukaryotic cell lines, refer to cells which can be, or have been, used as recipients for a recombinant vector or other transferred DNA, and include the progeny of the cell which has been transfected. Host cells may be cultured as unicellular or multicellular entities (e.g., tissue, organs, or organoids comprising the recombinant vector).

[0082] A “coding sequence” or a sequence that “encodes” 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 can be determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxy) terminus. A coding sequence can include, but is 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.

[0083] Typical “control elements,” include, but are not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located 3′ to the translation stop codon), sequences for optimization of initiation of translation (located 5′ to the coding sequence), and translation termination sequences.

[0084] “Operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. For example, a given promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when the proper enzymes are present. The promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence. In another example, a degron operably linked to a polypeptide is capable of promoting degradation of the polypeptide when the proper 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.

[0085] “Encoded by” refers to a nucleic acid sequence which codes for a polypeptide sequence, wherein the polypeptide sequence or a portion thereof contains an amino acid sequence of at least 3 to 5 amino acids, more preferably at least 8 to 10 amino acids, and even more preferably at least 15 to 20 amino acids from a polypeptide encoded by the nucleic acid sequence.

[0086] “Expression cassette” or “expression construct” refers to an assembly which is capable of directing the expression of the sequence(s) or gene(s) of interest. An expression cassette generally includes control elements, as described above, such as a promoter which is operably linked to (so as to direct transcription of) the sequence(s) or gene(s) of interest, and often includes a polyadenylation sequence as well. Within certain embodiments of the present disclosure, the expression cassette 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 which allows the plasmid construct to exist as single stranded DNA (e.g., a M1 3 origin of replication), at least one multiple cloning site, and a “mammalian” origin of replication (e.g., a SV40 or adenovirus origin of replication).

[0087] “Purified polynucleotide” refers to a polynucleotide of interest or fragment thereof which is essentially free, e.g., contains less than about 50%, preferably less than about 70%, and more preferably less than about at least 90%, of the protein with which the polynucleotide is naturally associated. Techniques for purifying polynucleotides of interest are well-known in the art and include, for example, disruption of the cell containing the polynucleotide with a chaotropic agent and separation of the polynucleotide(s) and proteins by ion-exchange chromatography, affinity chromatography and sedimentation according to density.

[0088] The term “transfection” is used to refer to the uptake of foreign DNA by a cell. A cell has been “transfected” when exogenous DNA has been introduced inside the cell membrane A number of transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (2001) Molecular Cloning, a laboratory manual, 3.sup.r 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. Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells. The term refers to both stable and transient uptake of the genetic material, and includes uptake of peptide- or antibody-linked DNAs.

[0089] A “vector” is capable of transferring nucleic acid sequences to target cells (e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes). Typically, “vector construct.” “expression vector,” and “gene transfer vector,” mean any nucleic acid construct capable of directing the expression of a nucleic acid of interest and which can transfer nucleic acid sequences to target cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors.

[0090] The terms “variant,” “analog” and “mutein” refer to biologically active derivatives of the reference molecule that retain desired activity, such as fluorescence or oligomerization characteristics. 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 conservative in nature) and/or deletions, relative to the native molecule, so long as the modifications do not destroy biological activity and which are “substantially homologous” to the reference molecule as defined below. In general, the amino acid sequences of such analogs will have a high degree of sequence homology to the reference sequence, e.g., amino acid sequence homology of more than 50%, generally more than 60%-70%, even more particularly 80%-85% or more, such as at least 90%-95% or more, when the two sequences are aligned. Often, the analogs will include the same number of amino acids but will include substitutions, as explained herein. The term “mutein” further includes polypeptides having one or more amino acid-like molecules including but not limited to compounds comprising 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 linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring (e.g., synthetic), cyclized, branched molecules and the like. The term also includes molecules comprising one or more N-substituted glycine residues (a “peptoid”) and other synthetic amino acids or peptides. (See, e.g., U.S. Pat. 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 descriptions of peptoids). Methods for making polypeptide analogs and muteins are known in the art and are described further below.

[0091] As explained above, analogs generally include substitutions that are conservative in nature, i.e., those substitutions that take place within a family of amino acids that are related in their side chains. Specifically, amino acids are generally divided 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, it is reasonably predictable that an isolated replacement of leucine with isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar conservative replacement of an amino acid with a structurally related amino acid, will not have a major effect on the biological activity. For example, the polypeptide of interest may include 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 5-25, so long as the desired function of the molecule remains intact. One of skill in the art may readily determine regions of the molecule of interest that can tolerate change by reference to Hopp/Woods and Kyte-Doolittle plots, well known in the art.

[0092] “Gene transfer” or “gene delivery” refers to methods or systems for reliably inserting DNA or RNA of interest into a host cell. Such methods can result in transient expression of non-integrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g., episomes), or integration of transferred genetic material into the genomic DNA of host cells. Gene delivery expression vectors include, but are not limited to, vectors derived from bacterial plasmid vectors, viral vectors, non-viral vectors, alphaviruses, pox viruses and vaccinia viruses.

[0093] The term “derived from” is used herein to identify the original source of a molecule but is not meant to limit the method by which the molecule is made which can be, for example, by chemical synthesis or recombinant means.

[0094] A polynucleotide “derived from” a designated sequence refers to a polynucleotide sequence which comprises 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, i.e., identical or complementary to, a region of the designated nucleotide sequence. The derived polynucleotide will not necessarily be derived physically from the 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 the information provided by the sequence of bases in the region(s) from which the polynucleotide is derived. As such, it may represent either a sense or an antisense orientation of the original polynucleotide.

[0095] The term “heterologous” as it relates to nucleic acid sequences such as coding sequences and control sequences, denotes sequences that are not normally joined together, and/or are not normally associated with a particular cell. Thus, a “heterologous” region of a nucleic acid construct or a 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 could include a coding sequence flanked by sequences not found in association with the coding sequence in nature. Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., synthetic sequences having codons different from the native gene). Similarly, a cell transformed with a construct which is not normally present in the cell would be considered heterologous for purposes of the present disclosure.

[0096] By “recombinant virus” is meant a virus that has been genetically altered, e.g., by the addition or insertion of a heterologous nucleic acid construct into the particle.

[0097] “Recombinant virion,” as used herein, refers to a viral particle containing a recombinant viral vector (e.g., conditionally replicating viral vector encoding a degron fusion protein). Generally, a recombinant virion comprises one or more structural proteins and the viral vector. The recombinant virion may also contain a nucleocapsid structure, and in some cases, a lipid envelope derived from the host cell membrane.

[0098] The terms “subject” refers to any invertebrate or vertebrate subject, including, without limitation, humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. The term does not denote a particular age. Thus, both adult and newborn individuals are intended to be covered.

[0099] “Recombinant animal” refers to a nonhuman subject which has been a recipient of a recombinant vector or other transferred DNA, and also includes the progeny of a recombinant

[00001]$\mathrm{animal}.$

Other Interpretational Conventions

[0100] Ranges recited herein are understood to be shorthand for all of the values within the range, inclusive of the recited endpoints. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range 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.

[0101] Unless otherwise indicated, reference to a compound that has one or more stereocenters intends each stereoisomer, and all combinations of stereoisomers, thereof.

Overview

[0102] 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 as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the present disclosure only, and is not intended to be limiting.

[0103] 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.

[0104] The present disclosure is based on the discovery that certain mutations within an immunodominant epitope of a protease of the present disclosure, 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 may be used to reduce the immunogenicity and modulate the activity of the protease when used in therapeutic applications, such as with small molecule-assisted shutoff (SMASh) techniques, in which a polypeptide of interest is fused to a 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 on subsequently synthesized protein copies. The degron when attached causes rapid degradation of the linked protein. Alternatively, a protease of the present disclosure may be fused to a polypeptide of interest with a functional domain, or in the case of a multi-domain polypeptide between domains such that addition of a protease inhibitor can control one or more functions of the polypeptide of interest. As disclosed herein use of such a repressible protease allows for reversible and dose-dependent shutoff of various proteins with high dynamic range in multiple cell types.

Fusion Proteins

[0105] Certain aspects of the present disclosure relate to fusion proteins comprise a variant protease (e.g., a variant HCV NS3 protease) fused to a selected polypeptide of interest and a cognate protease cleavage site in an arrangement designed to control function and/or production of the polypeptide of interest. The cleavage site is capable of being recognized by the protease of the fusion protein in order to allow cleavage of one or more domains within the polypeptide of interest. The position of the cleavage site in the fusion is preferably chosen to allow for controlled function and/or expression of the polypeptide of interest. The fusion proteins of the present disclosure may be designed with N-terminal or C-terminal attachment of the protease to the polypeptide of interest. The fusion protein may also contain sequences exogenous to the protease, cognate cleavage site, and polypeptide of interest. For example, the fusion may include targeting or localization sequences, or tag sequences. In addition, the fusion protein may comprise a detectable label (e.g., fluorescent, bioluminescent, chemiluminescent, colorimetric, or isotopic label) to facilitate monitoring production and degradation of the polypeptide of interest.

Variant Proteases

[0106] Certain aspects of the present disclosure relate to a fusion protein comprising a variant protease, wherein the variant protease comprises one or more mutations the decrease immunogenicity and/or modulate protease activity when the fusion protein is expressed in a mammalian cell.

[0107] Variant proteases 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 a variant protease of the present disclosure. When a protease is selected, its cognate cleavage site and protease inhibitors known in the art to bind and inhibit the protease can be used in a combination. Exemplary combinations for the use are provided below in Table 1. Representative sequences of the proteases are available from public database including UniProt through the uniprot.org website. UniProt accession numbers for the proteases are also provided below in Table 1.

