WO2012139112A1

WO2012139112A1 - Ubiquitin fusions for improving the efficacy of cytosolic acting targeted toxins

Info

Publication number: WO2012139112A1
Application number: PCT/US2012/032762
Authority: WO
Inventors: Christopher H. BACHRAN; Stephen H. Leppla; Shihui Liu; Thomas MORLEY
Original assignee: The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services
Priority date: 2011-04-08
Filing date: 2012-04-09
Publication date: 2012-10-11

Abstract

The present invention provides chimeric molecules having a ubiquitin moiety attached to a cytosol-targeting moiety and an effector moiety. The ubiquitin can be modified to replace one or more lysine residues with another amino acid to reduce the amount of ubiquitination and proteasomal degradation of the chimeric molecule. The modified ubiquitin results in increased stability of the effector moiety in the cytosol of the target cell. The invention also provides methods of using the ubiquitin-containing chimeric molecules to target diseased cells, such as tumor cells and HIV-infected cells.

Description

UBIQUITIN FUSIONS FOR IMPROVING THE EFFICACY OF

CYTOSOLIC ACTING TARGETED TOXINS

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 61/473,450, filed on April 8, 201 1 , the contents of which are incorporated by reference in the entirety.

BACKGROUND OF THE INVENTION

[0002] The targeting of drugs in biomedical sciences is a concept followed on for several decades. Various drugs were developed and a number were approved for therapeutic purposes. Antibodies are the most commonly used for targeting, however, other molecules and concepts were investigated in numerous studies. Many targeted drugs were applied for the treatment of cancer such as immunotoxins (ITs). They are targeted drugs studied mostly in the context of tumor therapies. ITs usually consist of an antibody or a fragment thereof coupled to a cytotoxic compound, e.g., a protein toxin. The term targeted toxin (TT) is more applicable to the variety of different targeted drugs. Protein toxins are of great interest for these studies due to their potency once inside the target cell. The targeted delivery of TTs to tumor cells is often of low efficacy and protein toxins are a way to overcome the limitations of inefficient delivery. Thus, having an enzyme as cytotoxic substance enables the drug to kill the tumor cells even with suboptimal drug delivery. These fusion proteins are to a smaller extent used in other fields, e.g., treatment of human immunodeficiency virus-infected cells (Berger and Pastan (2010) PLoS Pathog. 6, el000803) or the elimination of specific neurons in the brain in neurological studies (Baskin et al. (2010) Endocrinology 151, 4207-4213). The most prominent drug in this category is the FDA-approved denileukin diftitox for the treatment of cutaneous T-cell lymphoma. This drug proved to be valuable in a variety of further conditions (reviewed in Manoukian and Hagemeister (2009) Expert. Opin. Biol. Ther. 9, 1445-1451). Despite the problems associated with the use of highly potent drugs for targeted tumor therapy, the work on TTs is ongoing. Problems associated with TTs are usually high nonspecific toxicity (Onda et al. (2001) Cancer Res. 61, 5070-5077), immunogenicity (Onda et al. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 1 131 1 - 1 1316), and inefficient delivery of the cytotoxic compound into tumor tissue and the desired subcellular location (Traini et al. (2010) Mol. Cancer Ther. 9, 2007-2015). Several approaches were followed in order to improve the therapeutic index of TTs (reviewed in Hetzel et al. (2009) Curr. Pharm. Des. 15, 2700-2711) or to increase the uptake of TTs into the cytosol of tumor cells (Bachran et al. (2009) J Immunother. 32, 713-725).

[0003] Another TT for tumor therapy is described by Liu et al. to overcome the issues of nonspecific toxicity and inefficient toxin delivery to the cytosol by utilizing and modifying anthrax toxin (Liu et al. 2000 Cancer Res. 60, 6061-6067). The N-terminal 254 amino acids of anthrax toxin lethal factor (LFn) from Bacillus anthracis were fused to the catalytic domain of Pseudomonas exotoxin A (PEIII) from Pseudomonas aeruginosa. Once in the cytosol, PEIII will transfer ADP-ribose on to eukaryotic elongation factor-2 (eEF-2), resulting in protein synthesis inhibition, and initiating programmed cell death. Protective antigen (PA), the cell receptor-binding domain of anthrax toxin, was modified to be activated by tumor-specific proteases, e.g., urokinase plasminogen activator. Internalization of LFn-PEIII will then occur after binding of PA to its receptors, urokinase plasminogen activator cleavage and activation of PA, heptamerization of activated PA, and subsequent binding of the LFn domain to the heptamer. The complex is endocytosed and acidification results in pore formation by the PA heptamer and translocation of LFn-PEIII into the cytosol, where PEIII inhibits protein synthesis. This system proved to be highly effective in terms of cytosolic delivery and tumor-specific activation and was further investigated on several tumor cells (Abi-Habib et al. (2006) Mol. Cancer Ther. 5, 2556-2562). In a follow-up study, Gupta et al. described the relevance of the N- terminal amino acids for the potency of this TT (2008, PLoS. ONE 3, e3130). This indicates involvement of the N-end rule by determining susceptibility of the TT to ubiquitination and proteasomal degradation.

[0004] Ubiquitin is a small protein in eukaryotic cells that plays a mayor role in protein recycling. Proteins to be degraded are conjugated to ubiquitin by ubiquitin ligases. Ubiquitin consists of a di-glycine motif at its C-terminus, which is conjugated to the epsilon amine of a lysine within the target protein. Ubiquitination occurs on proteins with specific destabilizing N- terminal residues (Tasaki and Kwon (2007) Trends Biochem. Sci. 32, 520-528) and

ubiquitination may also occur on several sites within one protein. Ubiquitinated proteins, apparently with a minimum of four ubiquitins, are targeted for degradation by the 26S proteasome system (Thrower et al. (2000) EMBO J. 19, 94-102). Ubiquitin itself may be ubiquitinated after its conjugation to a target protein. The resulting polyubiquitin chains are conjugated to all seven lysine residues within ubiquitin; however the residue 48 is most often used for ubiquitination (Kim et al. (2007) J Biol. Chem. 282, 17375-17386). Furthermore, polyubiquitination of lysine residue 63 instead of residue 48 seems to result in less protein degradation (Jacobson et al., J. Biol. Chem. 284, 35485-35494). Ubiquitination is a balanced process with deubiquitinating enzymes (DUBs) counteracting the ubiquitination. They recognize the C-terminal di-glycine motif of ubiquitin and release ubiquitin from labeled proteins.

[0005] Targeted toxins are an important tool in modern medicine. There exists a strong need to provide new, more potent, and more durable targeted toxins to enhance efficacy of various targeted therapeutic schemes. The present invention fulfills this and other related needs.

BRIEF SUMMARY OF THE INVENTION

[0006] The present invention provides chimeric molecules that are useful for delivering effector molecules to the cytosol of a target cell. The chimeric molecules have a cytosol targeting moiety that binds directly or indirectly to a cell and functions to deliver the chimeric molecule to the cytosol of the cell. The chimeric molecules also have an effector moiety that comprises a molecule that alters the metabolism or physiology of the target cell. The chimeric molecules further have a ubiquitin moiety that functions to release the effector moiety from the chimeric molecule into the cytosol of the target cell, thus increasing the stability and/or activity of the effector moiety. [0007] Thus, in one aspect, the invention provides a chimeric molecule comprising (1 ) a cytosol targeting moiety, (2) an effector moiety, and (3) a ubiquitin moiety located between the cytosol targeting moiety and the effector moiety, wherein the ubiquitin moiety is a wild-type ubiquitin or a modified ubiquitin. In some embodiments, the modified ubiquitin comprises an amino acid substitution at one or more lysine residues. One, two, three, four, five, six or seven lysine (Lys or K) residues in ubiquitin can be substituted with another amino acid. In one embodiment, the modified ubiquitin comprises an amino acid substitution at all lysine residues except the lysine at residue number 48. In another embodiment, the modified ubiquitin comprises an amino acid substitution at all lysine residues except the lysine at residue number 63. In some embodiments, the modified ubiquitin comprises an amino acid substitution at all seven lysine residues. In some embodiments, the one or more lysine residues are substituted with arginine (Arg or R) residues. [0008] In some embodiments, the cytosol targeting moiety comprises anthrax toxin lethal factor from Bacillus anthracis. In some embodiments, the cytosol targeting moiety comprises the N-terminal 254 amino acids of anthrax toxin lethal factor (LFn).

[0009] In some embodiments, the effector moiety comprises a toxin. In some embodiments, the toxin comprises the catalytic domain of Pseudomonas exotoxin A (PEIII). In one embodiment, the toxin comprises the effector domain of cytolethal distending toxin (CdtB). In one embodiment, the toxin comprises saporin. In some embodiments, the toxin is a cytostatic agent or mitotic toxin.

[0010] In some embodiments, the effector moiety comprises a peptide nucleic acid (PNA). In - one embodiment, the effector moiety comprises an RNAse.

[0011] In some embodiments, the chimeric molecule comprises LFn, ubiquitin having an amino acid substitution at a lysine residue, and PEIII. In some embodiments, the chimeric molecule is a fusion polypeptide or fusion protein.

[0012] In one embodiment, the chimeric molecule further comprises an amino acid sequence recognized by an HIV protease. The HIV protease recognition sequence can be located between the ubiquitin moiety and the effector moiety.

[0013] In another aspect, the invention provides a polynucleotide sequence that encodes a chimeric molecule of the invention. Thus, in some embodiments, the polynucleotide sequence encodes a fusion protein having a cytosol-targeting moiety, a ubiquitin moiety, and an effector moiety. In some embodiments, an expression cassette is provided that comprises the polynucleotide sequence encoding a chimeric molecule of the invention. In some embodiments, a host cell containing the expression cassette is provided.

[0014] In some embodiments, a composition comprising a chimeric molecule described herein is provided. In one embodiment, the composition further comprises a physiologically acceptable excipient.

[0015] In another aspect, the invention also provides methods for recombinantly producing a chimeric molecule described herein. In some embodiments, the method comprises culturing a host cell comprising an expression cassette under conditions that are suitable for expressing the chimeric molecule, and isolating the chimeric molecule. [0016] In some embodiments, the invention provides a method for delivering an effector moiety to a target cell, the method comprising contacting the target cell with a chimeric molecule described herein.

[0017] In some aspects, the invention provides a method for improving the efficacy of a conjugate intended for cytosol delivery to a target cell, wherein the conjugate comprises a cytosol-targeting moiety and an effector moiety. In some embodiments, the method comprises inserting a ubiquitin moiety between the cytosol-targeting moiety and the effector moiety, the ubiquitin moiety being a wild-type ubiquitin or modified ubiquitin that comprises an amino acid substitution at a lysine residue. [0018] Also provided are methods for killing target cells. In some embodiments, a method for killing tumor cells is provided, the method comprising contacting a tumor cell directly or indirectly with a chimeric molecule of the invention. For example, in some embodiments, the method comprises contacting a tumor cell with a chimeric molecule having a cytosol-targeting moiety, a ubiquitin moiety and an effector moiety, where the effector moiety is a toxin or antineoplastic agent that kills the tumor cell. In some embodiments, the effector moiety is a cytostatic agent or mitotic toxin that inhibits cell division of a tumor cell. In one embodiment, the cytostatic agent is auristatin. In some embodiments, the cytosol-targeting moiety binds to PA or a modified PA. Thus, in some embodiments, the method further comprises contacting the tumor cell with PA or a modified PA that is capable of being cleaved by a tumor-specific protease, thereby providing tumor cell specificity. In some embodiments, the tumor cell is contacted with a modified PA that is capable of being cleaved and activated by a tumor specific protease, and the tumor cell is contacted with a chimeric molecule having a cytosol-targeting moiety that binds PA.

[0019] In other embodiments, a method for killing cells infected with HIV is provided, the method comprising directly or indirectly contacting an HIV-infected cell with a chimeric molecule described herein. For example, in some embodiments, the HIV-infected cell is contacted with a chimeric molecule having a cytosol-targeting moiety, a ubiquitin moiety and an effector moiety, where the effector moiety is a toxin or other agent that kills the cell. In some embodiments, the cytosol-targeting moiety is capable of directly binding a cell-surface receptor or protein that is expressed on an HIV-infected cell. In some embodiments, the cytosol-targeting moiety binds to PA or a modified PA. Thus, in some embodiments, the method further comprises contacting the HIV-infected cell with a modified PA that is capable of binding a cell- surface receptor or protein that is expressed on an HIV-infected cell. In some embodiments, the modified PA comprises the translocation domain and LF binding domain of native PA and a ligand domain that specifically binds to a protein expressed on the surface of an HIV-infected cell. In one embodiment, the C-terminal domain of PA is replaced with the extracellular domains of CD4 that are capable of binding to viral gpl20 envelope proteins displayed on HIV- infected cells. In some embodiments, the chimeric molecule further comprises a sequence recognized by an HIV protease. In some embodiments, the method comprises contacting an HIV infected cell with a chimeric molecule comprising an HIV viral protein.