TABLE-US-00001 TABLE 1 UniProt Accession Cognate cleavage Specific Protease Number/Sequence site Inhibitors HCVNS3 APITAYAQQTRGLLGCIITSLT ADLEVVTSTWL Simeprevir, GRDKNQVEGEVQIVSTATQTFL (NS3/NS4A) Danoprevir, ATCINGVCWAVYHGAGTRTIA (SEQ ID NO: 8) Asunaprevir, SPKGPVIQMYTNVDQDLVGWP CMSADLEVVTSTW Ciluprevir, APQGSRSLTPCTCGSSDLYLVT VLVGGVL Boceprevir, RHADVIPVRRRGDSRGSLLSPR (NS3/NS4A) Sovaprevir, PISYLKGSSGGPLLCPAGHAVG (SEQ ID NO: 4) Paritaprevir, LFRAAVCTRGVAKAVDFIPVE DEMEECSQHL Telaprevir, NLETTMRSPVFTD (NS4A/NS4B) Grazoprevir, (SEQ ID NO: 2) (SEQ ID NO: 9) Glecaprevir, APITAYAQQT RGLLGCIITS YQEFDEMEECSQH Voxiloprevir LTGRDKNQVE GEVQIVSTAA LPYIEQG QTFLATCING VCWTVYHGAG (NS4A/NS4B) TRTIASSKGP VIQMYTNVDQ (SEQ ID NO: 5) DLVGWPAPQG ARSLTPCTCG ECTTPCSGSWL SSDLYLVTRH ADVIPVRRRG (NS4B/NS5A) DGRGSLLSPR PISYLKGSSG (SEQ ID NO: 10) GPLLCPAGHA VGIFRAAVCT WISSECTTPCSGSW RGVAKAVDFI PVEGLETTMR LRDIWD SPVFSD (SEQ ID NO: 12) (NS4B/NS5A) (SEQ ID NO: 6) EDVVPCSMG (NS5A/NS5B) (SEQ ID NO: 11) GADTEDWCCSMSYSW TGAL (NS5A/NS5B)  (SEQ ID NO: 7) HIV-1 PQVTLWQRPLVTIKIGGQLKEA Amprenavir, protease LLDTGADDTVLEEMSLPGRWK Atazanavir, PKMIGGIGGFIKVRQYDQILI Darunavir, EICGHKAIGTVLVGPTPVNII Fosamprenavir, GRNLLTQIGCTLNF Indinavir, (SEQ ID NO: 13) Lopinavir, Nelfmavir, Ritonavir, Saquinavir, Tipranavir Signal P67812, P15367, preference of peptidase P00804, P0803 eukaryotic signal peptidase for  cleavage after residue 20 (Xaa.sup.20↓) of pre(Apro)apoA-II: Ala, Cys > Gly > Ser, Thr > Pro > Asn, Val, Ile, Leu, Tyr, His, Arg, Asp. proprotein Q16549, Q8NBP7, (R/K)-X-(hydrophobic)-X↓, convertases Q92824, P29120, where cleaving at Q6UW60, P29122, X is any amino acid hydrophobic Q9QXV0 residues (e.g., Leu, Phe, Val, or Met); proprotein Q16549, Q8NBP7, Q92824, K/R)-(X)n-(K/R)↓, convertases P29120, Q6UW60, P29122 where n is 0, cleaving at 2, 4 or 6 and X is small amino any amino acid acid residues such as Ala or Thr; proopiomelanoc Q9UO77615, 0776133 Cleavage at paired ortin converting basic residues enzyme (PCE); in certain prohormones, either between them, or on the carboxyl side chromaffin lends to cleave granule aspartic dipeptide bonds protease that have hydrophobic (CGAP); residues as well as a beta- methylene group prohormone P07154, P07711, thiol protease P06797, P25975, (cathepsin L1) Q28944 carboxypeptidases Q9M099, P15169, cleaves a peptide (e.g., Q04609, P08819, bond at the carboxvpeptidase P08818, O77564, carboxy-terminal E/H, P70627, 035409, (C-terminal) end carboxypeptidase P07519, Q8VZU3, of a protein or D and P22792, P15087, peptide carboxypeptidase P16870, Q9JHH6, Z); Q96IY4, Q7L8A9 aminopeptidases cleaves a peptide (e.g., arginine bond at the aminopeptidase, amino-terminal  lysine (N-terminal) end aminopeptidase, of a protein or aminopeptidase peptide B); prolyl Q12884, P48147, Hydrolysis of Pro-|-Xaa >> endopeptidase; P97321, Q4J6C6, Ala-|-Xaa in oligopeptides. Release of an N-terminal dipeptide, Xaa-Yaa-|-Zaa-, from a polypeptide, preferentially when Yaa is Pro. provided Zaa is neither Pro nor hydroxyproline. aminopeptidase P97449, P15144, Release of an N-terminal N; P15145, P15684 Amino acid, Xaa-|-Yaa- from a peptide, amide or arylamide. Xaa is preferably Ala, but may be most amino acids including Pro (slow action). When a terminal hydrophobic residue is followed by a prolyl residue, the two may be released as an intact Xaa-Pro dipeptide insulin P14735, P35559, Degradation of insulin, degrading Q9JHR7, Glucagon and other enzyme; P22817, Q24K02 polypeptides. No action on proteins. Cleaves multiple short polypeptides that vary considerably in sequence calpain; 008529, P17655, No specific amino acid sequence Q07009, Q27971, is uniquely recognized by P20807, P07384, calpains. Amongst protein O35350, O14815, substrates, tertiary structure P04632, Q9Y6Q1, elements rather than primary O15484, Q9HC96, amino acid sequences appear to be A6NHC0, Q9UMQ6 responsible for directing cleavage to a specific substrate. Amongst peptide and small-molecule substrates, the most consistently reported specificity is for small, hydrophobic amino acids (e.g., leucine, valine and isoleucine) at the P2 position, and large hydrophobic amino acids (e.g., phenylalanine and ty rosine) at the P1 position. One fluorogenic calpain substrate is (EDANS-Glu- Pro-Lcu-Phe═Ala-Glu-Arg-Lys- DABCYL) (EDANSEPLFAERKDABCYL, SEQ ID NO: 14), with cleavage occurring at the Phe═Ala bond. caspase 1 P29466, P29452 Strict requirement for an Asp residue at position P1 and has a preferred cleavage sequence of Tyr-Val-Ala-Asp-|- (YVAD, SEQ ID NO: 15). caspase 2 P42575, P29594 Strict requirement for an Asp residue at P1, with 316-asp being essential for proteolytic activity and has a preferred cleavage sequence of Val-Asp-Val-Ala- Asp-|- (YDVAD, SEQ ID NO: 16) caspase 3 P42574, P70677 Strict requirement for an Asp residue at positions P1 and P4. It has a preferred cleavage sequence of Asp-Xaa-Xaa-Asp-|- with a hydrophobic amino-acid residue at P2 and a hydrophilic amino-acid residue at P3, although Val or Ala are also accepted at this position. caspase 4 P70343, P49662 Strict requirement for Asp at the P1 position. It has a preferred cleavage sequence of Tyr-Val- Ala-Asp-|- (YVAD, SEQ ID NO: 15) but also cleaves at Asp-Glu- Val-Asp-|- (DEVD; SEQ ID NO: 17) caspase 5 P51878 Strict requirement for Asp at the P1 position. It has a preferred cleavage sequence of Tyr-Val- Ala-Asp-|- (YVAD, SEQ ID NO: 15) but also cleaves at Asp-Glu- Val-Asp-|- (DEVD; SEQ ID NO: 17). caspase 6 P55212 Strict requirement for Asp at position P1 and has a preferred cleavage sequence of Val-Glu- His-Asp-|- (VEHD; SEQ ID NO: 18). caspase 7 P97864, P55210 Strict requirement for an Asp residue at position P1 and has a preferred cleavage sequence of Asp-Glu-Val-Asp-KDEVD; SEQ ID NO: 17). caspase 8 Q8IRY7, 089110, Strict requirement for Asp at Q14790 position P1 and has a preferred cleavage sequence of (Leu/Asp/Val)-Glu-Thr-Asp-|- (Gly/Ser/Ala). caspase 9 P55211, Q8C3Q9, Strict requirement for an Asp Q5IS54 residue at position P1 and with a marked preference for His at position P2. It has a preferred cleavage sequence of Leu-Gly- His-Asp-|-Xaa (LGHD (SEQ ID NO: 19) -|- Xaa). caspase 10 Q92851 Strict requirement for Asp at position P1 and has a preferred cleavage sequence of Leu-Gln- Thr-Asp-|-Gly (LQTDG, SEQ ID NO: 20). puromycin P55786, Q11011, Release of an N-terminal amino acid, sensitive preferentially alanine, from a aminopeptidase: wide range of peptides, amides and arvlamides. angiotensin P12821, P09470, Release of a C-terminal dipeptide, Benazepril converting Q9BYF1 oligopeptide-|-Xaa-Yaa, when Xaa (Lotensin), enzyme (ACE); MGAASGRRGP GLLLPLPLLL is not Pro, and Yaa is neither Asp Captopril, LLPPQPALAL DPGLQPGNFS nor Glu. Enalapril ADEAGAQLFA QSYNSSAEQV (Vasotec), LFQSVAASWA HDTNITAENA Fosinopril, RRQEEAALLS QEFAEAWGQK Lisinopril AKELYEPIWQ NFTDPQLRRI (Prinivil, IGAVRTLGSA NLPLAKRQQY Zestril), NALLSWMSRI YSTAKVCLPN Moexipril, KTATCWSLDP DLTNILASSR Perindopril SYAMLLFAWE GWHNAAGIPL (Aceon), KPLYEDFTAL SNEAYKQDGF Quinapril TDTGAYWRSW YNSPTFEDDL (Accupril), SHLYQQLEPL YLNLHAFVRR Ramipril ALHRRYGDRY INLRGPIPAH (Altace), LLGDMWAQSW ENIYDMVVPF Trandolapril PDKPNLDVTS TMLQQGWNAT (Mavik), HMFRVAEEFF TSLELSPMPP Zofenopril, EFWEGSMLEK PADGREVVCH ASAWDFYNRK DPRIKQCTRV TMDQLSTVHH EMGHIQYYLQ YKDLPVSLRR GANPGFHEAI GDYLALSVST PEHLHKIGLL DRVTNDTESD INYLLKMALE KIAFLPFGYL VDQWRWGVFS GRTPPSRYNF DWWYLRTKYQ GICPPVTRNE THFDAGAKFH VPNVTPYIRY FVSFVLQFQF HEALCKEAGY EGPLHQCDIY RSTKAGAKLR KVLQAGSSRP WQEVLKDMVG LDALDAQPLL KYFQPVTQWL QEQNQQNGEV LGWPEYQWHP PLPDNYPEGI DLVTDEAEAS KFVEEYDRTS QVVWNEYAEA NWNYNTNITT ETSKILLQKN MQIANHTLKY GTQARKFDVN QLQNTTIKRI IKKVQDLERA ALPAQELEEY NKILLDMETT YSVATVCHPN GSCLQLEPDL TNVMATSRKY SDLLWAWEGW RDKAGRAILQ FYPKYVELIN QAARLNGYVD AGDSWRSMYE TPSLEQDLER LFQELQPLYL NLHAYVRRAL HRHYGAQHIN LEGPIPAHLL GNMWAQTWSN IYDLVVPFPS APSMDTTSAM LKQGWTPRRM FKEADDFFTS LGLLPVPPEF WNKSMLEKPT DGREVVCHAS AWDFYNGKDF RIKQCTTVNL EDLVVAHHEM GHIQYFMQYK DLPVALREGA NPGFHSAIGD VLALSVSTPK HLHSLNLLSS EGGSDEHDIN FLMKMALDKI AFIPFSYLVD QWRWRVFDGS ITKENYNQEW WSLRLKYQGL CPPVPRTQGD FDPGAKFHIP SSVPYIRYFV SFIIQFQFHE ALCQAAGHTG PLHKCDIYQS KEAGQRLATA MKLGFSRPWP EAMQLITGQP NMSASAMLSY FKPLLDWLRT ENELHGEKLG WPQYNWTPNS ARSEGPLPDS GRVSFLGLDL DAQQARVGQW LLLFLGIALL VATLGLSQRL FSIRHRSLHR HSHGPQFGSE VELRHS (SEQ ID NO: 21) pyroglutamyl Q9NXJ5 Release of the N-terminal peptidase II; pyroglutamyl group from pGlu-- His-Xaa tripeptides and pGlu-- His-Xaa-Gly tetrapeptides dipeptidyl P27487, P14740, Release of an N-terminal peptidase IV; P28843 dipeptide, Xaa-Yaa-|-Zaa-, from a polypeptide, preferentially when Yaa is Pro, provided Zaa is neither Pro nor hydroxyproline. N-arginine O43847, Q8BHG1 Hydrolysis of polypeptides, dibasic preferably at -Xaa-|-Arg-Lys-, convertase; And less commonly at -Arg-|-Arg-Xaa-, in which Xaa is not Arg or Lys. endopeptidase P52888, P24155 Preferential cleavage of bonds 24.15 (thimet with hydrophobic residues at P1, oligopeptidase) P2 and P3′ and a small residue at P1′ in substrates of 5 to 15 residues. endopeptidase Q9BYT8, Q91YP2 Preferential cleavage in 24.16 neurotensin: 10-Pro-|-Tyr-11 (neurolysin) amyloid P05067, P12023, Endopeptidase of broad precursor Q9Y5Z0, P56817 specificity. protein secretase alpha amyloid P05067, P12023, Broad endopeptidase specificity. precursor Q9Y5Z0, P56817 Cleaves Glu-Val-Asn-Leu-|-Asp- protein Ala-Glu-Phe (EVNLDAEF, SEQ secretase ID NO: 22) in the beta Swedish variant of AlzhFeimer's amyloid precursor protein. amyloid P05067, P12023, intramembrane cleavage of precursor Q9Y5Z0, P56817 integral membrane proteins protein secretase gamma MMP 1 P03956, Q9EPL5uy Cleavage of the triple helix of SB-3CT collagen at about three-quarters of p-OH  the length of the molecule from SB-3CT the N-terminus, at 775-Gly-|-Ile- O-phosphate 776 in the alpha-1(I) chain. Cleaves synthetic substrates and alpha-macroglobulins at bonds SB-3CT where P1′ is a hydrophobic RXP470.1 residue. MMP 2 P08253, P33434 Cleavage of gelatin type I and SB-3CT collagen types IV, V, VII, X. p-OH SB-3CT Cleaves the collagen-like O-phosphate sequence Pro-Gln-Gly-|-Ile-Ala- SB-3CT Gly-Gln (PQGIAGQ, SEQ ID RXP470.1 NO: 23). MMP 3 P08254, P28862 Preferential cleavage where P1′, SB-3CT P2′ and P3′ are hydrophobic p-OH SB-3CT residues. O-phosphate SB-3CT RXP470.1 MMP 7 P09237, Q10738 Cleavage of 14-Ala-|-Leu-15 and SB-3CT 16-Tyr-|-Leu-17 in B chain of p-OH SB-3CT insulin. No action on collagen O-phosphate types I, II, IV, V. Cleaves gelatin SB-3CT chain alpha-2(I) > alpha-1(1). RXP470.1 MMP 8 P22894, O70138 Can degrade fibrillar type I, II, SB-3CT and III collagens. p-OH SB-3CT Cleavage of interstitial collagens O-phosphate in the triple helical domain. SB-3CT Unlike EC 3.4.24.7, this enzyme RXP470.1 cleaves type III collagen more slowly than type I. MMP 9 P14780, P41245 Cleavage of gelatin ty pes I and V SB-3CT and collagen types IV and V. p-OH SB-3CT Cleaves KiSS1 at a Gly-|-Leu O-phosphate bond. SB-3CT Cleaves type IV and type V RXP470.1 collagen into large C-terminal three quarter fragments and shorter N-tenninal one quarter fragments. Degrades fibronectin but not laminin or Pz-peptide. MMP 10 P09238, O55123 Can degrade fibroncctin, gelatins SB-3CT of type I, III, IV, and V; weakly p-OH SB-3CT collagens III, IV, and V. O-phosphate SB-3CT RXP470.1 MMP 11 P24347, Q02853 A(A/Q)(N/A)↓(L/Y) SB-3CT (T/V/M/R)(R/K) p-OH SB-3CT G(G/A)E1LR O-phosphate ↓ denotes the cleavage site SB-3CT RXP470.1 MMP 12 P39900, P34960 Hydrolysis of soluble and SB-3CT insoluble elastin. Specific p-OH SB-3CT cleavages arc also produced at 14- O-phosphate Ala-|-Leu-15 and 16-Tyr-|-Leu-17 SB-3CT in the B chain of insulin has  RXP470.1 significant elastolytic activity. Can accept large and small amino acids at the P1′ site, but has a preference for leucine. Aromatic or hydrophobic residues are preferred at the P1 site, with small hydrophobic residues (preferably alanine) occupying P3. MMP 13 P45452, P33435 Cleaves triple helical collagens, SB-3CT including type I, type II and type p-OH SB-3CT III collagen, but has the highest O-phosphate activity with soluble type II SB-3CT collagen. Can also degrade RXP470.1 collagen type IV, type XIV and type X. MMP 14 P50281, P53690 Activates progclalinase A by SB-3CT cleavage of the propeptide at 37- p-OH SB-3CT Asn-|-Leu-38. Other bonds O-phosphate hydrolyzed include 35-Gly-|-Ile- SB-3CT 36 in the propeptide of RXP470.1 collagenase 3. and 341-Asn-|-Phe- 342, 441-Asp-|-Leu-442 and 354- Gln-|-Thr-355 in the aggrecan interglobular domain. urokinase P00749, P06869 Specific cleavage of Arg-|-Val Plasminogen plasminogen bond in plasminogen to form activator activator (uPA) plasmin. inhibitors (PAI) tissue P00750, P11214 Specific cleavage of Arg-|-Val Plasminogen plasminogen bond in plasminogen to form activator activator (tPA) plasmin. inhibitors (PAI) plasmin P00747, P20918 Preferential cleavage: Lys-|-Xaa > α-2- Arg-|-Xaa, higher selectivity than antiplasmin trypsin. Converts fibrin into (AP) soluble products. thrombin P00734, P19221 Cleaves bonds after Arg and Lvs Converts fibrinogen to fibrin and activates factors V, VII, VIII, XIII, and, in complex with thrombomodulin, protein C. BMP-1 P13497, P98063 Cleavage of the C-terminal (procollagen C- propeptide at Ala-|-Asp in type I peptidase) and II procollagens and at Arg-|- Asp in type III. ADAM Q9P0K1, Q9UKQ2, Q9JLN6, SB-3CT O14672, Q13444, P78536, p-OH SB-3CT Q13443, O43184, P78325, O-phosphatc Q9UKF5, Q9BZ11, Q9H2U9, SB-3CT Q99965, O75077, Q9H013, RXP470.1 O43506 granzyme A P12544, P11032 Preferential cleavage: -Arg-|-Xaa-, -Lys-|-Xaa->>-Phe-|-Xaa- in small molecule substrates. granzyme B P10144, P04187 Preferential cleavage: -Asp-|-Xaa->>-Asn-|-Xaa- > -Met-|-Xaa-, -Ser-|-Xaa-. granzyme C/ P08882, P20718 Preference for bulky and aromatic granzyme H residues at the P1 position and acidic residues at the P3′ and P4′ sites. granzyme M P51124, Q03238 Cleaves peptide substrates after methionine, leucine, and norleucine. tobacco Etch P04517, P0CK09 E-Xaa-Xaa-Y -Xaa-Q-(G/S), with virus (TEV) cleavage occurring between Q and protcase G/S. The most common sequence is ENLYFQS (SEQ ID NO: 24). chymotrypsin- P08217, Q9UNI1, Q91X79, -Thermobifida like serine P08861, P09093, P08218 fusca protease Thermopin -Pyrobaculum aerophilum Aeropin -Thermococciis kodakaraensis Tk-serpin -Alteromonas sp. Marinostatin -Streptomyces misionensis SMTI -Streptomyces sp. chymostatin alphavirus P08411, P03317, P13886, proteases Q8JUX6, Q86924, Q4QXJ8, 08QL53, P27282, Q5XXP4 chymotrypsin- Q86TL0, Q14790, Q99538, -Thermobifida like cysteine O15553 fusca proteases Thermopin -Pyrobaculum aerophilum Aeropin -Thermococcus kodakaraensis Tk-serpin -Alteromonas sp. Marinostatin -Streptomyces misionensis SMTI -Streptomyces sp. chymostatin papain-like P25774, P53634, Q96K76 cysteine protcascs picomavirus P03305, P03311, P13899 leader proteases HIV proteases P04585, P03367, P04584, P03369, P12497, P03366, P04587 Herpesvirus P10220, Q2HRB6, O40922, proteases O69527 adenovirus P03252, P24937, Q83906, proteases P68985, P09569, P11825, P10381 Streptomyces P00776 griseus protease A (SGPA) Streptomyces P00777 griseus protcase B (SGPB) alpha-lytic P85142, P00778 protease serine P48740, P98064, Q9UL52, proteases P05981, O60235 cysteine Q86TL0, Q14790, Q8WYN0, proteases Q96DT6, P55211 aspartic Q9Y5Z0, P56817, Q00663, protcascs Q53RT3, P0CY27 threonine Q9UI38, Q16512, Q9H6P5, proteases Q8IWU2, Mast cell (MC) NM_001836 Abz-HPFHL (SEQ ID NO: 25)- BAY 1142524 chymase Lys(Dnp)-NH2 (SEQ ID NO: 56) SUN13834 (CMA1) Rat mast cell NM_017145, NM_172044, Abz-HPFHL (SEQ ID NO: 25)- TY-51469 protcase NM_001170466, Lys(Dnp)-NH2 (SEQ ID NO: 56) −1,−2, NM_019321, −3, −4, −5 NM_013092 Rat vascular O70500 Abz-HPFHL chymase (SEQ ID NO: 25)- (RVCH) Lys(Dnp)-NH2  (SEQ ID NO: 56) DENV NS3pro >sp|P33478|1475-2093 A strong preference for basic Anthraquinone (NS2B/NS3) SGVLWDTPSPPEVERAVLDDGI amino acid residues (Arg/Lys) at BP13944 YRIMQRGLLGRSQVGVGVFQD the P1 positions was observed, ZINC04321905 GVFHTMWHVTRGAVLMYQG whereas the preferences for the MB21 KRLEPSWASVKKDLISYGGGW P2-4 sites were in the order of Policresulen RFQGSWNTGEEVQVIAVEPGK Arg > Thr > Gln/Asn/Lys for P2, SK-12 NPKNVQTAPGTFKTPEGEVGAI Lys > Arg > Asn for P3, and Nle > NSC135618 ALDFKPGTSGSPIVNREGKIVG Leu > Lys > Xaa for P4. The Biliverdin LYGNGWTTSGTYVSAIAQAK prime site substrate specificity ASQEGPLPEIEDEVFRKRNLTI was for small and polar amino MDLHPGSGKTRRYLPAIVREAI acids in P1 and P3. RRNVRTLILAPTRVVASEMAE ALKGMPIRYQTTAVKSEHTGK EIVDLMCHATFTMRLLSPVRVP NYNMIIMDEAHFTDPASIARRG YISTRVGMGEAAAIFMTATPPG SVEAFPQSNAVIQDEERDIPERS WNSGYEWITDFPGKTVWFVPS IKSGNDIANCLRKNGKRVIQLS RKTFDTEYQKTKNNDWDYVV TTDISEMGANFRADRVIDPRRC LKPVILKDGPERVILAGPMPVT VASAAQRRGRIGRNQNKEGDQ YVYMGQPLNNDEDHAHWTEA KMLLDNINTPEGIIPALFEPERE KSAAIDGEYRLRGEARKTFVEL MRRGDLPVWLSYKVASEGFQ YSDRRWCFDGERNNQVLEEN MDVEMWTKEGERKKLRPRWL DARTYSDPLALREFKEFAAGR R (SEQ ID NO: 26) >sp|P14340|1476-2093 AGVLWDVPSPPPVGKAELEDG AYRIKQKGILGYSQIGAGVYKE GTFHTMWHVTRGAVLMHKGK RIEPSWADVKKDLISYGGGWK LEGEWKEGEEVQVLALEPGKN PRAVQTKPGLFKTNAGTIGAVS LDFSPGTSGSPIIDKXGKVVGL YGNGVVTRSGAYVSAIAQTEK SIEDNPEIEDDIFRKRKLTIMDL HPGAGKTKRYLPAIVREAIKRG LRTLILAPTRVVAAEMEEALRG LPIRYQTPAIRAEHTGREIVDL MCHATFTMRLLSPVRVPNYNL IIMDEAHFTDPASIAARGYISTR VEMGEAAGIFMTATPPGSRDPF PQSNAPIMDEEREIPERSWSSG HEWVTDFKGKTVWFVPSIKAG NDIAACLRKNGKKVIQLSRKTF DSEYVKTRTNDWDFVVTTDIS EMGANFKAERVIDPRRCMKPV ILTDGEERVILAGPMPVTHSSA AQRRGRIGRNPKNENDQYIYM GEPLENDEDCAHWKEAKMLLD NINTPEGIIPSMFEPEREKVDA IDGEYRLRGEARKTFVDLMRR GDLPVWLAYRVAAEGINYADR RWCFDGIKNNQILEENVEVEI WTKEGERKKLKPRWLDAKIYS DPLALKEFKEFAAGRK (SEQ ID NO: 27) >sp|Q99D3511474-2092 SGVLWDVPSPPETQKAELEEG VYRIKQQGIFGKTQVGVGVQK EGVFHTMWHVTRGAVLTHNG KRLEPNWASVKKDLISYGGGW RLSAQWQKGEEVQVIAVEPGKN PKNFQTMPGIFQTTTGEIGAIA LDFKPGTSGSPIINREGKVVGL YGNGVVTKNGGYVSGIAQTNA EPDGPTPELEEEMFKKRNLTIM DLHPGSGKTRKYLPAIVREAIK RRLRTLILAPTRVVAAEMEEAL KGLPIRYQTTATKSEHTGREIV DLMCHATFTMRLLSPVRVPNYN LIIMDEAHFTDPASIAARGYIS TRVGMGEAAAIFMTATPPGTAD AFPQSNAPIQDEERDIPERSW NSGNEWITDFVGKTVWFVPSIK AGNDIANCLRKNGKKVIQLSR KTFDTEYQKTKLNDWDFWTTD ISEMGANFKADRVIDPRRCLK PVILTDGPERVILAGPMPVTVA SAAQRRGRVGRNPQKENDQYI FMGQPLNKDEDHAHWTEAKMLL DNINTPEGIIPALFEPEREKSA AIDGEYRLKGESRKTFVELMR RGDLPVWLAHKVASEGIKYTD RKWCFDGERNNQILEENMDVE IWTKEGEKKKLRPRWLDARTY SDPLALKEFKDFAAGRK (SEQ ID NO: 28) >sp|Q5UCB8|1475-2092 SGALWDVPSPAATQKAALSEG VYRIMQRGLFGKTQVGVGIHIE GVFHTMWHVTRGSVICHETGR LEPSWADVRNDMISYGGGWR LGDKWDKEEDVQVLAIEPGKN PKHVQTKPGLFKTLTGEIGAVT LDFKPGTSGSPIINRKGKVIGLY GNGWTKSGDYVSAITQAERIG EPDYEVDEDIFRKKRLTIMDLH PGAGKTKRILPSIVREALKRRL RTLILAPTRWAAEMEEALRGL PIRYQTPAVKSEHTGREIVDLM CHATFTTRLLSSTRVPNYNLIV MDEAHFTDPSSVAARGYISTRV EMGEAAAIFMTATPPGTTDPFP QSNSPIEDIEREIPERSWNTGFD WITDYQGKTVWFVPSIKAGND IANCLRKSGKKVIQLSRKTFDT EYPKTKLTDWDFWTTDISEM GANFRAGRVIDPRRCLKPVILP DGPERVILAGPIPVTPASAAQR RGRIGRNPAQEDDQYVFSGDP LKNDEDHAHWTEAKMLLDNI YTPEGIIPTLFGPEREKTQAIDG EFRLRGEQRKTFVELMRRGDL PVWLSYKVASAGISYKDREWC FTGERNNQILEENMEVEIWTRE GEKKKLRPKWLDARVYADPM ALKDFKEFASGRK (SEQ ID NO: 29)