DEFINITIONS

[0020] The term "cytosol-targeting moiety" or "cytosol-targeting domain"refers to the portion of a chimeric molecule that directly or indirectly targets the molecule to a cell of interest and facilitates the transportation of the molecule into the cytosolic portion of the target cell. A cytosol-targeting moiety typically has the ability to specifically bind to a molecule present on the target cell surface or can bind a second molecule that specifically binds a molecule present on the cell surface and subsequently participates in an internalization process of the chimeric molecule for relocation to cytosol. A cytosol-targeting moiety may be an antibody or a ligand for a cell surface receptor, or it may be a molecule that binds such antibody or ligand, thereby indirectly targeting to the surface of a cell of choice. One example of such a cytosol-targeting moiety is the N-terminal section (254 amino acids) of anthrax toxin lethal factor (LFn) from Bacillus anthracis.

[0021] The term "effector moiety" or "effector domain" refers to the portion of a chimeric molecule that carries out the purpose of the chimeric molecule, e.g., for a therapeutic or diagnostic purpose. For example, the effector moiety can be any molecule that perturbs cellular metabolism in a way that may be beneficial to a subject, such as an animal or human subject. For instance, an effector moiety may be a therapeutic agent that imparts cytotoxicity against a target cell or a label that provides detectability. Suitable therapeutic agents include such compounds as nucleic acids, proteins, peptides, amino acids or derivatives, glycoproteins, radioisotopes, lipids, carbohydrates, low molecular mass organic compounds, or recombinant viruses. Nucleic acid therapeutic and diagnostic moieties include antisense nucleic acids, derivatized oligonucleotides for covalent cross-linking with single or duplex DNA, and triplex forming oligonucleotides. In one embodiment, the effector moiety is a peptide nucleic acid (PNA). One of skill in the art will appreciate that therapeutic agents may include various antineoplastic agents, cytotoxins, such as native or modified Pseudomonas exotoxin A or diphtheria toxin, and radioactive agents such as ¹²⁵1, ³ P, ¹⁴C, ³H and ⁵S. A therapeutic agent may be an encapsulation system, such as a liposome or micelle that contains a therapeutic composition. In some embodiments, the effector moiety is a cytotoxin. A number of plant and bacterial toxins are known and can be used for this purpose, such as the bacterial toxin known as Pseudomonas exotoxin A ("PE") or an active portion thereof. Other possible cytotoxins include diphtheria toxin ("DT"), cholix toxin ("CT"), cholera exotoxin ("CET"), shiga toxin, ricin toxin, and pokeweed antiviral protein (PAP) toxin. In one particular embodiment, the effector moiety is the catalytic domain of Pseudomonas exotoxin A (PEIII). In one embodiment, the effector moiety is cytolethal distending toxin B from Haemophilus ducreyi (CdtB). In one embodiment, the effector moiety is a mitotic toxin such as auristatin. In one embodiment, the effector moiety is an RNAse. In some embodiments, the effector moiety is a fluorophore that can be used for imaging target cells.

[0022] Native Pseudomonas exotoxin A ("PE") is an extremely active monomeric protein (molecular weight 66 kD), secreted by Pseudomonas aeruginosa. PE inhibits protein synthesis in eukaryotic cells. The native 613-amino acid sequence of PE is provided in U.S. Patent No. 5,602,095. The mechanism of action is inactivation of the ADP-ribosylation and inactivation of eukaryotic elongation factor 2 (EF-2). The exotoxin contains three structural domains that act in concert to cause cytotoxicity. Domain la (amino acids 1 -252) mediates cell binding. Domain II (amino acids 253-364) is responsible for translocation into the cytosol and domain III (amino acids 400-613) mediates ADP ribosylation of EF-2. The function of domain lb (amino acids 365-399) remains undefined, although a large part of it, amino acids 365-380, can be deleted without loss of cytotoxicity. See Siegall, et al, J. Biol. Chem. 264: 14256- 14261 (1989).

[0023] "Ubiquitin" ("Ub") is a small protein that exists in all eukaryotic cells. The human ubiquitin protein consists of 76 amino acids and has a molecular mass of about 8.5 kDa. Key features of ubiquitin include its C-terminal tail (which is cleaved by cytosolic deubiquitinating enzymes or DUBs) and the 7 lysine residues:

MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNI QKESTLHLVLRLRGG (SEQ ID NO: l) [0024] As used herein, "polypeptide", "peptide" and "protein" are used interchangeably and include reference to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms also apply to polymers containing conservative amino acid substitutions such that the protein remains functional.

[0025] The term "conjugating," "joining," "bonding," or "linking" refers to connecting two or more molecules to form a chimeric molecule, e.g., joining three polypeptides (such as the N- terminal 254 amino acids of anthrax toxin lethal factor (LFn) from Bacillus anthracis as a cytosol-targeting moiety, the catalytic domain of Pseudomonas exotoxin A (PEIII) as an effector moiety is, and a modified ubiquitin with at least one or all lysines replaced by arginines as a ubiquitin moiety) from separate origins into one contiguous fusion polypeptide molecule. The connection can be either by chemical or recombinant means. Chemical means refers to carrying out a chemical reaction between two moieties such that there is a covalent bond formed between the two components. Examples of chemical reactions for conjugating two molecules include reactions involving maleimides or iodoacetyl groups. Examples of linkers for conjugating two polypeptide molecules include peptide bonds and disulfide bonds. Peptide linkers can also be included in recombinant nucleic acid constructs that encode fusion polypeptides such that the peptide linker is translated and incorporated into the fusion polypeptide.

[0026] As used herein, "recombinant" includes reference to a protein produced using cells that do not have, in their native state, an endogenous copy of the D A encoding the protein. The cells produce the recombinant protein after they have been genetically altered by the introduction of the appropriate isolated nucleic acid sequence. The term also includes reference to a cell, or nucleic acid, or vector, that has been modified by the introduction of a heterologous nucleic acid or the alteration of a native nucleic acid to a form not native to that cell, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell, express mutants of genes that are found within the native form, or express native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

[0027] As used herein, "nucleic acid" or "polynucleotide sequence" includes reference to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence includes the complementary sequence thereof as well as conservative variants, i.e., nucleic acids present in wobble positions of codons and variants that, when translated into a protein, result in a conservative substitution of an amino acid.

[0028] As used herein, "encoding" with respect to a specified nucleic acid, includes reference to nucleic acids that comprise the information for translation into the specified protein. The information is specified by the use of codons. Typically, the amino acid sequence is encoded by the nucleic acid using the "universal" genetic code. However, variants of the universal code, such as is present in some plant, animal, and fungal mitochondria, the bacterium Mycoplasma capricolum (Proc. Nat 'l Acad. Sci. USA 82:2306-2309 (1985), or the ciliate Macronucleus, may be used when the nucleic acid is expressed in using the translational machinery of these organisms.

[0029] The phrase "fusing in frame" refers to joining two or more polynucleotide sequences that encode polypeptides so that the joined nucleic acid sequence translates into a single chain polypeptide comprising the original polypeptides fused together. For example, the individual polynucleotide sequences encoding the N-terminal 254 amino acids of anthrax toxin lethal factor (LFn) from Bacillus anthracis as a cytosol-targeting moiety, the catalytic domain of

Pseudomonas exotoxin A (PEIII) as an effector moiety is, and a ubiquitin moiety that is a modified ubiquitin with at least one or all lysines replaced by arginines can be fused in frame to form a longer coding sequence for a chimeric molecule of this invention, which is a fusion polypeptide in this instance.

[0030] An "expression cassette" is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular polynucleotide sequence in a host cell. An expression cassette may be part of a plasmid, viral genome, or nucleic acid fragment. Typically, an expression cassette includes a polynucleotide to be transcribed, operably linked to a promoter. Other elements that may be present in an expression cassette include those that enhance transcription {e.g., enhancers) and terminate transcription (e.g., terminators), as well as those that confer certain binding affinity or antigenicity to the recombinant protein produced from the expression cassette. [0031] As used herein, the term "expression" includes reference to transcription of a nucleic acid sequence into RNA or translation of an encoding polynucleotide sequence into a polypeptide. Proteins may be expressed and remain intracellular, become a component of the cell surface membrane or be secreted into the extracellular matrix or medium.

[0032] A "host cell" is a cell that can support the replication or expression of the expression vector. Host cells may be prokaryotic cells, such as E. coli, or eukaryotic cells, such as yeast, insect, amphibian, or mammalian cells.

[0033] A "physiologically acceptable carrier" typically does not have any therapeutic effects per se but may contain a physiologically acceptable compound that acts, for example, to stabilize the antibody or chimeric molecule of the invention. A physiologically acceptable carrier or excipient can include, for example, carbohydrates (such as glucose, sucrose or dextrans), antioxidants (such as ascorbic acid or glutathione), chelating agents, low molecular weight proteins or other stabilizers or excipients. Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives, which are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. Examples of carriers, stabilizers or adjuvants can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985).

[0034] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g. , hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. "Amino acid mimetics" refers to chemical compounds having a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. [0035] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, "conservatively modified variants" refers to those nucleic acids that encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

[0036] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.

[0037] The following eight groups each contain amino acids that are conservative substitutions for one another:

1 ) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins, W. H. Freeman and Co., N. Y. (1984)).

[0038] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical

Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0039] In the present application, amino acid residues are numbered according to their relative positions from the left most residue, which is numbered 1, in an unmodified wild-type polypeptide sequence.

[0040] As used in herein, the terms "identical" or percent "identity," in the context of describing two or more polynucleotide or amino acid sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same. For example, a protein sequence can have at least 80%, 85%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, to a reference sequence, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. With regard to polynucleotide sequences, this definition also refers to the complement of a test sequence. Preferably, the identity exists over a region that is at least about 50 amino acids or nucleotides in length, or more preferably over a region that is 75-100 amino acids or nucleotides in length.

[0041] The phrase "substantially identical," used in the context of two nucleic acids or polypeptides, refers to a sequence that has at least 60% sequence identity with a reference sequence. Alternatively, percent identity can be any integer from 60% to 100%. Some embodiments include at least: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94% 95%, 96%, 91%, 98%, or 99%, compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described below.

[0042] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. For sequence comparison of nucleic acids and proteins, the BLAST and BLAST 2.0 algorithms and the default parameters discussed below are used.

[0043] A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well- known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et ah, eds. 1995 supplement)).

[0044] Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul e/ a/., (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available at the National Center for Biotechnology Information website, ncbi.nlm.nih.gov. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits acts as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=l, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0045] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karl in & A ltschul, iVoc. Nai l Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01 , and most preferably less than about 0.001.

[0046] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1. Domains structures and sequences of ubiquitin fusion proteins. Six different TTs were used for the study on the effects of ubiquitin. (A) LFn-PEIII (FP59) is the basic TT with PA-binding LFn domain fused to the catalytic domain PEIII. All other TTs contain ubiquitin between these two domains, with either an uncleavable alanine sequence (UbuN) or with several or all of the lysines of ubiquitin mutated to arginines (Ub_K48, Ub_K63, and Ubidiuii)- All the ubiquitin mutants have the cleavable di-glycine motif. (B) The amino acid sequences of the variable portions of the proteins are shown (SEQ ID NOS:6-l 1). All PEIII domains end in the native PE sequence REDLK (SEQ ID NO: 15) (not shown). All constructs contained an N- terminal HMAGG (SEQ ID NO: 16) sequence. [0048] Figure 2. Analysis of purified TTs. (A) All TTs were analyzed on a Tris-glycine polyacrylamide gel by Coomassie staining. Each lane contained 1 μg of protein. (B) The enzymatic activity of PEIII within all TTs was studied by in vitro ADP-ribosylation. Each TT (20 ng) was incubated with eEF2 and biotin-labeled NAD⁺ and the ADP-ribosylated eEF2 was detected after western blotting by streptavidin detection. [0049] Figure 3. Deubiquitinating enzyme (DUB) cleavage of TTs. (A-C) Each TT (150 ng) was incubated with HN6 cell lysate for 0, 10 and 120 min. Additional samples were incubated for 120 min in lysates that were preincubated with the DUB inhibitor ubiquitin aldehyde.

Samples were analyzed by anti-LF and anti-PE immunodetection following Western blotting.

[0050] Figure 4. DUB cleavage of TTs in cells. (A-C) Each TT (0.25 μg/ml) was incubated in the presence of 1 μg/ml PA on CHO TEM8 T4 cells for 0 h and 1 h. Additional samples were preincubated for 1 h with 50 μΜ PYR-41 or incubated with PAAFF instead of PA. 35 ng of Ub, Ub_K48, and Ub_Knuii were loaded as controls. Cytosolic fractions were isolated and analyzed by simultaneous anti-LF and anti-PE immunodetection following Western blotting.