[0108] Exemplary proteases which can be used in fusion proteins of the present disclosure include hepatitis C virus proteases (e.g., NS3 and NS2-3); signal peptidase, proprotein convertases of the subtilisin/kexin family (furin, PCI, PC2, PC4, PACE4, PC5, PC), proprotein convertases cleaving at hydrophobic residues (e.g., Leu, Phe, Val, or Met), proprotein convertases cleaving at small amino acid residues such as Ala or Thr, proopiomelanocortin converting enzyme (PCE); chromaffin granule aspartic protease (CGAP); prohormone thiol protease; 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 protease; and, caspases 1, 2, 3, 4, 5, 6, 7, 8, and 9. Other proteases include, but are not limited to, 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 secretases alpha, beta and gamma; angiotensin converting enzyme secretase; TGF alpha secretase; T F alpha secretase; FAS ligand secretase, TNF receptor-I and -II secretases; CD30 secretase; KL1 and KL2 secretases; IL6 receptor secretase; CD43, CD44 secretase; CD 16-1 and CD 16-11 secretases; L-selectin secretase; Folate receptor secretase; MMP 1, 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); ADAM 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11; and, granzymes A, B, C, D, E, F. G, and H. The protease chosen for use in the fusion protein is preferably highly selective for the cleavage site in the cleavable linker. Additionally, protease activity is preferably inhibitable with inhibitors that are cell-permeable and not toxic to the cell or subject under study. For a discussion of proteases, see, e.g., V. Y. H. Hook, Proteolytic and cellular mechanisms in prohormone and proprotein processing, RG Landes Company. Austin, Tex., USA (1998); N. M. Hooper et al., Biochem. J. 321: 265-279 (1997); Z. Werb, Cell 91: 439-442 (1997); T. G. Wolfsberg et al., J. Cell Biol. 131: 275-278 (1995): K. Murakami and J. D. Etlinger, Biochem. Biophys. Res. Comm. 146: 1249-1259 (1987): T. Berg et al., Biochem. J. 307: 313-326 (1995): M. J. Smyth and J. A. Trapani, Immunology Today 16: 202-206 (1995); R. V. Talanian et al., J. Biol. Chem. 272: 9677-9682 (1997), and N A. Thomberry et al, J Biol Chem. 272: 17907-17911 (1997), the disclosures of which are incorporated herein.

[0109] In certain embodiments, the protease used in the fusion protein is derived from hepatitis C virus (HCV). In some embodiments, the protease is an 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 the HCV nonstructural protein 4A (NS4A co-factor), which activates proteolytic activity. An NS3 protease may comprise the entire NS3 protein or a proteolytically active fragment thereof and may further comprise an activating NS4A co-factor region. Advantages of using an NS3 protease include that it is highly selective and can be well-inhibited by a number of non-toxic, cell-permeable drugs, which are currently clinically available. NS3 protease inhibitors that can be used in the practice of the present disclosure include, but are not limited to, simeprevir, danoprevir, asunaprevir, ciluprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and voxiloprevir.

[0110] When an NS3 protease is used in a fusion protein, the cleavable linker of the fusion protein may comprise an NS3 protease cleavage site (e.g., a cognate cleavage site). Exemplary NS3 protease cleavage sites, which can be used in the cleavable linker, include the four junctions between nonstructural (NS) proteins of the HCV polyprotein normally cleaved by the NS3 protease during HCV infection, including the NS3/NS4A, NS4A/NS4B, NS4B/NS5A, and NS5A/NS5B junction cleavage sites. For a description of NS3 protease and representative sequences of its cleavage sites for various strains of HCV, see, e.g., Hepatitis C Viruses: Genomes and Molecular Biology (S. L. Tan ed., Taylor & Francis, 2006), Chapter 6, pp. 163-206; herein incorporated by reference in its entirety.

[0111] NS3 nucleic acid and protein sequences may be derived from HCV, including any isolate of HCV having any genotype (e.g., seven genotypes 1-7) or subtype. A number of NS3 nucleic acid and protein sequences are known. A representative NS3 sequence is presented in Table 1. Additional representative sequences are listed in the National Center for Biotechnology Information (NCBI) database See, for example, NCBI entries: Accession Nos. 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, AHH29575, AIZ00747, AIZ00744, AB136969, ABN05226, KF516075, KF516074, KF516056, AB826684. AB826683, JX171009, JX171008, JX171000, EU847455, EF154714, GU085487, JX171065, JX171063, all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference. Any of these sequences or a variant thereof comprising a sequence 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 a fusion protein or a recombinant polynucleotide encoding such a fusion protein, as described herein.

[0112] NS4A nucleic acid and protein sequences may be derived from HCV, including any isolate of HCV having any genotype (e.g., seven genotypes 1-7) or subtype. A number of NS4 A 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 date of filing of this application) are herein incorporated by reference Any of these sequences or a variant thereof comprising a sequence 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 a fusion protein or a recombinant polynucleotide encoding such a fusion protein, as described herein.

[0113] HCV polyprotein nucleic acid and protein sequences may be derived from HCV, including any isolate of HCV having any genotype (e.g., seven genotypes 1-7) or subtype. A number of 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, for example, NCI 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 EF 108306; all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference. Any of these sequences or a variant thereof comprising a sequence 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 a fusion protein or a recombinant polynucleotide encoding such a fusion protein, as described herein.

[0114] 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):

TABLE-US-00002         10         20         30         40         50 MSTNPKPQKK NKRNTNRRPQ DVKFPGGGQI VGGVYLLPRR GPRLGVRATR         60         70         80         90        100 KTSERSQPRG RRQPIPKARR PEGRTWAQPG YPWPLYGNEG CGWAGWLLSP        110        120        130        140        150 RGSRPSWGPT DPRRRSRNLG KVIDTLTCGF ADLMGYIPLV GAPLGGAARA        160        170        180        190        200 LAHGVRVLED GVNYATGNLP GCSFSIFLLA LLSCLTVPAS AYQVRNSTGL        210        220        230        240        250 YHVTNDCPNS SIVYKAADAI LHTPGCVPCV REGNASRCWV AMTPTVATRD        260        270        280        290        300 GKLPATQLRR HIDLLVGSAT LCSALYVGDL CGSVFLVGQL FTFSPRRHWT        310        320        330        340        350 TQGCNCSIYP GHITGHRMAW DMMMNWSPTT ALVMAQLLRI PQAILDMIAG        360        370        380        390        400 AHWGVLAGIA YFSMVGNWAK VLVVLLLFAG VDAETHVTGG SAGHTVSGFV        410        420        430        440        450 SLLAPGAKQN VQLINTNGSW HLNSTALNCN DSLNTGWLAG LFYHHKENSS        460        470        480        490        500 GCPERLASCR PLTDFDQGWG PISYANGSGP DQRPYCWHYP PKPCGIVPAK        510        520        530        540        550 SVCGPVYCFT PSPVVVGTTD RSGAPTYSWG ENDTDVFVLN NTRPPLGNWF        560        570        580        590        600 GCTWMNSTGF TKVCGAPPCV IGGAGNNTLH CPTDCFRKHP DATYSRCGSG        610        620        630        640        650 PWITPRCLVD YPYRLWHYPC TINYTIFKIR MYVGGVEHRL EAACMWTRGE        660        670        680        690        700 RCDLEDRDRS ELSPLLLTTT QWQVLPCSFT TLPALSTGLI HLHQNIVDVQ        710        720        730        740        750 YLYGVGSSIA SWAIKWEYVV LLFLLLADAR VCSCLWMMLL ISQAEAALEN        760        770        780        790        800 LVILNAASLA GTHGLVSFLV FFCFAWYLKG KWVPGAVYTF YGMWPILLLL        810        820        830        840        850 LALPQRAYAL DTEVAASCGG VVLVGLMALT LSPYYKEYIS WCLWWLQYFL        860        870        880        890        900 TRVEAQLHVW IPPLNVRGGR DAVILLMCAV HPTLVEDITK LLLAVFGPLW        910        920        930        940        950 ILQASLLKVP YFVRVQGLLR FCALARKMIG GHYVQMVIIK LGALTGTYVY        960        970        980        990       1000 NHLTPLRDWA HNGLRDLAVA VEPVVFSQME TKLITWGADT AACGDIINGL       1010       1020       1030       1040       1050 PVSARRGREI LLGPADGMVS KGWRLLAPIT AYAQQTRGLL GCIITSLTGR       1060       1070       1080       1090       1100 DKNQVEGEVQ IVSTAAQTFL ATCINGVCWT VYHGAGTRTI ASPKGPVIQM       1110       1120       1130       1140       1150 YTYVDQDLVG WPAPQGSRSL TPCTCGSSDL YLVTRHADVI PVRRRGDSRG       1160       1170       1180       1190       1200 SLLSPRPISY LKGSSGGPLL CPAGHAVGIF RAAVCTRGVA KAVDFIPVEN       1210       1220       1230       1240       1250 LETTMRSPVF TDNSSPPVVP QSFQVAHLHA PTGSGKSTKV PAAYAAQGYK       1260       1270       1280       1290       1300 VLVLNPSVAA TLGFGAYMSK AEGIDPNIRT GVRTITTGSP ITYSTYGKFL       1310       1320       1330       1340       1350 ADGGCSGGAY DIIICDECHS TDATSILGIG TVLDQAETAG ARLVVLATAT       1360       1370       1380       1390       1400 PPGSVTVPHP NIEEVALSTT GEIPFYGKAI PLEVIKGGRH LIFCHSKKKC       1410       1420       1430       1440       1450 DELAAKLVAL GINAVAYYRG LDVSVIPTSG DVVVVATDAL MTGYTGDFDS       1460       1470       1480       1490       1500 VIDCNTCVTQ TVDFSLDPTF TIETITLPQD AVSRTQRRGR TGRGKPGIYR       1510       1520       1530       1540       1550 FVAPGERPSG MFDSSVLCEC YDAGCAWYEL TPAETTVRLR AYMNTPGLPV       1560       1570       1580       1590       1600 CQDHLEFWEG VYTGLTHIDA HFLSQTKQSG ENLPYLVAYQ ATVCARAQAP       1610       1620       1630       1640       1650 PPSWDQMWKC LIRLKPTLHG PTPLLYRLGA VQNEITLTHP VTKYIMTCMS       1660       1670       1680       1690       1700 ADLEVVTSTW VLVGGVLAAL AAYCLSTGCV VIVGRVVLSG KPAIIPDREV       1710       1720       1730       1740       1750 LYREFDEMEE CSQHLPYIEQ GMMLAEQFKQ KALGLLQTAS RQAEVIAPAV       1760       1770       1780       1790       1800 QTMWQKLETF WAKHMWMFIS GIQYLAGLST LPGNPAIASL MAFTAAVTSP       1810       1820       1830       1840       1850 LTTSQTLLFN ILGGWVAAQL AAPGAATAFV GAGLAGAAIG SVGLGKVLID       1860       1870       1880       1890       1900 ILAGYGAGVA GALVAFKIMS GEVPSTEDLV NLLPAILSPG ALVVGVVCAA       1910       1920       1930       1940       1950 ILRRHVGPGE GAVQWMNREI AFASRGNHVS PTHYVPESDA AARVTAILSS       1960       1970       1980       1990       2000 LTVTQLLRRL HQWISSECTT PCSGSWLRDI WDWICEVLSD FKTWLKAELM       2010       2020       2030       2040       2050 PQLPGIPFVS CQRGYKGVWR VDGIMHTRCE CGAEITGHVK NGTMRIVGPR       2060       2070       2080       2090       2100 TCRNMWSGTF PINAYTTGPC TPLPAPNYTF ALWRVSAEEY VEIRQVGDFH       2110       2120       2130       2140       2150 YVTGMTTDNL KCPCQVPSPE FFTELDGVRL HRFAPPCKPL LREEVSFRVG       2160       2170       2180       2190       2200 LHEYPVGSQL PCEPEPDVAV LTSMLTDPSH ITAEAAGRRL ARGSPPSVAS       2210       2220       2230       2240       2250 SSASQLSAPS LKATCTANHD SPDAELIEAN LLWRQEMGGN ITRVESENRV       2260       2270       2280       2290       2300 VILDSFDPLV AEEDEREISV PAEILRKERR FAQALPVWAR PDYNPPLVET       2310       2320       2330       2340       2350 WKKPDYEPPV VHGCPLPPPK SPPVPPPRKK RTVVLTESTL STALAELATR       2360       2370       2380       2390       2400 SFGSSSTSGI TGDNTTTSSE PAPSGCPPDS DAESYSSMPP LEGEPGDPDL       2410       2420       2430       2440       2450 SDGSWSTVSS EANAEDVVCC SMSYSWTGAL VTPCAAEEQK LPINALSNSL       2460       2470       2480       2490       2500 LRHHNLVYST TSRSACQRQK KVTFDRLQVL DSHYQDVLKE VKAAASKVKA       2510       2520       2530       2540       2550 NLLSVEEACS LTPPHSAKSK FGYGAKDVRC HARKAVTHIN SVWKDLLEDN       2560       2570       2580       2590       2600 VTPIDTTIMA KNEVECVQPE KGGRKPARII VFPDLGVRVC EKMALYDVVT       2610       2620       2630       2640       2650 KLPLAVMGSS YGFQYSPGQR VEFLVQAWKS KKTPMGESYD TRCFDSTVTE       2660       2670       2680       2690       2700 SDIRTEEAIY QCCDLDPQAR VAIKSLTERL YVGGPLTNSR GENCGYRRCR       2710       2720       2730       2740       2750 ASGVLTTSCG NTLTCYIKAR AACRAAGLQD CTMLVCGDDL VVICESAGVQ       2760       2770       2780       2790       2800 EDAASLRAFT EAMTRYSAPP GDPPQPEYDL ELITSCSSNV SVAHDGAGKR       2810       2820       2830       2840       2850 VYYLTRDPTT PLARAAWETA RHTPVNSWLG NIIMFAPTLW ARMILMTHFF       2860       2870       2880       2890       2900 SVLIARDQLE QALDCEIYGA CYSIEPLDLP PlIQRLHGLS AFSLHSYSPG       2910       2920       2930       2940       2950 EINRVAACLR KLGVPPLRAW RHRARSVRAR LLARGGRAAI CGKYLFNWAV       2960       2970       2980       2990       3000 RTKLKLTPIA AAGQLDLSGW FTAGYSGGDI YHSVSHARPR WIWFCLLLLA       3010 AGVGIYLLPN R

[0115] In some embodiments, a fusion proteins of the present disclosure comprise a variant NS3 protease derived from the 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 decrease 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, 10 or more mutations, 11 or more mutations, 12 or more mutations, 13 or more mutations, 14 or more mutations, 15 or more mutations, 16 or more mutations, 17 or more mutations, 18 or more mutations, 19 or more mutations, or 20 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, 1 mutations, 12 mutations, 13 mutations, 14 mutations, 15 mutations, 16 mutations, 17 mutations, 18 mutations, 19 mutations, or 20 mutations. In some embodiments the one or more mutations are amino acid substitutions.