[0051] Figure 5. Cytotoxicity of TTs on HN6 human head and neck cancer cells. HN6 cells (10,000/well) were exposed to different concentrations of TTs for 48 h (A) or 2 h (B). All samples contained a fixed concentration of 250 ng/ml PA and varying TT concentrations. Viable cells were quantitated in an MTT assay. Relative survival was calculated as the percentage of living cells after treatment in relation to untreated cells. Error bars indicate S.E.M. of 6 (A) and 4 (B) independent experiments performed in triplicate. [0052] Figure 6. Cytotoxicity of TTs on RAW264.7 cells in the presence of the El ubiquitin- activating enzyme inhibitor PYR-41. RAW264.7 cells (15,000 cells/well) were pre-incubated with or without 21 μΜ PYR-41 for 1 h and further exposed to the TTs for 1 8 h. TTs were added to the cells in varying concentrations with a fixed concentration of 250 ng/ml PA. Relative survival was calculated as in Figure 5. Error bars indicate S.E.M. of two independent experiments performed in triplicate. Each panel presents the relative survival data for the indicated TT. (A) TT with wild-type Ub; (B) TT with uncleavable Ub (Ubu_N); (C) TT with all lysines except lysine 48 substituted with arginine (ΙΛ> 48); (D) TT with all lysines except lysine 63 substituted with arginine (ΙΛκ₄₆₃); (E) TT with all lysines substituted with arginine (Ub_Knuii); (F) FP59. [0053] Fig. 7A. Cytotoxicity of CdtB, LFn-CdtB, LFn-Ub-CdtB, and LFn-Ub_Knuii-CdtB. The toxins were incubated in the presence of 0.25 μg/ml PA on RAW267.4 (murine macrophage cell line) for 72 h and cell survival was measured in a MTT assay. Relative cell survival was determined in comparison to untreated cells. CdtB shows no cytotoxicity and LFn-Ub-CdtB shows only moderate cytotoxicity at high concentrations. LFn-CdtB is most toxic with a 50% survival concentration (SI₅₀) in the low pM range. LFn-UbKnuii-CdtB is about l Ofold less cytotoxic. The result demonstrates the improved stability of mutated ubiquitin in comparison to fast degraded wildtype ubiquitin.

[0054] Fig. 7B. Cytotoxicity of CdtB, LFn-CdtB, LFn-Ub-CdtB, and LFn-Ub_Knuii-CdtB. The toxins were incubated in the presence of 0.25 μg/ml PA on HN6 cells (human head and neck cancer cell line) for 72 h. This cell line showed higher resistance to LFn-CdtB and LFn-Ubi nuii- CdtB. SI50 values are around 100 pM and 10 nM, respectively.

[0055] Fig. 7C. Cytotoxicity of CdtB, LFn-CdtB, LFn-Ub-CdtB, and

The toxins were incubated in the presence of 0.25 μg/ml PA on HeLa cells (human cervical carcinoma cell line) for 72 h. This cell line was more succeptible to LFn-Ub-CdtB than the previously tested cells. LFn-CdtB and LFn-UbKnuii-CdtB are nevertheless much more cytotoxic with SI50 values of approximately 10 pM and 100 pM, respectively.

[0056] Fig. 7D. Cytotoxicity of CdtB, LFn-CdtB, LFn-Ub-CdtB, and LFn-Ub_Knuii-CdtB. The toxins were incubated in the presence of 0.25 μg/ml PA on CHO WTP4 cells (Chinese hamster ovary cell line) for 72 h. This cell line showed higher succeptibility for LFn-CdtB and LFn- UbKnuii-CdtB. SI50 values are approximately 1 pM and 10 pM, respectively. LFn-Ub-CdtB shows some initial growth inhibitory effect but fails to kill the cells even at very high concentrations. DETAILED DESCRIPTION OF THE INVENTION

CHIMERIC MOLECULES

[0057] The present invention is directed to chimeric molecules that comprise a ubiquitin moiety and an effector moiety. The effector moiety can be a molecule that alters the normal physiology, metabolism, or functions of the target cell. For example, in some embodiments, the effector molecule is a toxin or a functional domain of a toxin that can be used to kill the target cell. In some embodiments, the effector moiety is a polypeptide or protein. In some embodiments, the effector moiety is a nucleic acid.

[0058] The invention also provides chimeric molecules that comprise a ubiquitin moiety and a cytosol targeting moiety. The cytosol targeting moiety can be a molecule that functions to allow access of the chimeric molecules described herein to the cytosol or cytoplasm of a cell. The cytosol targeting moiety can bind directly to the target cell, or it can bind to another molecule that directly binds a target cell. In some embodiments, the cytosol targeting moiety is a polypeptide or protein. In some embodiments, the cytosol targeting moiety is derived from anthrax toxin lethal factor. In one embodiment, the cytosol targeting moiety comprises the N- terminal 254 amino acids of anthrax toxin lethal factor (LFn). In one embodiment, the cytosol targeting moiety comprises an amino acid sequence that is substantially identical to the N- terminal 254 amino acids of anthrax toxin lethal factor.

[0059] The invention also provides chimeric molecules that comprise a ubiquitin moiety, an effector moiety, and a cytosol targeting moiety. The combination of the three moieties provides chimeric molecules that can deliver an effector moiety to the cytosol of a target cell.

Representative examples of the different moieties are described in more detail below.

I. Effector Moieties

[0060] The chimeric molecules described herein comprise an effector moiety that alters the normal cellular physiology or function of the target cell. In some embodiments, the effector moiety alters the cellular metabolism of a target cell to the benefit of the subject. For example, the effector moiety can be a therapeutic agent. In some embodiments, the therapeutic agent is a molecule that kills a target cell, such as a cancer cell or a cell infected with a pathogen, e.g., a virus. Suitable therapeutic agents include compounds of virtually any chemical nature such as nucleic acids (including antisense nucleic acids), proteins, peptides, antigens, amino acids or derivatives, amino acids or derivatives, glycoproteins, radioisotopes, lipids, carbohydrates, low molecular mass organic compounds, toxins, or recombinant viruses. One of skill in the art can select the effector moiety based on its desired functional characteristics.

[0061] In some embodiments, the effector moiety comprises a toxin or a functional fragment of a toxin. Any toxin that disrupts the normal physiology or function of a cell once the toxin is in the cytosol can be used. For example, any toxin that causes cell death when the toxin is present in the cytosol can be used. Thus, in one embodiment, the toxin comprises the C-terminal 216 amino acid catalytic domain of Pseudomonas exotoxin A (PEIII) (SEQ ID NO: 13). In some embodiments, the effector moiety is substantially identical to SEQ ID NO: 13. In some embodiments, the toxin comprises the effector domain of cytolethal distending toxin (CdtB) (Guerra et al., Toxins (Basel), 3(3): 172 (201 1)). Thus, in some embodiments, the effector moiety is substantially identical to SEQ ID NO:22. In another embodiment, the toxin comprises saporin (Maras, Biochem Int., 21(5):831 (1990)).

[0062] In some embodiments, the effector moiety comprises a peptide nucleic acid (PNA). PNAs are well known in the art. They typically comprise a nucleic acid mimic that can function as an antisense nucleic acid tool for altering gene expression, pre-mRNA splicing, etc. The development of PNAs as reagents and therapeutics has been limited by the difficulty of delivering or introducing them into the cell. A reactive cysteine residue containing a SH group can be used to conjugate the PNA to the ubiquitin moiety. Thus, in some embodiments, the chimeric molecule comprises a ubiquitin moiety and a reactive cysteine residue conjugated to PNA. It will be understood that the reactive cysteine residue can be conjugated to other effector molecules that are desired to be delivered into cells. Thus, in some embodiments, the reactive cysteine residue is conjugated to small molecule toxins. In some embodiments, the small molecule toxin is a mitotic toxin or cytostatic agent that is an effective anti-tumor agent. In one embodiment, the small molecule toxin is an antimitotic agent that blocks the polymerization of tubulin, such as auristatin.

[0063] In some embodiments, the effector moiety is an RNAse. Examples of RNAses are well known in the art, and include endoribonucleases and exoribonucleases. The RNAse can be cytotoxic or can be useful for degrading RNA viruses such as HIV. In one embodiment, the RNAse is angiogenin. [0064] In some embodiments, the effector moiety comprises a fluorophore. The fluorophore can be conjugated to ubiquitin using a reactive cysteine linker. The use of a fluorophore allows the delivery of a molecule into the cytosol to be monitored by fluorescence imaging, and thus allows the effectiveness of the chimeric molecules described herein to be monitored. For example, in combination with tumor-specific PA molecules that are activated by tumor-specific proteases, a chimeric molecule comprising a fluorophore effector moiety can be used as a diagnostic tool. The fluorophore is expected to be released in the cytosol, which enriches the signal and can increase sensitivity. The fluorophore can be used as a diagnostic marker for chimeric molecules that target other cell types as well.

II. Ubiquitin Moieties

[0065] The ubiquitin moiety of the chimeric molecule of this invention can be a wild-type ubiquitin or a modified ubiquitin. Wild-type ubiquitin (amino acid sequence provided in SEQ ID NO: l ) is involved in protein recycling and protein degradation. Ubiquitin itself can be ubiquitinated at one or more of the seven lysine residues in the protein, which targets ubiquitin for degradation in the proteasome. Thus, conjugation of a wild-type ubiquitin to a protein of interest to can result in the degradation of the protein of interest in the cell. [0066] On the other hand, ubiquitin can be modified such that it is not as easily ubiquitinated, which results in the protein of interest conjugated to the modified ubiquitin being protected from degradation. Thus, in some embodiments, the chimeric molecule comprises a modified ubiquitin that is not efficiently ubiquitinated. In some embodiments, the modified ubiquitin comprises an amino acid substitution at a lysine residue. In some embodiments, the modified ubiquitin has at least one lysine residue substituted with another amino acid. In some embodiments, the modified ubiquitin has one, two, three, four, five, six, or seven lysine residue substituted with another amino acid, which may be either identical or different for all substituted lysine residues. In one embodiment, the modified ubiquitin has all lysine residues except the lysine at residue 48 substituted with another amino acid. In one embodiment, all lysine residues except the lysine residue at position 48 are substituted with arginine (SEQ ID NO:2) (referred to as Ub_K48). In one embodiment, the modified ubiquitin has all lysine residues except the lysine at residue 63 substituted with another amino acid. In one embodiment, all lysine residues except the lysine residue at position 63 are substituted with arginine (SEQ ID NO:3) (referred to as Ub^)- In some embodiments, the modified ubiquitin has all seven lysine residues substituted with another amino acid. In one embodiment, all seven lysine residues are substituted with arginine (SEQ ID NO:4) (referred to as Ubj nuii)- Examples of fusion proteins comprising ubiquitin or modified ubiquitin are shown in Figure 1.

[0067] In some embodiments, the ubiquitin comprises an amino acid sequence that is substantially identical to SEQ ID NOS: 1, 2, 3, or 4, i.e., having an amino acid sequence that is at least 80%, 85%, 90%, 95% or greater identity to SEQ ID NOS: 1 , 2, 3, or 4. In one example, at least one, two, three, four, five, six, and up to seven of the lysine residues in SEQ ID NO: l are replaced by another amino acid while a certain percentage overall sequence identity to SEQ ID NO: l is retained.

[0068] The amino acid sequence of ubiquitin comprises a di-glycine motif at the C-terminus. In the chimeric molecule of this invention, the ubiquitin moiety is typically conjugated at its C- terminal end to the epsilon amine of a lysine located at the N-terminal end of the effector moiety. Once in the target cell cytosol, the ubiquitin moiety is cleaved at the di-glycine motif by cytosolic deubiquitinating enzymes or DUBs, which releases the ubiquitin moiety from the effector moiety that is conjugated to the C-terminal end of ubiquitin. Once cleaved by DUBs, the effector moiety is released into the cytosol, where it is more stable due to lack of

ubiquitination. In some embodiments, the effector moiety is a protein that is resistant to ubiquitination, thereby increasing the stability or half-life of the effector moiety in the cytosol. This increased stability allows the effector moiety to alter the metabolism or physiology of the cell. Thus, the present invention provides chimeric molecules with increased activity by increasing the stability of the effector moiety in the cytosol of the target cell. In some embodiments, the effector moiety is a protein with a relatively low lysine content.

[0069] Mutation of the di-glycine motif of ubiquitin can prevent cleavage by DUBs, resulting in altered toxicity of the effector moiety. While not being bound by theory, it is believed that removal of the DUB cleavage motif prevents the release of the C-terminal effector moiety and results in rapid ubiquitination and proteosomal degradation of the chimeric molecule. In one embodiment, the C-terminal glycines (Gly or G) of ubiquitin are mutated to alanines (Ala or A) (SEQ ID NO: 5) (referred to as UbuN), which prevents efficient cleavage of the effector moiety from the chimeric molecule.

[0070] In some embodiments, the ubiquitin molecule comprises additional mutations that serve to destabilize ubiquitin and facilitate unfolding in the endosome to improve delivery of the chimeric molecule to the cytosol. In some embodiments, the mutations include F4H, T12H, E24H, A28H, I30A, F45W, L50E, L56E, L67A, H68N (described in Ralat et al., J. Mol. Biol. 406:454-466, 2011), E34G (see Ermolenko et al., J. Mol. Biol. 322, 123-135, 2002), V5A, and/or V17A (see Loladze et al., J. Mol. Biol. 320:343-357, 2002), either alone or in various

combinations. Other mutations can be introduced to destabilize ubiquitin and improve delivery of the chimeric molecule to the cytosol, and are considered within the scope of this invention.