[0116] The variant protease may include one or more mutations within an immunodominant epitope that results in a reduction in immunogenicity of the protease and/or within an epitope that that results in modulation of the catalytic activity of the protease (see e.g., Söerholm J, et al. Gut. 2006 February; 55(2):266-74; Soumana D et al. ACS Chem Biol. 2014 Nov. 21; 9(11):2485-90; and Wertheimer A M et al. Hepatology. 2003 March; 37(3):577-89). For example, the one or more mutations may be within a region 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. In some embodiments, the one or more mutations may be within a region selected from GLLGCIITSL (SEQ ID NO: 30), GEVQIVSTAAQTFLATCINGVCWTVY (SEQ ID NO: 31), GEVQIVSTAAQTFLA (SEQ ID NO. 32), QTFLATCINGVCWTV (SEQ ID NO: 33), CINGVCWTVY (SEQ ID NO: 34), SSDLYLVTRHADVIP (SEQ ID NO: 35), YLVTRHAD (SEQ ID NO: 36), LLCPAGHAV (SEQ ID NO: 37), AVDFIPVEGLETTMR (SEQ ID NO: 38), KIDTKYIMTCMSADL (SEQ ID NO. 39), and any combination thereof.

[0117] In some embodiments, the one or more mutations are one or more amino acid substitutions selected from 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 1074 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.

[0118] In some embodiments, the one or more mutations are one or more amino acid substitutions 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, 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 comprise 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 comprise 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 comprise 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.

[0119] In some embodiments, the variant protease may comprise one or more additional mutations, such as amino acid substitutions, that tune or otherwise modulate the enzymatic activity of the protease. In some embodiments, the variant protease comprises 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 10 or more additional mutations. In some embodiments, the variant protease comprises 1 additional mutation, 2 additional mutations, 3 additional mutations, 4 additional mutations, 5 additional mutations, 6 additional mutations, 7 additional mutations, 8 additional mutations, 9 additional mutations, or 10 additional mutations. In some embodiments the one or more additional mutations are amino acid substitutions. In some embodiment, the one or more additional mutations are amino acid substitutions at one 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 embodiment, the one or more additional mutations decrease the enzymatic activity of the protease. In some embodiments, the one or more additional mutations that decrease the enzymatic activity of the protease are one or more additional amino acid substitutions selected from an lie 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 embodiment, 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 that include a Cys to Ala substitution at a position corresponding to position 1078 of SEQ ID NO: 1.

[0120] In some embodiments, a fusion protein of the present disclosure comprise a variant NS3 protease derived from the HCV NS3 protease having an amino acid sequence of:

TABLE-US-00003 (SEQ ID NO. 2) APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTATQTFLATC INGVCWAVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGSRSL TPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLLSPRPISYLKGSSG GPLLCPAGHAVGLFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTD.

[0121] In some embodiments, the fusion protein further comprises an HCV NS4A co-factor. In some embodiments, the NS4A co-factor has the amino acid sequence of

TABLE-US-00004 (SEQ ID NO: 3) TWVLVGGVLAALAAYCLSTGCVVIVGRWLSGKPAEPDREVLY.

Cognate Protease Cleavage Sites

[0122] Certain aspects of the present disclosure relate to a fusion protein comprising a variant protease and a cognate cleavage site recognized by the protease. When a protease is selected, its cognate cleavage site and protease inhibitors known in the art to bind and inhibit the protease may be used in a 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 below in Table 1.

[0123] When an NS3 protease is used, the cognate cleavage site comprises an NS3 protease cleavage site Exemplary NS3 protease cleavage sites include the four junctions between nonstructural (NS) proteins of the HCV polyprotein normally cleaved by the NS3 protease during HCV infection, including the NS3/NS4A, NS4A/NS4B, NS4B/NS5A, and NS5A/NS5B junction cleavage sites. For a description of NS3 protease and representative sequences of its cleavage sites for various strains of HCV, see, e.g., Hepatitis C Viruses Genomes and Molecular Biology (S. L. Tan ed., Taylor & Francis, 2006), Chapter 6, pp. 163-206; herein incorporated by reference in its entirety. For example, the sequences of HCV NS3/4A protease cleavage sites, HCV NS4A/4B protease cleavage sites (SEQ ID NO. 9, 44); HCV NS4B/5A protease cleavage sites; and HCV NS5A/5B protease cleavage sites (SEQ ID NO: 11, 45) are provided in Table 1.

[0124] In some embodiments, cognate cleavage sites for NS3 protease include those listed in Table 1. In some embodiments, a cognate cleavage site for an NS3 protease, such as a variant NS3 protease of the present disclosure, is selected from CMSADLEVVTSTWVLVGGVL (SEQ ID NO: 4), YQEFDEMEECSQHLPYIEQG (SEQ ID NO: 5), WISSECTTPCSGSWLRDIWD (SEQ ID NO: 6), and GADTEDVVCCSMSYSWTGAL (SEQ ID NO: 7). In some embodiment, a cognate cleavage site for an NS3 protease, such as a variant NS3 protease of the present disclosure, is selected from ADLEVVTSTWL (SEQ ID NO: 8), DEMEECSQHL (SEQ ID NO: 9), ECTTPCSGSWL (SEQ ID NO: 10), and EDVVPCSMG (SEQ ID NO: 11). In some embodiments, the cognate cleavage site comprises one or more mutations, such as one or more amino acid substitutions. In some embodiments, mutations in the cognate cleavage site can tune, or otherwise modulate, 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.

Degrons

[0125] Certain aspects of the present disclosure relate to a fusion protein comprising a polypeptide of interest, a protease, a cognate protease cleavage site, and that further comprises a degron or a self-excising degron.

[0126] Degrons of the present disclosure may comprise a sequence of amino acids, which provides a degradation signal that directs a polypeptide for cellular degradation. The degron may promote degradation of an attached polypeptide through either the proteasome or autophagy-lysosome pathways. In a fusion protein of the present disclosure, the degron must be operably linked to the polypeptide of interest, but need not be contiguous with it as long as the degron still functions to direct 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 9(7): 1100-1103, Varshavsky (2012) Methods Mol. Biol. 832: 1-11, and Fayadat et al. (2003) Mol Biol Cell. 14(3): 1268-1278, herein incorporated by reference.

[0127] Degrons with degradation sequences known in the art may be used for various embodiments of the present disclosure. In some embodiments, a degron of the present disclosure may be derived from a degron identified from an organism, or a modification thereof. Such a degron includes, but not limited to, an HCV NS4 degron, a PEST (Two copies of residues 277-307 of IκBα(human) (SEQ ID NO: 46), a GRR (residues 352-408 of p105 (human) (SEQ ID NO: 47), a DRR (residue 210-295 of Cdc34 (yeast) (SEQ ID NO: 48), an SNS (tandem repeat of SP2 and NB (SP2-NB-SP2) (Influenza A and B) (SEQ ID NO 49), an RPB (four copies of residues 1688-1702 of RPB1 (yeast) (SEQ ID NO: 50), an SPmix (tandem repeat of SP1 and SP2 (SP2-SP1-SP2-SP1-SP2) (Influenza A virus M2 protein) (SEQ ID NO 51), an NS2 (three copies of residue 79-93 of Influenza A virus NS protein) (SEQ ID NO: 52), an ODC (residue 106-142 of ornithine decarboxylase) (SEQ ID NO: 53), a Nek2A (human), an mODC (amino acids 422-461 (moue), an mODC_DA (amino acids 422-461 of mODC (D433A, D434A point mutations (mouse)) (SEQ ID NO: 54), an APC/C degrons (e.g., D box, KEN box and ABBA motif), a COP1 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 (e.g., Nbox, or UBRbox), a hydroxyproline modification in hypoxia signaling, a phytohormone-dependent SCF-LRR-binding degron, an SCF ubiquitin ligase binding phosphodegron, a phytohormone-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.

[0128] In some embodiments the degron comprises portions of the HCV nonstructural proteins NS3 and NS4A. In one embodiment, the degron comprises the amino acid sequence of PITKIDTKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLST (SEQ ID NO: 40) or a variant thereof comprising a sequence 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, wherein the degron is capable of promoting degradation of a polypeptide. It is to be understood that degrons comprising the residues corresponding to the reference sequence of SEQ ID NO. 40 in I-iCV nonstructural proteins NS3 and NS4A obtained from other strains of HCV are also intended to be encompassed by the present disclosure.

[0129] In the fusion protein, the degron may be linked to the N-terminus or the C-terminus of the polypeptide of interest. For example, the fusion protein can be represented by the formula NH.sub.2-P-D-L-X-COOH or NH.sub.2-X-L-P-D-COOH, wherein: P is an amino acid sequence of a protease; D is an amino acid sequence of a degron; L is an amino acid sequence of a linker comprising a cleavage site for the protease; and X is an amino acid sequence of a selected polypeptide of interest. The cleavable linker between the polypeptide of interest and the degron is designed for selective cleavage by the particular protease included in the fusion protein. The cleavage site of the linker includes the specific amino acid sequence recognized by the protease during proteolytic cleavage and typically includes the surrounding one to six amino acids on either side of the scissile bond, which bind to the active site of the protease and are needed for recognition as a substrate. The cleavable linker may contain any protease recognition motif known in the art and is typically cleavable under physiological conditions.

[0130] The polypeptides included in the fusion construct may be connected directly to each other by peptide bonds or may be separated by intervening amino acid sequences. The fusion polypeptides may also contain sequences exogenous to the protease or the selected protein of interest. For example, the fusion protein may include targeting or localization sequences, tag sequences, or sequences of fluorescent or bioluminescent proteins.

[0131] In certain embodiments, tag sequences are 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 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.

[0132] 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 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-protein interaction motif sequence. Examples of targeting sequences include those targeting the nucleus (e.g., KKKRK, SEQ ID NO: 41), mitochondrion (e.g., MLRT S SLFTRRVQP SLFRNILRLQ ST, SEQ ID NO. 42), endoplasmic reticulum (e.g., KDEL, SEQ ID NO. 43), peroxisome (e.g., SKL), synapses (e.g., S/TDV or fusion to GAP 43, kinesin or tau), plasma membrane (e.g., CaaX) where “a” is an aliphatic amino acid, CC, CXC, CCXX at C-terminus), or protein-protein interaction motifs (e.g., SH2, SH3, PDZ, WW, RGD, Src homology domain, DNA-binding domain, SLiMs).

[0133] In certain embodiments, the fusion protein comprises a detectable label. The detectable label may comprise any molecule capable of detection. Detectable labels that may be used in the practice of the present disclosure include, but are not limited to, radioactive isotopes, stable (non-radioactive) heavy isotopes, fluorescers, chemiluminescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin or haptens) and the like. Particular examples of labels that may be used with the present disclosure include, but are 3 125 35 14 32 not limited to radiolabels (e.g., H, I, S, C, or P), stable (non-radioactive) heavy isotopes (e.g., .sup.13C or .sup.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, acradimum 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), 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, chloramphenical acetyl transferase, and urease Enzyme tags are used with their cognate substrate. The terms also include color-coded microspheres of known fluorescent light intensities (see e.g., microspheres with xMAP technology produced by Luminex (Austin, Tex.); microspheres containing quantum dot nanocrystals, for example, containing different ratios and combinations of quantum dot colors (e.g., Qdot nanocrystals produced by Life Technologies (Carlsbad, Calif.); glass coated metal nanoparticles (see e.g., SERS nanotags produced by Nanoplex Technologies. Inc. (Mountain View, Calif.); barcode materials (see e.g., sub-micron sized striped metallic rods such as Nanobarcodes produced by Nanoplex Technologies, Inc.), encoded microparticles with colored bar codes (see e.g., CellCard produced by Vitra Bioscience, vitrabio.com), and glass microparticles with digital holographic code images (see e.g, CyVera microbeads produced by Illumina (San Diego, Calif.). As with many of the standard procedures associated with the practice of the present disclosure, skilled artisans will be aware of additional labels that can be used.

Polypeptides of Interest

[0134] 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 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 a 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.

[0135] 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 thrombolytics.

[0136] 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 immunoreceptors, antibody-coupled T cell receptors (ACTRs), T cell receptor fusion constructs (TRUCs), and chimeric antigen receptors (CARs).

[0137] In some embodiments the polypeptide of interest is a cytokine, such as a proinflammatory 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.

Inducible Receptors

[0138] In one aspect, a polypeptide of interest of the present disclosure is an inducible cell receptor, which 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 a operably linked to the fusion protein. In another aspect, a polypeptide of interest of the present disclosure is an inducible cell receptor comprising (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.

ON and OFF Switches

[0139] In some embodiments, the present disclosure provides a fusion protein with an “OFF switch,” wherein the polypeptide of interest is an inducible receptor that is selectively inactivated in the presence of a protease inhibitor. An exemplary OFF switch, as provided herein, may be a cell receptor that comprises (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, wherein components (a)-d) are configured such that the cell receptor is inactivated (does not transmit an intracellular signal) when the repressible protease is repressed. In some embodiments, the is located at the C-terminal (carboxy-terminal) end of the polypeptide of interest, at the N-terminal (amino-terminal) end of the polypeptide of interest, or located within domains of the polypeptide of interest. With OFF switches, cleavage by the protease removes the, thereby preserving structural integrity of the receptor, and addition of the protease inhibitor causes degradation of the receptor.

[0140] In some embodiments, the present disclosure provides a fusion protein with an “ON switch,” wherein the polypeptide of interest is an inducible receptor that is selectively activated in the presence of a protease inhibitor. An exemplary ON switch, as provided herein, may be a cell receptor that comprises (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, wherein components (a)-(e) are configured such that the cell receptor is activated (transmits an intracellular signal) when the protease is repressed. Unlike the OFF switches above, the ON switches do not include a. Rather, with ON switches, cleavage by the protease removes a functional element of the cell receptor (e.g., a signaling domain or a protein-binding domain), and addition of the protease inhibitor preserves structural integrity of the receptor.

[0141] The protease and the cognate cleavage site of an ON switch may be located between any two domains of the cell receptor. For example, the protease and the cognate cleavage site may be located between the extracellular protein binding domain and the transmembrane domain. In some embodiments, the protease and the cognate cleavage site are located between the transmembrane domain and the intracellular signaling domain. In other embodiments, the protease and the cognate cleavage site are located between two co-signaling domains. In some embodiments, a domain of the cell receptor further comprises a ligand operably linked to the ligand-binding domain (e.g., an extracellular protein binding domain). In this case, the protease and the cognate cleavage site can be located between the ligand and the ligand-binding domain.

[0142] In some embodiments, the inducible cell receptor comprises two polypeptides (e.g., a multichain receptor). In such embodiments, recruitment domains can be used to bring the two polypeptides together to activate the receptor. Recruitment domains are protein domains that bind to each other and thus, can bring together two different polypeptides, each comprising one of a pair of recruitment domains. A pair of recruitment domains are considered to assemble with each other if the two domains bind directly to each other, or if the two domains bind to the same (intermediate) molecule. Non-limiting examples of pairs of recruitment domains 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 associated 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.

[0143] In some embodiments of the OFF switches, one polypeptide comprises a protein binding domain, a transmembrane domain, a signaling domain, and a first recruitment domain. In some embodiments, the second polypeptide comprises a second recruitment domain that assembles with the first recruitment domain. In some embodiments, a is located in the first polypeptide or in the second polypeptide. In some embodiments, the protease may be located in one (a first) polypeptide, while the cognate cleavage site and are located in the other (a second) polypeptide.