III. Cytosol Targeting Moieties

[0071] The chimeric molecules of the invention can also comprise a moiety that targets the chimeric molecule to the cytosol of a cell. Any molecule that can bind to a cell and mediate delivery of the chimeric molecule into the cytosol of the cell can be used. In some embodiments, the cytosol targeting moiety is a protein or polypeptide. Thus, in some embodiments, the chimeric molecule comprises a second protein of interest that targets the chimeric molecule to the cytosol of a cell. In some embodiments, the cytosol targeting moiety or protein is attached to the N-terminus of ubiquitin.

[0072] In some embodiments, the cytosol targeting moiety binds to endogenous cellular receptors, which mediate internalization or endocytosis into a target cell. In some embodiments, the cytosol targeting moiety binds to another molecule that in turn binds to a receptor on the target cell. In some embodiments, the cytosol targeting moiety is derived from anthrax toxin, as described below.

A. Anthrax Toxin Lethal Factor system [0073] Anthrax toxin is a three-part toxin secreted by Bacillus anthracis consisting of protective antigen (PA, 83 kDa), lethal factor (LF, 90 kDa) and edema factor (EF, 89 kDa) (Smith et al., J. Gen. Microbiol, 29:517-521 (1962); Leppla, Sourcebook of Bacterial Protein Toxins, p. 277-302 (1991); Leppla, Handb. Nat. Toxins, 8:543-572 (1995)), which are individually non-toxic. The mechanism by which individual toxin components interact to cause toxicity was recently reviewed (Leppla, Handb. Nat. Toxins, 8:543-572 (1995)). Protective antigen, recognized as the central, receptor-binding component, binds to cellular receptors (Escuyer et al, Infect. Immun., 59:3381 -3386 (1991)). There are two cellular receptors for PA: capillary morphogenesis protein 2 (CMG2) and tumor endothelium marker-8 (TEM8). PA is cleaved at the sequence RKKR₁₆₇ (SEQ ID NO: 17) by cell-surface furin or furin-like proteases (Klimpel et al, Proc. Natl. Acad. Sci. USA, 89:10277-10281 (1992); Molloy et al, J. B. Chem., 267:16396-16402 (1992)) into two fragments: PA63, a 63 kDa C-terminal fragment, which remains receptor-bound; and PA20, a 20 kDa N-terminal fragment, which is released into the medium (Klimpel et al, Mol. Microbiol, 13: 1094-1 100 (1994)). Dissociation of PA20 allows PA63 to form a heptamer (Milne et al, J. Biol. Chem., 269:20607-20612 (1994); Benson et al, Biochemistry, 37:3941-3948 (1998)) and also bind LF or EF (Leppla et al, Bacterial Protein Toxins, p.11 1-1 12 (1988)). The resulting hetero-oligomeric complex is internalized by endocytosis (Gordon et al, Infect. Immun., 56: 1066-1069 (1988)), and acidification of the vesicle causes insertion of the PA63 heptamer into the endosomal membrane to produce a channel through which LF or EF translocate to the cytosol (Friedlander, J. Biol. Chem.,

261 :7123-7126 (1986)), where LF and EF induce cytotoxic events.

[0074] Thus, the combination of PA+LF, named anthrax lethal toxin, kills animals and certain cultured cells, due to intracellular delivery and action of LF, a zinc-dependent metalloprotease that is known to cleave nearly all mitogen-activated protein kinase kinases (1 , 2, 3, 5, 6, and 7) (Tonello and Montecucoo Mol. Aspects Med., 2009, 30(6):431 -8). The combination of PA+EF, named edema toxin, disables phagocytes and probably other cells, due to the intracellular adenylate cyclase activity of EF (Leppla, Proc. Natl Acad. Sci. USA., 79:3162-3166 (1982)).

[0075] LF and EF have substantial sequence homology in amino acids 1 -250 (Leppla, Handb. Nat. Toxins, 8:543-572 (1995)), and a mutagenesis study showed this region constitutes the PA- binding domain (Quinn et al, J. Biol. Chem., 166:20124-20130 (1991)). Systematic deletion of LF fusion proteins containing the catalytic domain of Pseudomonas exotoxin A established that LF aa 1 -254 (LFn) are sufficient to achieve translocation of "passenger" polypeptides to the cytosol of cells in a PA-dependent process (Arora et al, J. Biol Chem., 267: 15542-15548 (1992); Arora et al, J. Biol Chem., 268:3334-3341 (1993)). A highly cytotoxic LFn fusion to the ADP-ribosylation domain of Pseudomonas exotoxin A, named FP59, has been developed (Arora et al, J. Biol. Chem., 268:3334-3341 (1993)). When combined with PA, FP59 kills any cell type that contains receptors for PA by the mechanism of inhibition of protein synthesis through ADP ribosylating inactivation of elongation factor 2 (EF-2), whereas native LF is highly specific for macrophages (Leppla, Handb. Nat. Toxins, 8:543-572 (1995)). For this reason, FP59 is an example of a potent therapeutic agent when specifically delivered to the target cells with a target-specific PA.

[0076] Anthrax toxin proteins constitute a highly efficient system for delivering cytotoxic enzymes to the cytosol of tumor cells. However, the ultimate anti-tumor efficacy of these agents depends on their resistance to inactivation and degradation within the cells. It is evident from the low lysine to arginine ratios in many bacterial toxins that avoidance of ubiquitination is a key factor controlling their potency. Thus, in some embodiments, the present invention provides fusion proteins containing ubiquitin or modified ubiquitins having C-terminal fusions to the catalytic domain of Pseudomonas Exotoxin A (PEIII). The potency of these proteins was highly dependent on the number of lysines retained with the ubiquitin domain, and on whether they retained the C-terminal ubiquitin sequence cleaved by cytosolic deubiquitinating enzymes (DUBs). In particular, fusions in which all seven native lysines of ubiquitin were retained whereas the DUB site was removed were non-toxic, apparently due to rapid ubiquitination and proteasomal degradation. In contrast, fusions in which all lysines of ubiquitin were substituted by arginine and the DUB site was retained had high potency, exceeding that of a simple fusion lacking ubiquitin. It appears that rapid cytosolic release of a cytotoxic enzyme {e.g. , PEIII) that is itself resistant to ubiquitination, is an effective strategy for enhancing the potency of tumor- targeting toxins.