[0144] In some embodiments of the ON switches, a first polypeptide may comprise a protein binding domain, a transmembrane domain, a signaling domain, a first recruitment domain, and the cognate cleavage site. In some embodiments, the second polypeptide comprises the protease and a second recruitment domain that assembles with (binds directly or indirectly to) the first recruitment domain.

[0145] Also provided herein are methods of regulating activity of a cell receptor (e.g., OFF switches). In some embodiments of the OFF switches, the methods comprise providing a cell comprising cell receptor that includes (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 components (a)-(f) are configured such that the cell receptor is inactivated when the protease is repressed, and contacting the cell with a protease inhibitor (e.g., simeprevir, danoprevir, asunaprevir, ciluprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, or voxiloprevir) that represses activity of the protease, thereby inactivating the cell receptor.

[0146] In other embodiments of the ON switches, the methods comprise providing a cell comprising a cell receptor that includes (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 cell receptor is activated when the repressible protease is repressed, and contacting the cell with a protease inhibitor (e.g., simeprevir, danoprevir, asunaprevir, ciluprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, or voxiloprevir) that represses activity of the protease, thereby activating the cell receptor.

Chimeric Antigen Receptors (CARs)

[0147] In one aspect, a polypeptide of interest of the present disclosure is a chimeric antigen receptor (CAR) CARs, generally, are artificial immune cell receptors engineered to recognize and bind to an antigen expressed by tumor cells. CARs may typically include an antibody fragment as an antigen-binding domain, a spacer domains, a hydrophobic alpha helix 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 can 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 in the C-terminus of the CAR.

[0148] Generally, a CAR is designed for a T cell, or NK cell, and is a chimera of a signaling domain of the T-cell receptor (TCR) complex and an antigen-recognizing domain (e.g., a single chain fragment (scFv) of an antibody) (Enblad et al., Human Gene Therapy. 2015: 26(8):498-505). A T cell that expresses a CAR is known in the art as a CAR T cell.

[0149] There are at least four generations of CARs, each of which contains different components. First generation CARs join an antibody-derived scFv to the CD3zeta (ζ or z) intracellular signaling domain of the T-cell receptor through hinge and transmembrane domains. Second generation CARs incorporate an additional domain, e.g., CD28, 4-1BB (41BB), or ICOS, to supply a costimulatory signal. Third-generation CARs contain two costimulatory domains fused with the TcR CD3-ζ chain. Third-generation costimulatory domains may include, e.g., a combination of CD3z, CD27, CD28, 4-1BB, ICOS, or OX40. CARs, in some embodiments, contain an ectodomain (e.g., CD3ζ), commonly derived from a single chain variable fragment (scFv), a hinge, a transmembrane domain, and an endodomain with one (first generation), two (second generation), or three (third generation) signaling domains derived from CD3Z and/or co-stimulatory molecules (Maude et al., Blood 2015, 125(26):4017-4023, Kakarla and Gottschalk, Cancer J. 2014; 20(2):151-155).

[0150] In some embodiments, a chimeric antigen receptor (CAR) is a T-cell redirected for universal cytokine killing (TRUCK), also known as a fourth generation CAR. TRUCKs are CAR-redirected T-cells used as vehicles to produce and release a transgenic cytokine that accumulates in the targeted tissue, e.g., a targeted tumor tissue. The transgenic cytokine is released upon CAR engagement of the target. TRUCK cells may deposit a variety of therapeutic cytokines in the target. This may result in therapeutic concentrations at the targeted site and avoid systemic toxicity.

[0151] CARs typically differ in their functional properties. The CD3ζ signaling domain of the T-cell receptor, when engaged, will activate and induce proliferation of T-cells but can lead to anergy (a lack of reaction by the body's defense mechanisms, resulting in direct induction of peripheral lymphocyte tolerance). Lymphocytes are considered anergic when they fail to respond to a specific antigen. The addition of a costimulatory domain in second-generation CARs improved replicative capacity and persistence of modified T-cells. Similar antitumor effects are observed in vitro with CD28 or 4-1BB CARs, but preclinical in vivo studies suggest that 4-1BB CARs may produce superior proliferation and/or persistence. Clinical trials suggest that both of these second-generation CARs are capable of inducing substantial T-cell proliferation in vivo, but CARs containing the 4-1BB costimulatory domain appear to persist longer. Third generation CARs combine multiple signaling domains (costimulatory) to augment potency. Fourth generation CARs are additionally modified with a constitutive or inducible expression cassette for a transgenic cytokine, which is released by the CAR T-cell to modulate the T-cell response. See, for example, Enblad et al., Human Gene Therapy. 2015; 26(8):498-505; Chmielewski and Hinrich, Expert Opinion on Biological Therapy 2015; 15(8) 1145-1154.

[0152] In some embodiments, a chimeric antigen receptor of the present disclosure is a first generation CAR. In some embodiments, a chimeric antigen receptor of the present disclosure is a second generation CAR. In some embodiments, a chimeric antigen receptor of the present disclosure is a third generation CAR. In some embodiments, a chimeric antigen receptor of the present disclosure is a fourth generation CAR.

[0153] In some embodiments, a spacer domain or a hinge domain is located between an extracellular domain (e.g., comprising the antigen binding domain) and a transmembrane domain of a CAR, or between a cytoplasmic signaling domain and a transmembrane domain of the CAR. A spacer domain is any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular domain and/or the cytoplasmic signaling domain in the polypeptide chain. A hinge domain is any oligopeptide or polypeptide that functions to provide flexibility to the CAR, or domains thereof, or to prevent steric hindrance of the CAR, or domains thereof. In some embodiments, a spacer domain or hinge domain may comprise up to 300 amino acids (e.g., 10 to 100 amino acids, or 5 to 20 amino acids). In some embodiments, one or more spacer domain(s) may be included in other regions of a CAR.

[0154] In some embodiments, a CAR is an antigen-specific inhibitory CAR (iCAR), which may be used, for example, to avoid off-tumor toxicity (Fedorov, V D et al. Sci. Transl. Med. 2013, incorporated herein by reference). iCARs contain an antigen-specific inhibitory receptor, for example, to block nonspecific immunosuppression, which may result from extra-tumor target expression. iCARs may be based, for example, on inhibitory molecules CTLA-4 or PD-1. In some embodiments, these iCARs block T cell responses from T cells activated by either their endogenous T cell receptor or an activating CAR. In some embodiments, this inhibiting effect is temporary.

[0155] In some embodiments, CARs may be used in adoptive cell transfer, wherein immune cells are removed from a subject and modified so that they express receptors specific to an antigen, e.g., a tumor-specific antigen. The modified immune cells, which may then recognize and kill the cancer cells, are 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).

Multipart CARs

[0156] In some embodiments, a polypeptide of interest of the present disclosure is a single chain (polypeptide) cell receptor or a multichain (and thus multipart) receptor. Thus, an ON switch or an OFF switch may comprise a single polypeptide, or at least two polypeptides.

[0157] In some embodiments of an OFF switch, a CAR is a multipart 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 signaling domain and a second recruitment domain that assembles with the first recruitment domain, wherein a is located in the first polypeptide and/or the second polypeptide. In some embodiments, the is located in the C-terminus of the first polypeptide and/or the second polypeptide.

[0158] In other embodiments of an 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 an 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 in the first polypeptide, and the cognate cleavage site and a are located in the second polypeptide, or wherein the protease is located in the second polypeptide, and the cognate cleavage site and are located in the first polypeptide. In some embodiments, the is located in the C-terminus of the first polypeptide and/or the second polypeptide.

[0159] In some embodiments of an ON switch, a 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 the protease and a second recruitment domain that assembles with the first recruitment domain, wherein the cognate cleavage site is located between the antibody fragment and the transmembrane domain, between the transmembrane domain and first intracellular signaling domain, or between the first intracellular signaling domain and the second intracellular signaling domain.

[0160] In other embodiments of an ON switch, a 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 the protease and a second recruitment domain that assembles with the first recruitment domain, wherein the cognate cleavage site is located between the antibody fragment and the transmembrane domain, between the transmembrane domain and first intracellular signaling domain, or between the first intracellular signaling domain and the second intracellular signaling domain.

[0161] Additional CAR-Regulation Switches

[0162] In some embodiments, a (e.g., OFF switch) and/or a protease/cognate cleavage site (e.g., ON switch) may be combined with orthogonal CAR-regulating switches to yield logic gates (e.g., AND, OR, NOR, and conditional ON gates) with, for example, at least 2 agent (e.g., drug) inputs that perform higher order functionalities.

[0163] 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 (g) a cognate cleavage site, and a second polypeptide comprising a signaling domain and 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, methods of regulating activity of the CAR comprise contacting a cell comprising the CAR with (a) a protease inhibitor that represses 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.

[0164] 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 signaling domain and a second recruitment domain that assembles with the first recruitment domain unless in the CAR is contacted with an agent that prevents assembly of the first recruitment domain with the second recruitment domain. In some embodiments, methods of regulating activity of the CAR comprise contacting a cell comprising the CAR with (a) a protease inhibitor that represses 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.

[0165] In yet other embodiments, a 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, and (e) a protease and a cognate cleavage site, wherein the protease and cognate cleavage site are located between the signaling domain and the first recruitment domain, and a second polypeptide comprising a signaling domain and 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, methods of regulating activity of the CAR comprise contacting a cell comprising the CAR with (a) a protease inhibitor that represses 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.

[0166] In still other embodiments, a 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 required for assembly of the first recruitment domain with the second recruitment domain, wherein the CAR further comprises a, a protease, a cognate cleavage site, and wherein the cognate cleavage site and are located at the C-terminus of the first polypeptide and the protease is located at the C-terminus of the second polypeptide. In some embodiments, methods of regulating activity of the CAR comprise 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 methods may further comprise contacting the cell with a protease inhibitor that represses activity of the protease, thereby inactivating the CAR.

[0167] In some embodiments, a 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, methods of regulating activity of the CAR comprise 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 methods may further comprise contacting the cell with a protease that represses activity of the protease, thereby inactivating the CAR.

[0168] The ability of constructs to produce fusion proteins can be empirically determined (e.g., detecting fusion proteins labeled with EGFP or AIA by fluorescence microscopy or immunoblotting, respectively).

[0169] Additionally, production and, in certain embodiments, the degradation ofa polypeptide of interest in the presence and absence of protease inhibitors can be monitored. Because the presence of a protease inhibitor prevents accumulation of new protein copies without affecting old copies, the overall levels of a polypeptide of interest after adding the protease inhibitor depend on its degradation rate. Accordingly, the half-life of the polypeptide of interest in a cell can be readily calculated by monitoring its decay. Additionally, the turnover of the polypeptide of interest can be determined by measuring amounts of the polypeptide of interest in a transformed cell before and after contacting the cell with a protease inhibitor and calculating the turnover of the polypeptide of interest based on the amounts of the polypeptide of interest in the cell before and after adding the protease inhibitor. The amount of the polypeptide of interest in the cell can be measured either continuously or periodically over a period of time by any suitable method (e.g., immunoblotting or microscopy).

Production of Fusion Proteins

[0170] Fusion proteins of the present disclosure can be produced using recombinant techniques well known in the art. One of skill in the art can readily determine nucleotide sequences that encode the desired polypeptides 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 and, e.g., restriction enzymes employed to truncate the gene at desired portions of the full-length sequence. Similarly, sequences of interest can be isolated directly from cells and tissues containing the same, using known techniques, such as phenol extraction and the sequence further manipulated to produce the desired truncations See, e.g., Sambrook et al., supra, for a description of techniques used to obtain and isolate DNA.

[0171] The sequences encoding polypeptides can also be produced synthetically, for example, based on the known sequences. The nucleotide sequence can be designed with the appropriate codons for the particular 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, e.g., 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.

[0172] Recombinant techniques are readily used to clone sequences encoding polypeptides useful in the claimed fusion proteins that can then be mutagenized in vitro by the replacement of the appropriate base pair(s) to result in the codon for the desired amino acid. Such a change can include as little as one base pair, effecting a change in a single amino acid, or can encompass several base pair changes.

[0173] Alternatively, the mutations can be affected using a mismatched primer that hybridizes to the parent nucleotide sequence (generally cDNA corresponding to the RNA sequence), at a temperature below the melting temperature of the mismatched duplex. The primer can be made specific by keeping primer length and base composition within relatively narrow limits and by keeping the mutant base centrally located. See, e.g., Innis et al, (1990) PCR Applications. Protocols for Functional Genomics; Zoller and Smith, Methods Enzymol. (1983) 100:468. Primer extension is affected using DNA polymerase, the product cloned and clones containing the mutated DNA, derived by segregation of the primer extended strand, selected.

[0174] Selection can be accomplished using the mutant primer as a hybridization probe. The technique is also applicable for generating multiple point mutations. See, e.g., Dalbie-McFarland et al. Proc. Natl. Acad. Sci USA (1982) 79:6409.

[0175] Once coding sequences have been isolated and/or synthesized, they can be cloned into any suitable vector or replicon for expression. As will be apparent from the teachings herein, a wide variety of vectors encoding modified polypeptides can be generated by creating expression constructs which operably link, in various combinations, polynucleotides encoding polypeptides having deletions or mutations therein.

[0176] Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. Examples of recombinant DNA vectors for cloning and host cells which they can transform include the bacteriophage λ (E. coli), pBR322 (E. coli), pACYC177 (E. coli), pKT230 (gram-negative bacteria), pGVI 106 (gram-negative bacteria), pLAFR1 (gram-negative bacteria), pME290 (non-E. coli gram-negative bacteria), pHV14 (E. coli and Bacillus subtilis), pBD9 (Bacillus), pU61 (Streptomyces), pUC6 (Streptomyces), YIp5 (Saccharomyces), YCp19 (Saccharomyces) and bovine papilloma virus (mammalian cells) See, generally, DNA Cloning Vols I & II, supra; Sambrook et al, supra; B. Perbal, supra.

[0177] Insect cell expression systems, such as baculovirus systems, can also be used and are known to those of skill in the art and described in, e.g., 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).

[0178] 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, e.g., Porta et al., Mol. Biotech (1996) 5:209-221; and Hackland et al., Arch. Virol. (1994) 139: 1-22.

[0179] Viral systems, such as a vaccinia based infection/transfection system, as 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 with the present disclosure. In this system, cells are first transfected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase. This polymerase displays exquisite specificity in that it only transcribes templates bearing T7 promoters. Following infection, cells are transfected with the DNA of interest, driven by a T7 promoter. The polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA that is then translated into protein by the host translational machinery. The method provides for high level, transient, cytoplasmic production of large quantities of RNA and its translation product(s).

[0180] The gene can be placed under the control of a promoter, 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 the host cell transformed by a vector containing this expression construction. The coding sequence may or may not contain a signal peptide or leader sequence. With the present disclosure, both the naturally occurring signal peptides and heterologous sequences can be used. Leader sequences can be removed by the host in post-translational processing. See, e.g., U.S. Pat. Nos. 4,431,739; 4,425,437, 4,338,397 Such sequences include, but are not limited to, the TPA leader, as well as the honeybee mellitin signal sequence.

[0181] Other regulatory sequences may also be desirable which allow for regulation of expression of the protein sequences relative to the growth of the host cell Such regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.

[0182] The control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector. Alternatively, the coding sequence can be cloned directly into an expression vector that already contains the control sequences and an appropriate restriction site.

[0183] In some cases, it may be necessary to modify the coding sequence so that it may be attached to the control sequences with the appropriate orientation; i.e., to maintain the proper reading frame. Mutants or analogs may be prepared by the deletion of a portion of the sequence encoding the protein, by insertion of a sequence, and/or by substitution of 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, e.g., Sambrook et al, supra; DNA Cloning, Vols I and II, supra; Nucleic Acid Hybridization, supra.

[0184] The expression vector is then used to transform an appropriate host cell. A number of mammalian cell lines are known in the art and include immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but not limited to, 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, as well as others. Similarly, bacterial hosts such as E. coli, Bacillus subtilis, and Streptococcus spp., will find use with the present expression constructs. Yeast hosts useful in the present disclosure include inter alia, 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, inter alia, Aedes aegypti, Autographa calif or nica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni.

[0185] Depending on the expression system and host selected, the fusion proteins of the present disclosure are produced by growing host cells transformed by an expression vector described above under conditions whereby the protein of interest is expressed. The selection of the appropriate growth conditions is within the skill of the art.

[0186] In one embodiment, the transformed cells secrete the polypeptide product into the surrounding media Certain regulatory sequences can be included in the vector to enhance secretion of the protein product, for example using a tissue plasminogen activator (TP A) leader sequence, an interferon (y or a) signal sequence or other signal peptide sequences from known secretory proteins. The secreted polypeptide product can then be isolated by various techniques described herein, for example, using standard purification techniques such as but not limited to, hydroxy apatite resins, column chromatography, ion-exchange chromatography, size-exclusion chromatography, electrophoresis, HPLC, immunoadsorbent techniques, affinity chromatography, immunoprecipitation, and the like. Alternatively, the transformed cells are disrupted, using chemical, physical or mechanical means, which lyse the cells yet keep the recombinant polypeptides substantially intact. Intracellular proteins can also be obtained by removing components from the cell wall or membrane, e.g., by the use of detergents or organic solvents, such that leakage of the polypeptides occurs. Such methods are known to those of skill in the art and are described in, e.g., Protein Purification Applications: A Practical Approach, (Simon Roe, Ed., 2001).

[0187] 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-thaw; desiccation; explosive decompression, osmotic shock; treatment with lytic enzymes including proteases such as trypsin, neuraminidase and lysozyme; alkali treatment; and the use of detergents and solvents such as bile salts, sodium dodecyl sulphate, Triton, P40 and CHAPS. The particular technique used to disrupt the cells is largely a matter of choice and will depend on the cell type in which the polypeptide is expressed, culture conditions and any pre-treatment used.

[0188] Following disruption of the cells, cellular debris is removed, generally by centrifugation, and the intracellularly produced polypeptides are further purified, using standard purification techniques such as but not limited to, column chromatography, ion-exchange chromatography, size-exclusion chromatography, electrophoresis, FIPLC, immunoadsorbent techniques, affinity chromatography, immunoprecipitation, and the like.

[0189] For example, one method for obtaining the 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). The choice of a suitable affinity resin is within the skill in the art. After affinity purification, the polypeptides can be further purified using conventional techniques well known in the art, such as by any of the techniques described above.

Polynucleotides Encoding Fusion Proteins

[0190] In another aspect, the present disclosure provides a polynucleotide encoding a fusion protein of the present disclosure, and a vector comprising such a polynucleotide. In some embodiments, the polynucleotide comprises a sequence encoding an inducible cell receptor (e.g., a CAR), wherein the sequence encoding an extracellular protein binding domain is contiguous with and in the same reading frame as a sequence encoding an intracellular signaling domain and a transmembrane domain.

[0191] The polynucleotide can be codon optimized for expression in a mammalian cell in some embodiments, the entire sequence of the polynucleotide has been codon optimized for expression in a mammalian cell. Codon optimization refers to the discovery that the frequency of occurrence of synonymous codons (i.e., codons that code for the same amino acid) in coding DNA is biased in different species. Such codon degeneracy allows an identical polypeptide to be encoded by a variety of nucleotide sequences. A variety of codon optimization methods is known in the art, and include, e.g., methods disclosed in at least U.S. Pat. Nos. 5,786,464 and 6,114,148

[0192] The 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 include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the polynucleotide can be produced synthetically, rather than cloned.

[0193] The polynucleotide can be cloned into a vector. In some embodiments, an expression vector known in the art is used. For example, polynucleotide described herein can be inserted into an expression vector to create an expression cassette capable of producing the degron fusion proteins in a suitable host cell (e.g. in a tissue, organ, organoid, or subject). Expression cassettes typically include control elements operably linked to the coding sequence, which allow for the expression of the gene in vivo in the subject species. For example, typical promoters for mammalian cell expression include the SV40 early promoter, a CMV promoter such as the CMV immediate early promoter, the mouse mammary tumor virus LTR promoter, the adenovirus major late promoter (Ad MLP), and the herpes simplex virus promoter, among others. Other nonviral promoters, such as a promoter derived from the murine metallothionein gene, will also find use for mammalian expression. Typically, transcription termination and polyadenylation sequences will also be present, located 3′ to the translation stop codon Preferably, a sequence for optimization of initiation of translation, located 5′ to the coding sequence, is also present. Examples of transcription terminator/polyadenylation signals include those derived from SV40, as described in Sambrook et al., supra, as well as a bovine growth hormone terminator sequence.

[0194] Enhancer elements may also be used herein to increase expression levels of mammalian constructs. Examples include the SV40 early gene enhancer, as described in Dijkema et al., EMPO J. (1985) 4 761, the enhancer/promoter derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus, as described in Gorman et al., Proc. Natl. Acad. Sci. USA (1982b) 79:6777 and elements derived from human CMV, as described in Boshart et al., Cell (1985) 41:521, such as elements included in the CMV intron A sequence.

[0195] Constructs encoding fusion proteins can be administered to a subject or introduced into cells, tissue, organs, or organoids using standard gene delivery protocols. Methods for gene delivery are known in the art. See, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466. Genes can be delivered either directly to a subject or, alternatively, delivered ex vivo, to cells derived from the subject and the cells reimplanted in the subject.

[0196] A number of viral based systems have been developed for gene transfer into mammalian cells. These include adenoviruses, retroviruses (y-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, herein incorporated by reference).

[0197] For example, retroviruses provide a convenient platform for gene delivery systems. Selected sequences can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems have been described (U.S. Pat. No. 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (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 able to infect both dividing and nondividing cells (see e.g., Lois et al (2002) Science 295:868-872; Durand et al. (2011) Viruses 3(2): 132-159; herein incorporated by reference).

[0198] A number of adenovirus vectors have also been described. Unlike retroviruses which integrate into the host genome, adenoviruses persist 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, K. L. BioTechniques (1988) 6:616-629; and Rich et al., Human Gene Therapy (1993) 4:461-476).

[0199] 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, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International Publication Nos WO 92/01070 (published 23 Jan. 1992) and WO 93/03769 (published 4 Mar. 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 Smith, Gene Therapy (1994) 1: 165-169; and Zhou et al., J. Exp. Med. (1994) 179: 1867-1875.

[0200] Another vector system useful for delivering the polynucleotides of the present disclosure is the enterically administered recombinant poxvirus vaccines described by Small, Jr., P. A., et al. (U.S. Pat. No. 5,676,950, issued Oct. 14, 1997, herein incorporated by reference).

[0201] Additional viral vectors which will find use for delivering the 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 the fusion proteins can be constructed as follows. The DNA encoding the particular fusion protein coding sequence is first inserted into an appropriate vector so that it is adjacent to a vaccinia promoter and flanking vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK) This vector is then used to transfect cells which are simultaneously infected with vaccinia. Homologous recombination serves to insert the vaccinia promoter plus the gene encoding the coding sequences of interest into the viral genome. The resulting TK-recombinant can be selected by culturing the cells in the presence of 5-bromodeoxyuridine and picking viral plaques resistant thereto.

[0202] Alternatively, avipoxviruses, such as the fowlpox and canarypox viruses, can also be used to deliver the genes. Recombinant avipox viruses, expressing immunogens from mammalian pathogens, are known to confer protective immunity when administered to non-avian species. The use of an avipox vector is particularly desirable in human and other mammalian species since members of the avipox genus can only productively replicate in susceptible avian species and therefore are not infective in mammalian cells. Methods for producing recombinant avipoxviruses are known in the art and employ genetic recombination, as described above with respect to the production of vaccinia viruses. See, e.g., WO 91/12882; WO 89/03429; and WO 92/03545.

[0203] 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. Nati. Acad. Sci. USA (1992) 89.6099-6103, can also be used for gene delivery.

[0204] Members of the Alphavirus genus, such as, but not limited to, vectors derived from the Sindbis virus (SIN), Semliki Forest virus (SFV), and Venezuelan Equine Encephalitis virus (VEE), will also find use as viral vectors for delivering the polynucleotides of the present disclosure. For a description of Sindbis-virus derived vectors useful for the practice of the instant methods, see, Dubensky et al. (1996) J. Virol. 70:508-519; and International Publication Nos. WO 95/07995, WO 96/17072; as well as, Dubensky, Jr., T. W., et al., U.S. Pat. No. 5,843,723, issued Dec. 1, 1998, and Dubensky, Jr., T. W., U.S. Pat. No. 5,789,245, issued Aug. 4, 1998, both herein incorporated by reference Particularly preferred are chimeric alphavirus vectors comprised of sequences derived from Sindbis virus and Venezuelan equine encephalitis vims. 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; herein incorporated by reference in their entireties.

[0205] A vaccinia based infection/transfection system can be conveniently used to provide for inducible, transient expression of the coding sequences of interest (for example, a fusion protein expression cassette) in a host cell. In this system, cells are first infected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase. This polymerase displays exquisite specificity in that it only transcribes templates bearing T7 promoters Following infection, cells are transfected with the polynucleotide of interest, driven by a 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 translational machinery. The method provides for high level, transient, cytoplasmic production of large quantities 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.

[0206] As an alternative approach to infection with vaccinia or avipox virus recombinants, or to the delivery of genes using other viral vectors, an amplification system can be used that will lead to high level expression following introduction into host cells. Specifically, a T7 RNA polymerase promoter preceding the coding region for T7 RNA polymerase can be engineered. Translation of RNA derived from this template will generate T7 RNA polymerase which in turn will transcribe more template. Concomitantly, there will be a cDNA whose expression is under the control of the T7 promoter. Thus, some of the T7 RNA polymerase generated from translation of the amplification template RNA will lead to transcription of the desired gene. Because some T7 RNA polymerase is required to initiate the amplification, T7 RNA polymerase can be introduced into cells along with the template(s) to prime the transcription reaction. The polymerase can be introduced as a protein or on a plasmid encoding the RNA polymerase. For a further discussion of T7 systems and their use for transforming 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. Pat. No. 5,135,855.

[0207] 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 prior to delivery to the subject or to cells derived therefrom. Lipid encapsulation is generally accomplished using liposomes which are able to stably bind or entrap and retain nucleic acid. The ratio of condensed DNA to lipid preparation can vary but will generally be around 1:1 (mg DNA:micromoles lipid), or more of lipid. For a review of the use ofliposomes as carriers for delivery of nucleic acids, see, e.g., Hug and Sleight, Biochim. Biophys. Acta (1991) 1097: 1-17, Straubinger et al, in Methods of Enzymology (1983), Vol 101, pp 512-527.

[0208] Liposomal preparations for use in the present disclosure include cationic (positively charged), anionic (negatively charged) and neutral preparations, with cationic liposomes particularly preferred. Cationic liposomes have been shown to mediate 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 functional form.

[0209] Cationic liposomes are readily available. For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N Y (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, e.g., 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.

[0210] Similarly, anionic and neutral liposomes are readily available, such as, from Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using readily available materials. Such materials include phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios Methods for making liposomes using these materials are well known in the art.

[0211] The liposomes can comprise multilammelar 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 See, e.g., 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., 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., Biochem. Biophys. Res. Commun. (1977) 76:836; Fraley et al., 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.

[0212] 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. Pat. Nos. 4,663,161 and 4,871,488.

[0213] The expression cassette of interest may also be encapsulated, adsorbed to, or associated with, particulate carriers Examples of particulate carriers include those derived from polymethyl methacrylate polymers, as well as microparticles derived from poly(lactides) and poly(lactide-co-glycolides), known as PLG. See, e.g., Jeffery et al., Pharm. Res (1993) 10.362-368; McGee J. P., et al., J Microencapsul. 14(2): 197-210, 1997; O'Hagan D. T., et al., Vaccine 11(2): 149-54, 1993.

[0214] Furthermore, other particulate systems and polymers can be used for the in vivo or ex vivo delivery of the nucleic acid of interest. For example, polymers such as polylysine, polyarginine, polyornithine, spermine, spermidine, as well as conjugates of these molecules, are useful for transferring a nucleic acid of interest. Similarly, DEAE dextran-mediated transfection, calcium phosphate precipitation or precipitation using other insoluble inorganic salts, such as strontium phosphate, aluminum silicates including bentonite and kaolin, chromic oxide, magnesium silicate, talc, and the like, will find use with the present methods. See, e.g., Feigner, P. L., Advanced Drug Delivery Reviews (1990) 5: 163-187, for a review of delivery systems useful for gene transfer Peptoids (Zuckerman, R N., et al., U.S. Pat. No. 5,831,005, issued Nov. 3, 1998, herein incorporated by reference) may also be used for delivery of a construct of the present disclosure.

[0215] Additionally, biolistic delivery systems employing particulate carriers such as gold and tungsten are especially useful for delivering synthetic expression cassettes of the present disclosure. The particles are coated with the synthetic expression cassette(s) to be delivered and accelerated to high velocity, generally under a reduced atmosphere, using a gun powder discharge from a “gene gun.” For a description of such techniques, and apparatuses useful therefore, see, e.g., U.S. Pat. Nos. 4,945,050; 5,036,006; 5,100,792; 5,179,022; 5,371,015; and 5,478,744 Also, needle-less injection systems can be used (Davis, H. L., et al, Vaccine 12 1503-1509, 1994; Bioject, Inc., Portland. Oreg.).

[0216] Recombinant vectors can be formulated into compositions for delivery to a vertebrate subject. The compositions will generally include one or more “pharmaceutically acceptable excipients or vehicles” such as water, saline, glycerol, polyethyleneglycol, hyaluronic acid, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents. pH buffering substances, surfactants and the like, may be present in such vehicles. Certain facilitators of nucleic acid uptake and/or expression can also be included in the compositions or coadministered.

[0217] Once formulated, the compositions of the present disclosure can be administered directly to the subject (e.g., as described above) or, alternatively, delivered ex vivo, to cells derived from the subject, using methods such as those described above. For example, methods for the ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and can include, e.g., dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, lipofectamine and LT-1 mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.

[0218] Direct delivery of synthetic expression cassette compositions in vivo will generally be accomplished with or without viral vectors, as described above, by injection using either a conventional syringe, needless devices such as Bioject™ or a gene gun, such as the Accell gene delivery system (PowderMed Ltd, Oxford, England).

[0219] The present disclosure also includes an RNA construct that can be directly transfected into a cell. A method for generating mRNA for use in transfection involves in vitro transcription (IVT) of a template with specially designed primers, followed by polyA addition, to produce a construct containing 3′ and 5′ untranslated sequence (“UTR”) (e.g., a 3′ and/or 5′ UTR described herein), a 5′ cap (e.g., a 5′ cap described herein) and/or Internal Ribosome Entry Site (IRES) (e.g., an IRES described herein), the nucleic acid to be expressed, and a polyA tail. RNA so produced can efficiently transfect different kinds of cells.

Cells

[0220] In one aspect, the present disclosure provides cells expressing a fusion protein of the present disclosure or comprising a polynucleotide or vector encoding the fusion protein. The cells can be stem cells, progenitor cells, and/or immune cells modified to express a fusion protein described herein. In some embodiments, a cell line derived from an immune cell is used. Non-limiting examples of cells, as provided herein, 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 T regulatory cells (CD4+, FOXP3+, CD25+)) and B cells. In some embodiments, the cell a stem cell, such as pluripotent stem cell, embryonic stem cell, adult stein cell, bone-marrow stem cell, umbilical cord stein cells, or other stem cell.

[0221] The cells can be modified to express a fusion protein provided herein. In some embodiment, the fusion protein comprises an inducible receptor. The inducible receptor can comprise a single chain receptor (i.e., a single fusion protein) or a multichain receptor (i.e., multiple fusion proteins). When the inducible cell receptor is a multichain receptor, the cells comprise multiple fusion proteins. Accordingly, the present disclosure provides a cell (e.g., a population of cells) engineered to express an inducible receptor, such as a chimeric antigen receptor (CAR), wherein the receptor comprises an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

Pharmaceutical Compositions

[0222] Pharmaceutical compositions of the present disclosure can comprise a fusion protein or a cell expressing the fusion protein (e.g., a plurality of fusion protein-expressing cells), as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions can comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0223] Pharmaceutical compositions of the present disclosure can be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration can be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.

[0224] In preferred embodiments, the pharmaceutical composition is substantially free of a contaminant, such as endotoxin, 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 cell or plasmid components, a bacterium and a fungus. The pharmaceutical composition can be free from bacterium such as Alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilus influenza, Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumonia, and Streptococcus pyogenes group A.

Method of Preparing Therapeutic Cells

[0225] In one aspect, the present disclosure provides a method of preparing a modified cell comprising a fusion protein for experimental or therapeutic use.

[0226] Ex vivo procedures for making therapeutic fusion protein-modified cells are well known in the art. For example, cells are isolated from a mammal (e.g, a human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a fusion protein disclosed herein. The fusion protein-modified cell can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient may be a human and the fusion protein-modified cell can be autologous with respect to the recipient. Alternatively, the cells can be allogeneic, syngeneic or xenogeneic with respect to the recipient. The procedure for ex vivo expansion of hematopoietic stem and progenitor cells is described in U.S. Pat. No. 5,199,942, incorporated herein by reference, can be applied to the cells of the present disclosure. Other suitable methods are known in the art, therefore the present disclosure is not limited to any particular method of ex vivo expansion of the cells.

Method of Use

[0227] In one aspect, the present disclosure provides a type of cell therapy where a population of cells 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 methods comprise culturing the population of cells (e.g. in cell culture media) to a desired cell density (e.g., a cell density sufficient for a particular cell-based therapy). In some embodiments, the population of cells are cultured in the absence of a protease inhibitor that represses activity of the protease or in the presence of a protease inhibitor that represses activity of the protease.

[0228] In another aspect, the present disclosure provides a type of therapy where a pharmaceutical composition comprising a fusion protein provided herein is administered to a subject in need thereof.

[0229] In some embodiments, the method comprises administering a protease inhibitor that represses activity of the protease after administration of the modified cells or the pharmaceutical composition. In some embodiments, the method further comprises withdrawing the protease inhibitor after administration of the modified cells or the pharmaceutical composition.

[0230] In some embodiments, administration of the protease inhibitor to a subject induces degradation of the polypeptide of interest. In some embodiments, administration of the protease inhibitor protects the polypeptide of interest from degradation. In some embodiments, withdrawal of the protease inhibitor from a subject induces degradation of the polypeptide of interest. In some embodiments, withdrawing the protease inhibitor from a subject protects the polypeptide of interest from degradation.

[0231] In some embodiments, administration of the protease inhibitor to a subject induces activation of the polypeptide of interest. In some embodiments, administration of the protease inhibitor induces inhibition of the polypeptide of interest. In some embodiments, withdrawing the protease inhibitor from a subject induces activation of the polypeptide of interest. In some embodiments, withdrawing the protease inhibitor from a subject induces inhibition of the polypeptide of interest.

[0232] In some embodiments, the population of cells are cultured in the presence of a protease inhibitor that represses activity of the protease to degrade the polypeptide of interest to produce an expanded population of cells. For example, in some embodiments the fusion protein comprises a positioned at the C-terminal end of the polypeptide of interest such that when the cells are cultured in the presence of the protease inhibitor, the protease is inactivated and unable to cleave the cognate cleavage site that separates, for example, the C-terminal end of the polypeptide of interest from the degron. Thus, the degron remains fused to the polypeptide of interest and promotes degradation of the polypeptide through either the proteasome or an autophagy-lysosome pathway. This is particularly advantageous, for example, if the polypeptide of interest is a product that is toxic to the cells or inhibits cell survival and/or proliferation/expansion of the cells.

[0233] In some embodiments, the population of cells is cultured for a period of time that results in the production of an expanded cell population that comprises at least 2-fold the number of cells of the starting population. In some embodiments, the population of cells is cultured for a period of time that results in the production of an expanded cell population that comprises at least 4-fold the number of cells of the starting population. In some embodiments, the population of cells is cultured for a period of time that results in the production of an expanded cell population that comprises at least 16-fold the number of cells of the starting population.

[0234] In some embodiments, the methods further comprise withdrawing the protease inhibitor from the expanded population of cells. The protease inhibitor may be removed, for example, by simply washing the cells with fresh culture media. In the absence of the protease inhibitor, the cells are able to produce the polypeptide of interest, e.g., in vivo following administration of the cells to a subject in need.

[0235] Thus, in some embodiments, the methods comprise delivering cells of the expanded population of cells 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 an autoimmune condition. In some embodiments, the subject in need has a cancer (e.g., a primary cancer or a metastatic cancer).

[0236] 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-coupled 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 thrombolytics.

[0237] The methods, in some embodiments, may comprise administering to the subject a protease inhibitor that represses activity of the protease to degrade the polypeptide of interest. The protease inhibitor may be administered any time following 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 depending on the health condition of the subject.

[0238] Also provided herein are methods of regulating activity of a protein of interest either in vivo or ex vivo. In some embodiments, the activity of the protein of interest is regulated in Pivo by delivering to a subject in need of a cell-based therapy a population of cells that comprise a polynucleotide that encodes a fusion protein of the present disclosure comprising the protein of interest fused to a sequence encoding a degron, and administering to the subject a protease inhibitor that represses activity of the protease to degrade the protein of interest. In some embodiments, the protein of interest is a therapeutic protein. In some embodiments, the method can comprise the step of withdrawing a protease inhibitor that represses activity of the protease from a subject. The protease inhibitor may be withdrawn any time following administration of the cell-based therapy (the expanded cells containing the gene of interest). In some embodiments, the protease inhibitor is withdrawn 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 withdrawn 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 protease inhibitor is withdrawn depending on the health condition of the subject.

[0239] In some embodiments, the activity of the protein of interest is regulated by providing a population of cells comprising a fusion protein of the present disclosure or a polynucleotide encoding the fusion protein and contacting the population of cells with a protease inhibitor that represses activity of the protease. In some embodiments, the method further comprises removing the protease inhibitor from the population of cells. The protease inhibitor may be removed any time following contacting of the population of cells 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 following contacting of the population of cells with the protease inhibitor. In some embodiments, the protease inhibitor is removed 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 population of cells is administered to a subject in need of a cell-based therapy.

Kits

[0240] Fusion proteins or nucleic acids encoding them as well as conditionally replicating viral vectors can be provided in kits with suitable instructions and other necessary reagents for preparing or using them, as described above. The kit may contain in separate containers fusion proteins, and/or recombinant constructs for producing fusion proteins, and/or conditionally replicating viral vectors, and/or cells (either already transfected or separate). Additionally, instructions (e.g., written, tape, VCR, CD-ROM, DVD, Blu-ray, flash drive, etc.) for using the fusion proteins or viral vectors may be included in the kit. The kit may further include a protease inhibitor, such as an HCV NS3 protease inhibitor, including, for example, simeprevir, danoprevir, asunaprevir, ciluprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, or voxiloprevir. The kit may also contain other packaged reagents and materials (e.g., transfection reagents, buffers, media, and the like).

EXAMPLES

Example 1: Single and Double Deimmunized Variants of NS3 Protease/NS4 Degron Fusion Protein

[0241] Four different fusion proteins containing the following were generated: 1) chimeric antigen receptor (CAR) polypeptide of interest, 2) variant HCV NS3 protease, 3) cognate protease cleavage site, and 4) HCV NS4 degron operably linked to the CAR polypeptide of interest. The four different fusion proteins differ from one another 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 controllability over the CAR. Specifically, the four fusion proteins have the following mutations in the variant HCV NS3 protease.

[0242] Fusion Protein 1 (T1080A): Includes a Thr to Ala substitution at a position corresponding to position 1080 of the sequence shown in SEQ ID NO: 1.

[0243] Fusion Protein 2 (T1080A, V1077A): Includes a Thr to Ala substitution at a position corresponding to position 1080 of the sequence shown in SEQ ID NO: 1 and a Val to Ala substitution at a position corresponding to position 1077 of the sequence shown in SEQ ID NO: 1.

[0244] Fusion Protein 3 (T1080A, W1079A). Includes a Thr to Ala substitution at a position corresponding to position 1080 of the sequence shown in SEQ ID NO: 1 and a Trp to Ala substitution at a position corresponding to position 1079 of the sequence shown in SEQ ID NO: 1.

[0245] Fusion Protein 4 (T1080A, V1081A) Includes a Thr to Ala substitution at a position corresponding to position 1080 of the sequence shown in SEQ ID NO: 1 and a Val to Ala substitution at a position corresponding to position 1081 of the sequence shown in SEQ ID NO: 1.

[0246] On Day 0, total pan 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 media was changed to remove lentivirus and LentiBlast media. On Day 4, Dynabeads® were removed. On Day 7, cell media was changed and the T-cells were treated. To test the controllability of the CAR polypeptide of the fusion proteins, a first population was treated with 2 μM asunaprevir, a small molecule inhibitor of hepatitis C whereas a second population was left unreated (No ASV). On Day 9, flow cytometry using YFP and myc-tag (Alexa647) fluorescent tags was performed on the ASV treated T-cells and the non-ASV treated T-cells to determine the level of CAR expression in the cells.

[0247] FIG. 1 depicts the normalized % CAR expression in cells transfected to express one of the four different fusion proteins. The cells were either treated with asunaprevir (+ASV) or untreated (No ASV). Each of the values were normalized to the CAR expression in T1080A variant expressing cells that were untreated. Notably, Fusion Protein 2 (T1080A, V1077A), Fusion Protein 3 (T1080A, W1079A), and Fusion Protein 4 (T1080A, V1081A) exhibited close to or higher levels (e.g., 100%-150%) of CAR expression in comparison to Fusion Protein 1 (T1080A), thereby indicating that the additional mutations do not compromise the degron functionality. For three of the fusion proteins, Fusion Protein 1 (T1080A), Fusion Protein 2 (T1080A, V1077A), and Fusion Protein 4 (T1080A, V1081A), asunaprevir treatment significantly reduced the relative percentage of CAR expression. This indicates that the inhibition of the HCV NS3 protease by the asunaprevir directly led to the reduced CAR expression levels. Altogether, these results demonstrate the controllability of the expression of a polypeptide of interest, such as a CAR, on deimmunized fusion proteins by using small molecule knockdowns (e.g., using asunaprevir).

TABLE-US-00005 SEQUENCES SEQ ID NO Identity Sequence SEQ ID HCV 1a         10         20         30         40         50 NO: 1 polyprotein MSTNPKPQKK NKRNTNRRPQ DVKFPGGGQI VGGVYLLPRR GPRLGVRATR         60         70         80         90        100 KTSERSQPRG RRQPIPKARR PEGRTWAQPG YPWPLYGNEG CGWAGWLLSP        110        120        130        140        150 RGSRPSWGPT DPRRRSRNLG KVIDTLTCGF ADLMGYIPLV GAPLGGAARA        160        170        180        190        200 LAHGVRVLED GVNYATGNLP GCSFSIFLLA LLSCLTVPAS AYQVRNSTGL        210        220        230        240        250 YKVTNDCPNS SIVYEAADAI LHTPGCVPCV REGNASRCWV AMTPTVATRD        260        270        280        290        300 GKLPATQLRR HIDLLVGSAT LCSALYVGDL CGSVFLVGQL FTFSPRRHWT        310        320        330        340        350 TQGCNCSIYP GHITGHRMAW DMMMNWSPTT ALVMAQLLRI PQAILDMIAG        360        370        380        390        400 AHWGVLAGIA YPSMVGNWAK VLVVLLLFAG VDAETHVTGG SAGHTVSGFV        410        420        430        440        450 SLLAPGAKQN VQLINTNGSW HLNSTALNCN DSLNTGWLAG LFYHHKFNSS        460        470        480        490        500 GCPERLASCR PLTDFDQGWG PISYANGSGP DQRPYCWHYP PKPCGIVPAK        510        520        530        540        550 SVCGPVYCFT PSPWVGTTD RSGAPTYSWG SNDTDVFVLN NTRPPLGNVVF        560        570        580        590        600 GCTWMNSTGF TKVCGAPPCV IGGAGNNTLH CPTDCFRKHP DATYSRCGSG        610        620        630        640        650 PWITPRCLVD YPYRLWHYPC TINYTIFKIR MYVGGVEHRL EAACNWTRGE        660        670        680        690        700 RCDLEDRDRS ELSPLLLTTT QWQVLPCSFT TLPALSTGLI HLHQNIVDVQ        710        720        730        740        750 YLYGVGSSIA SWAIKWEYVV LLFLLLADAR VCSCLWMMLL ISQAEAALEN        760        770        780        790        800 LVILNAASLA GTKGLVSFLV FFCFAWYLKG KWVPGAVYTF YGMWPLLLLL        810        820        830        840        850 LALPQRAYAL DTEVAASCGG WLVGLMALT LSPYYKRYIS WCLWWLQYFL        860        870        880        890        900 TRVEAQLHVW IPPLNVRGGR DAVILLMCAV HPTLVFDITK LLLAVFGPLW        910        920        930        940        950 ILQASLLKVP YFVRVQGLLR FCALARKMIG GHYVQMVIIK LGALTGTYVY        960        970        980        990       1000 NKLTPLRDWA HNGLRDLAVA VEPVVFSQME TKLITWGADT AACGDIINGL       1010       1020       1030       1040       1050 PVSARRGREI LLGPADGMVS KGWRLLAPIT AYAQQTRGLL GCIITSLTGR       1060       1070       1080       1090       1100 DKNQVEGEVQ IVSTAAQTFL ATCINGVCWT VYHGAGTRTI ASPKGPVIQM       1110       1120       1130       1140       1150 YTNVDQDLVG WPAPQGSRSL TPCTCGSSDL YLVTRHADVI PVRRRGDSRG       1160       1170       1180       1190       1200 SLLSPRPISY LKGSSGGPLL CPAGHAVGIF RAAVCTRGVA KAVDFIPVEN       1210       1220       1230       1240       1250 LETTMRSPVP TDNSSPPVVP QSFQVAHLHA PTGSGKSTKV PAAYAAQGYK       1260       1270       1280       1290       1300 VLVLNPSVAA TLGFGAYMSK AHGIDPNIRT GVRTITTGSP ITYSTYGKFL       1310       1320       1330       1340       1350 ADGGCSGGAY DIIICDECHS TDATSILGIG TVLDQAETAG ARLVVLATAT       1360       1370       1380       1390       1400 PPGSVTVPHP NIEEVALSTT GEIPFYGKAI PLEVIKGGRH LIFCKSKKKC       1410       1420       1430       1440       1450 DELAAKLVAL GINAVAYYRG LDVSVIPTSG DVVVVATDAL MTGYTGDFDS       1460       1470       1480       1490       1500 VIDCNTCVTQ TVDFSLDPTF TIETITLPQD AVSRTQRRGR TGRGKPGIYR       1510       1520       1530       1540       1550 FVAPGERPSG MFDSSVLCEC YDAGCAWYEL TPAETTVRLR AYMNTPGLPV       1560       1570       1580       1590       1600 CQDHLEFWEG VFTGLTHIDA HFLSQTKQSG ENLPYLVAYQ ATVCARAQAP       1610       1620       1630       1640       1650 PPSWDQMWKC LIRLKPTLHG PTPLLYRLGA VQNEITLTHP VTKYIMTCMS       1660       1670       1680       1690       1700 ADLEVVTSTW VLVGGVLAAL AAYCLSTGCV VIVGRVVLSG KPAIIPDREV       1710       1720       1730       1740       1750 LYREFDEMEE CSQHLPYIEQ GMMLAEQFKQ KALGLLQTAS RQAEVIAPAV       1760       1770       1780       1790       1800 QTNWQKLETF WAKHMWNFIS GIQYLAGLST LPGNPAIASL MAFTAAVTSP       1810       1820       1830       1840       1850 LTTSQTLLFN ILGGWVAAQL AAPGAATAFV GAGLAGAAIG SVGLGKVLID       1860       1870       1880       1890       1900 ILAGYGAGVA GALVAFKIMS GEVPSTEDLV NLLPAILSPG ALVVGVVCAA       1910       1920       1930       1940       1950 ILRRHVGPGE GAVQWMNRLI AFASRGNHVS PTHYVPESDA AARVTAILSS       1960       1970       1980       1390       2000 LTVTQLLRRL HQWISSECTT PCSGSWLRDI WDWICEVLSD FKTWLKAKLM       2010       2020       2030       2040       2050 PQLPGIPFVS CQRGYKGVWR VDGIMHTRCH CGAEITGKVK NGTMRIVGPR       2060       2070       2080       2090       2100 TCRNMWSGTF PINAYTTGPC TPLPAPNYTF ALWRVSAEEY VEIRQVGDFH       2110       2120       2130       2140       2150 YVTGMTTDNL KCPCQVPSPE FFTELDGVRL HRFAPPCKPL LREEVSFRVG       2160       2170       2180       2190       2200 LKEYPVGSQL PCEPEPDVAV LTSMLTDPSH ITAEAAGRRL ARGSPPSVAS       2210       2220       2230       2240       2250 SSASQLSAPS LKATCTANHD SPDAELIEAN LLWRQEMGGN ITRVESENKV       2260       2270       2280       2290       2300 VILDSFDPLV AEEDEREISV PAEILRKSRR FAQALPVWAR PDYNPPLVET       2310       2320       2330       2340       2350 WKKPDYEPPV VKGCPLPPPK SPPVPPPRKK RTVVLTESTL STALAELATR       2360       2370       2380       239O       2400 SFGSSSTSGI TGDNTTTSSE PAPSGCPPDS DAESYSSMPP LEGEPGDPDL       2410       2420       2430       2440       2450 SDGSWSTVSS EANAEDWCC SMSYSVVTGAL VTPCAAEEQK LPINALSNSL       2460       2470       2480       2490       2500 LRHHNLVYST TSRSACQRQK KVTFDRLQVL DSHYQDVLKE VKAAASKVKA       2510       2520       2530       2540       2550 NLLSVEEACS LTPPHSAKSK FGYGAKDVRC HARKAVTHIN SVWKDLLEDN       2560       2570       2580       2590       2600 VTPIDTTIMA KNEVFCVQPE KGGRKPARLI VFPDLGVRVC EKMALYDVVT       2610       2620       2630       2640       2650 KLPLAVMGSS YGFQYSPGQR VEFLVQAWKS KKTPMGFSYD TRCFDSTVTE       2660       2670       2680       2690       2700 SDIRTEEAIY QCCDLDPQAR VAIKSLTERL YVGGPLTNSR GENCGYRRCR       2710       2720       2730       2740       2750 ASGVLTTSCG NTLTCYIKAR AACRAAGLQD CTMLVCGDDL VVICESAGVQ       2760       2770       2780       2790       2800 EDAASLRAFT EAMTRYSAPP GDPPQPEYBL SLITSCSSNV SVAHDGAGKR       2810       2820       2830       2840       2850 VYYLTRDPTT PLARAAWETA RHTPVNSWLG NIIMFAPTLW ARMILMTHFF       2860       2870       2880       2890       2900 SVLIARDQLB QALDCEIYGA CYSIEPLDLP PIIQRLHGLS AFSLHSYSPG       2910       2920       2930       2940       2950 EINRVAACLR KLGVPPLRAW RHRARSVRAR LLARGGRAAI CGKYLFNWAV       2960       2970       2980       2990       3000 RTKLKLTPIA AAGQLDLSGW FTAGYSGGDI YHSVSHARPS WIWFCLLLLA       3010 AGVGIYLLPN R SEQ ID a variant NS3 APITAYAQQT RGLLGCIITS LTGRDKNQVE GEVQIVSTAT QTFLATCING NO: 2 protease is VCWAVYHGAG TRTIASPKGP VIQMYTNVDQ DLVGWPAPQG derived from SRSLTPCTCG SSDLYLVTRH ADVIPVRRRG DSRGSLLSPR PISYLKGSSG an HCV NS3 GPLLCPAGHA VGLFRAAVCT RGVAKAVDFI PVENLETTMR SPVFTD SEQ ID HCV NS4A TWVLVGGVLA ALAAYCLSTG CWIVGRIVL SGKPAIIPDR EVLY NO: 3 co-factor SEQ ID cognate CMSADLEVVTSTWVLVGGVL NO: 4 protease cleavage site SEQ ID cognate YQEFDEMEECSQHLPYIEQG NO: 5 protease cleavage site SEQ ID cognate WISSECTTPCSGSWLRDIWD NO: 6 protease cleavage site SEQ ID cognate GADTEDVVCCSMSYSWTGAL NO: 7 protease cleavage site SEQ ID cognate ADLEVVTSTWL NO: 8 protease cleavage site SEQ ID cognate DEMEECSQHL NO: 9 protease cleavage site SEQ ID cognate ECTTPCSGSWL NO: 10 protease cleavage site SEQ ID cognate EDVVPCSMG NO: 11 protease cleavage site SEQ ID HCV NS3 APITAYAQQT RGLLGCIITS LTGRDKNQVE GEVQIVSTAA QTFLATCING NO: 12 protease VCWTVYHGAG TRTIASSKGP VIQMYTNVDQ DLVGWPAPQG ARSLTPCTCG SSDLYLVTRH ADVIPVRRRG DGRGSLLSPR PISYLKGSSG GPLLCPAGHA VGIFRAAVCT RGVAKAVDFI PVEGLETTMR SPVFSD SEQ ID HIV-I PQVTLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGG NO: 13 protease FIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF SEQ ID fluorogenic EDANS-EPLFAERK-DABCYL NO: 14 calpain substrate SEQ ID Caspasc 1 YVAD NO: 15 cleavage sile SEQ ID Caspase 2 VDVAD NO: 16 cleavage site SEQ ID Caspase 4 DEVD NO: 17 cleavage site SEQ ID Caspase 6 VEHD NO: 18 cleavage sile SEQ ID Caspasc 9 LGHD NO: 19 cleavage site SEQ ID Caspasc 10 LQTDG NO: 20 cleavage site SEQ ID angiotensin MGAASGRRGP GLLLPLPLLL LLPPQPALAL DPGLQPGNFS ADEAGAQLFA NO: 21 converting QSYNSSAEQV LFQSVAASWA HDTNITAENA RRQEEAALLS QEFAEAWGQK enzyme AKELYEPIWQ NFTDPQLRRI IGAVRTLGSA NLPLAKRQQY NALLSNMSRI (ACE) YSTAKVCLPN KTATCWSLDP DLTNILASSR SYAMLLFAWE GWHNAAGIPL KPLYEDFTAL SNEAYKQDGF TDTGAYWRSW YNSPTFEDDL EHLYQQLEPL YLNLHAFVRR ALHRRYGDRY INLRGPIPAH LLGDMWAQSW ENIYDMVVPF PDKPNLDVTS TMLQQGWNAT HMFRVAEEFF TSLELSPMPP EFWEGSMLEK PADGREVVCH ASAWDFYNRK DFRIKQCTRV TMDQLSTVHH EMGHIQYYLQ YKDLPVSLRR GANPGFHEAI GDYLALSVST PEHLHKIGLL DRVTNDTESD INYLLKMALE KIAFLPFGYL VDQWRWGVFS GRTPPSRYNF DWWYLRTKYQ GICPPVTRNE THFDAGAKFH VPNVTPYIRY FVSFVLQFQF HEALCKEAGY EGPLHQCDIY RSTKAGAKLR KVLQAGSSRP WQEVLKDMVG LDALDAQPLL KYFQPVTQWL QEQNQQNGEV LGWPEYQWHP PLPDNYPEGI DLVTDEAEAS KFVEEYDRTS QVVWNEYAEA NWNYNTNITT ETSKILLQKN MQ1ANHTLKY GTQARKFDVN QLQNTTIKRI IKKVQDLERA ALPAQELEEY NKILLDMETT YSVATVGHPN GSCLQLEPDL TNVMATSRKY EDLLWAWEGW RDKAGRAILQ FYPKYVELIN QAARLNGYVD AGDSWRSMYE TPSLEQDLER LFQELQPLYL NLHAYVRRAL HRHYGAQHIN LEGPIPAHLL GNMWAQTWSN IYDLVVTFPS APSMDTTEAM LKQGWTPRRM FKEADDFFTS LGLLPVPPEF WNKSMLEKPT DGREVVCHAS AWDFYNGKDF RIKQCTTVNL EDLVVAHHEM GHIQYFMQYK DLPVALREGA NPGFHEAIGD VLALSVSTPK HLHSLNLLSS EGGSDEHDIN FLMKMALDKI AFIPFSYLVD QWRWRVFDGS iTKENYNQEW WSLRLKYQGL CPPVPRTQGD FDPGAKFHIP SSVPYIRYFV SFIIQFQFHE ALCQAAGHTG PLHKCDIYQS KEAGQRLATA MKLGFSRPWP EAMQLITGQP NMSASAMLSY FKPLLDWLRT ENELHGEKLG WPQYNWTPNS ARSEGPLPDS GRVSFLGLDL DAQQARVGQW LLLFLGIALL VATLGLSQRL FSIRHRSLHR HSHGPQFGSE VELRHS SEQ ID amyloid EVNLDAEF NO: 22 precursor protein secretase beta cleavage site SEQ ID MMP2 PQGIAGQ NO: 23 cleavage sile SEQ ID tobacco Etch ENLYFQS NO: 24 virus (TEV) protease cleavage site SEQ ID Cleavage site HPFHL NO: 25 SEQ ID DENV SGVLWDTPSPPEVERAVLDDGIYRIMQRGLLGRSQ NO: 26 NS3pro VGVGVFQDGVFHTMWHVTRGAVLMYQGKRLEPSWA (NS2B/NS3) SVKKDLISYGGGWRFQGSWNTGEEVQVIAVEPGKN PKNVQTAPGTFKTPEGEVGAIALDFKPGTSGSPIV NREGKIVGLYGNGVVTTSGTYVSAIAQAKASQEGP LPEIEDEVFRKRNLTIMDLHPGSGKTRRYLPAIVR EAIRRNVRTLILAPTRVVASEMAEALKGMPIRYQT TAVKSEHTGKEIVDLMCHATFTMRLLSPVRVPNYN MIIMDEAHFTDPASIARRGYISTRVGMGEAAAIFM TATPPGSVEAFPQSNAVIQDEERDIPERSWNSGYE WITDFPGKTVWFVPSIKSGNDIANCLRKNGKRVIQ LSRKTFDTEYQKTKNNDWDYVVTTDISEMGANFRA DRVIDPRRCLKPVILKDGPERVILAGPMPVTVASA AQRRGRIGRNQNKEGDQYVYMGQPLNNDEDHAHWT EAKMLLDNINTPEGIIPALFEPEREKSAAIDGEYR LRGEARKTFVELMRRGDLPVWLSYKVASEGFQYSD RRWCFDGERNNQVLEENMDVEMWTKEGERKKLRPR WLDARTYSDPLALREFKEFAAGRR SEQ ID DENV AGVLWDVPSPPPVGKAELEDGAYRIKQKGILGYSQ NO: 27 NS3pro IGAGVYKEGTFHTMWHVTRGAVLMHKGKRIEPSWA (NS2B/NS3) DVKKDLISYGGGWKLEGEWKEGEEVQVLALEPGKN PRAVQTKPGLFKTNAGTIGAVSLDFSPGTSGSPII DKKGKWGLYGNGVVTRSGAYVSAIAQTEKSIEDNP EIEDDIFRKRKLTIMDLHPGAGKTKRYLPAIVREA IKRGLRTLILAPTRWAAEMEEALRGLPIRYQTPAI RAEHTGREIVDLMCHATFTMRLLSPVRVPNYNLII MDEAHFTDPASIAARGYISTRVEMGEAAGIFMTAT PPGSRDPFPQSNAPIMDEEREIPERSWSSGHEWVT DFKGKTVWFVPSIKAGNDIAACLRKNGKKVIQLSR KTFDSEYVKTRTNDWDFWTTDISEMGANFKAERVI DPRRCMKPVILTDGEERVILAGPMPVTHSSAAQRR GRIGRNPKNENDQYIYMGEPLENDEDCAHWKEAKM LLDNINTPEGIIPSMFEPEREKVDAIDGEYRLRGE ARKTFVDLMRRGDLPVWLAYRVAAEGINYADRRWC FDGIKNNQILEENVEVEIWTKEGERKKLKPRWLDA KIYSDPLALKEFKEFAAGRK SEQ ID DENV SGVLWDVPSPPETQKAELEEGVYRIKQQGIFGKTQ NO: 28 NS3pro VGVGVQKEGVFHTMWHVTRGAVLTHNGKRLEPNWA (NS2B/NS3) SVKKDLISYGGGWRLSAQWQKGEEVQVIAVEPGKN PKNFQTMPGIFQTTTGEIGAIALDFKPGTSGSPII NREGKWGLYGNGVVTKNGGYVSGIAQTNAEPDGPT PELEEEMFKKRNLTIMDLHPGSGKTRKYLPAIVRE AIKRRLRTLILAPTRVVAAEMEEALKGLPIRYQTT ATKSEHTGREIVDLMCHATFTMRLLSPVRVPNYNL IIMDEAHFTDPASIAARGYISTRVGMGEAAAIFMT ATPPGTADAFPQSNAPIQDEERDIPERSWNSGNEW ITDFVGKTVWFVPSIKAGNDIANCLRKNGKKVIQL SRKTFDTEYQKTKLNDWDFWTTDISEMGANFKADR VIDPRRCLKPVTLTDGPERVILAGPMPVTVASAAQ RRGRVGRNPQKENDQYIFMGQPLNKDEDHAHWTEA KMLLDNINTPEGIIPALFEPEREKSAAIDGEYRLK GESRKTFVELMRRGDLPVWLAHKVASEGIKYTDRK WCFDGERNNQILEENMDVEIWTKEGEKKKLRPRWL DARTYSDPLALKEFKDFAAGRK SEQ ID DENV SGALWDVPSPAATQKAALSEGVYRIMQRGLFGKTQ NO: 29 NS3pro VGVGIHIEGVFHTMWHVTRGSVICHETGRLEPSWA (NS2B/NS3) DVRNDMISYGGGWRLGDKWDKEEDVQVLAIEPGKN PKHVQTKPGLFKTLTGEIGAVTLDFKPGTSGSPI INRKGKVIGLYGNGVVTKSGDYVSAITQAERIGEP DYEVDEDIFRKKRLTIMDLHPGAGKTKRILPSIVR EALKRRLRTLILAPTRVVAAEMEEALRGLPIRYQT PAVKSEHTGREIVDLMCHATFTTRLLSSTRVPNYN LIVMDEAHFTDPSSVAARGYISTRVEMGEAAAIFM TATPPGTTDPFPQSNSPIEDIEREIPERSWNTGFD WITDYQGKTVWFVPSIKAGNDIANCLRKSGKKVIQ LSRKTFDTEYPKTKLTDWDFVVTTDISEMGANFRA GRVTDPRRCLKPVILPDGPERVTLAGPIPVTPASA AQRRGRIGRNPAQEDDQYVFSGDPLKNDEDHAHWT EAKMLLDNIYTPEGIIPTLFGPEREKTQAIDGEFR LRGEQRKTFVELMRRGDLPVWLSYKVASAGISYKD REWCFTGERNNQILEENMEVEIWTREGEKKKLRPK WLDARVYADPMALKDFKEFASGRK SEQ ID Sub-sequence GLLGCIITSL NO: 30 of HCV 1a polyprotein SEQ ID Sub-sequence GEVQIVSTAAQTFLATCINGVCWTVY NO: 31 of HCV 1a polyprotein SEQ ID Sub-sequence GEVQIVSTAAQTFLA NO: 32 of HCV 1a polyprotein SEQ ID Sub-sequence QTFLATCINGVCWTV NO: 33 of HCV 1a polyprotein SEQ ID Sub-sequence CINGVCWTVY NO: 34 of HCV 1a polyprotein SEQ ID Sub-sequence SSDLYLVTRHADVIP NO: 35 of HCV 1a polyprotein SEQ ID Sub-sequence YLVTRHAD NO: 36 of HCV 1a polyprotein SEQ ID Sub-sequence LLCPAGHAV NO: 37 of HCV 1a polyprotein SEQ ID Sub-sequence AVDFIPVEGLETTMR NO: 38 of HCV 1a polyprotein SEQ ID Sub-sequence KIDTKYIMTCMSADL NO: 39 of HCV 1a polyprotein SEQ ID Degradation PITKIDTKYIMTCMSADLEVVTSTWVLVGGVLAALA NO: 40 sequences AYCLST SEQ ID Targeting KKKRK NO: 41 sequence SEQ ID Targeting MLRT S SLFTRRVQP SLFRNILRLQ ST NO: 42 sequence SEQ ID Targeting KDEL NO: 43 sequence SEQ ID C-terminal DEMEECSQHLPGAGSSGDIMDYKDDDDKGSSGTGS NO: 44 degradation GSGTSAPITAYAQQTRGLLGCIITSLTGRDKNQVE signal with GEVQIVSTATQTFLATCINGVCWAVYHGAGTRTIA NS4A/4B SPKGPVIQMYTNVDQDLV protease GWPAPQGSRSLTPCTCGSSDLYLVTRHADVIPVRR cleavage site RGDSRGSLLSPRPISYLKGSSGGPLLCPAGHAVGL FRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNS SPPAVTLTHPITKIDTKYIMTCMSADLEWTSTWVL VGGVLAALAAYCLSTGCWIVGRIVLSGKPAIIPDR EVLY SEQ ID N-terminal MDYKDDDDKGSSGTGSGSGTSAPITAYAQQTRGLL NO: 45 degradation GCIITSLTGRDKNQVEGEVQIVSTATQTFLATCIN signal with GVCWAVYHGAGTRTIASPKGPVIQMYTNVDQDLVG HCV WPAPQGSRSLTPCTCGSSDLYLVTRHADVIPVRRR NS5A/5B GDSRGSLLSPRPISYLKGSSGGPLLCPAGHAVGLF protease RAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSS cleavage site PPAVTLTHPITKIDTKYIMTCMSADLEWTSTWVLV GGVLAALAAYCLSTGCWIVGRIVLSGKPAGS SGSSIIPDREVLYQEFEDWPCSMG SEQ ID PEST, Two LQMLPESEDEESYDTESEFTEFTEDELPYDDGSLQ NO: 46 copies of MLPESEDEESYDTESEFTEFTEDELPYDD residues 277- 307 of IκBα (human) SEQ ID GRR, EIKDKEEVQRKRQKLMPNFSDSFGGGSGAGAGGGG NO: 47 Residues MFGSGGGGGGTGSTGPGYSFPH 352-408 of p105 (human) SEQ ID DRR, IDDENGSVILQDDDYDDGNNHIPFEDDDVYNYNDN NO: 48 Residue 210- DDDDERIEFEDDDDDDDDSIDNDSVMDRKQPHKAE 295 of Cdc34 DESEDVEDVERVSKKD (yeast)) SEQ ID SNS, Tandem PESMREEYRKEGSKRIKCPDCEPFCNKRGSPESMR NO: 49 repeat of SP2 EEYRKE and NB (SP2- NB-SP2) SEQ ID RPB, (Four RSYSPTSPNYSPTSPSGSYSPTSPNYSPTSPSGGS NO: 50 copies of RSYSPTSPNYSPTSPSGSYSPTSPNYSPTSPSG residues 1688-1702 of RPB1 (yeast) SEQ ID SPmix, PESMREEYRKEGSSLLTEVETPGSPESMREEYRKE NO: 51 Tandem GSSLLTEVETPGSPESMREEYRKE repeat of SP1 and SP2 (SP2-SP1- SP2-SP1- SP2) (Influenza A virus M2 protein) SEQ ID Three copies LIEEVRHRLKTTENSGSLIEEVRHRLKTTENSGSL NO: 52 of residue 79- IEEVRHRLKTTENSGS 93 of Influenza A virus NS protein SEQ ID Residue 106- FPPEVEEQDDGTLPMSCAQESGMDRHPAACASARI NO: 53 142 of NV ornithine decarboxylase SEQ ID mODC DA, SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACAS NO: 54 amino acids ARINV 422-461 of mODC (D433A, D434A)