[0077] Thus, in some embodiments, the cytosol targeting moiety comprises a polypeptide that is efficiently ubiquitinated and rapidly degraded, whereas the effector moiety comprises a polypeptide that is resistant to ubiquitination and thus has a longer half-life. In some

embodiments, the cytosol targeting moiety comprises LFn (SEQ ID NO: 12) or a sequence substantially identical to LFn. In some embodiments, the cytosol targeting moiety is

substantially identical to SEQ ID NO: 12.

B. Other Cytosol Targeting Moieties

[0078] In some embodiments, the cytosol targeting moiety can be a protein that binds to a cell surface receptor. For example, the cytosol targeting moiety can be an antibody or fragment thereof, a cytokine, or a growth factor. The introduction of a modified ubiquitin molecule that is resistant to proteasomal degradation between these cytosol targeting moieties and the effector moiety could result in release of the effector moiety in the cytosol and increase the potency or activity of the effector moiety. See, for example, Weng et al., Mol. Oncol. (2012) and Lyu et al., Methods Enzymol. 502: 167-214 (2012). IV. Construction of the Chimeric Molecules

A. General Recombinant Technology

[0079] Basic texts disclosing general methods and techniques in the field of recombinant genetics include Sambrook and Russell, Molecular Cloning, A Laboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Ausubel et al, eds., Current Protocols in Molecular Biology (1994).

[0080] For nucleic acids, sizes are given in either kilobases (kb) or base pairs (bp). These are estimates derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences. For proteins, sizes are given in kilodaltons (kDa) or amino acid residue numbers. Proteins sizes are estimated from gel electrophoresis, from sequenced proteins, from derived amino acid sequences, or from published protein sequences.

[0081] Oligonucleotides that are not commercially available can be chemically synthesized, e.g., according to the solid phase phosphoramidite triester method first described by Beaucage & Caruthers, Tetrahedron Lett. 22: 1859-1 862 (1981 ), using an automated synthesizer, as described in Van Devanter et. al, Nucleic Acids Res. 12: 6159-6168 (1984). Purification of oligonucleotides is performed using any art-recognized strategy, e.g., native acrylamide gel electrophoresis or anion-exchange HPLC as described in Pearson & Reanier, J. Chrom. 255: 137-149 (1983).

[0082] The chimeric molecules described herein can be produced from a recombinant nucleic acid molecule that encodes each domain of the chimeric molecule, along with any peptide linkers, resulting in production of an in-frame fusion protein when the nucleic acid molecule is translated. Thus, in some embodiments, the invention provides isolated recombinant nucleic acids that encode the fusion proteins described herein. In some embodiments, the invention provides isolated fusion proteins encoded by the recombinant nucleic acids. [0083] The construction of chimeric molecules described herein can also be accomplished using gene synthesis techniques. The use of gene synthesis techniques allows for the individual components of the chimeric molecule to be assembled in various combinations. For example, each domain can be synthesized with optimum codon bias, substitution of lysine residues to reduce polyubiquitination, and reduced immunogenicity. Nucleic acids produced by gene synthesis techniques can encode fusion proteins described herein. [0084] The recombinant or synthesized polynucleotides that encode the chimeric molecules described herein can be transfected into host cells in order to express the chimeric fusion proteins. Standard transfection methods are used to produce bacterial, mammalian, yeast, insect, or plant cell lines that express large quantities of a recombinant fusion protein of this invention, which are then purified using standard techniques {see, e.g., Colley et al., J. Biol. Chem. 264: 17619- 17622 (1989); Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed., 1990)). Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques {see, e.g., Morrison, J. Bact. 132: 349-351 (1977); Clark- Curtiss & Curtiss, Methods in Enzymology 101 : 347-362 (Wu et al., eds, 1983). [0085] Any of the well known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, liposomes, microinjection, plasma vectors, viral vectors and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA, or other foreign genetic material into a host cell {see, e.g. , Sambrook and Russell, supra).

B. Synthesis of Chimeric Molecules by Chemical Methods

[0086] In some embodiments, the chimeric molecule is a polypeptide and can be synthesized using conventional peptide synthesis or other protocols well known in the art. For example, peptides may be synthesized by solid-phase peptide synthesis methods using procedures similar to those described by Merrifield et al., J. Am. Chem. Soc, 85:2149-2156 ( 1963); Barany and Merrifield, Solid-Phase Peptide Synthesis, in The Peptides: Analysis, Synthesis, Biology Gross and Meienhofer (eds.), Academic Press, N.Y., vol. 2, pp. 3-284 ( 1980); and Stewart et al., Solid Phase Peptide Synthesis 2nd ed., Pierce Chem. Co., Rockford, 111. (1984). During synthesis, N- -protected amino acids having protected side chains are added stepwise to a growing polypeptide chain linked by its C-terminal and to a solid support, i.e. , polystyrene beads. The peptides are synthesized by linking an amino group of an N-a-deprotected amino acid to an ot- carboxy group of an N-a-protected amino acid that has been activated by reacting it with a reagent such as dicyclohexylcarbodiimide. The attachment of a free amino group to the activated carboxyl leads to peptide bond formation. The most commonly used N-a-protecting groups include Boc, which is acid labile, and Fmoc, which is base labile. [0087] Materials suitable for use as the solid support are well known to those of skill in the art and include, but are not limited to, the following: halomethyl resins, such as chloromethyl resin or bromomethyl resin; hydroxymethyl resins; phenol resins, such as 4-(a-[2,4-dimethoxyphenyl]- Fmoc-aminomethyl)phenoxy resin; tert-alkyloxycarbonyl-hydrazidated resins, and the like. Such resins are commercially available and their methods of preparation are known by those of ordinary skill in the art.

[0088] Briefly, the C-terminal N-a-protected amino acid is first attached to the solid support. The N-a-protecting group is then removed. The deprotected a-amino group is coupled to the activated a-carboxylate group of the next N-a-protected amino acid. The process is repeated until the desired peptide is synthesized. The resulting peptides are then cleaved from the insoluble polymer support and the amino acid side chains deprotected. Longer peptides can be derived by condensation of protected peptide fragments. Details of appropriate chemistries, resins, protecting groups, protected amino acids and reagents are well known in the art and so are not discussed in detail herein (See, Atherton et al., Solid Phase Peptide Synthesis: A Practical Approach, IRL Press (1989), and Bodanszky, Peptide Chemistry, A Practical Textbook, 2nd Ed., Springer- Verlag (1993)).

C. Chemical conjugation and Linkers

[0089] The cytosol targeting moiety, the ubiquitin moiety, and the effector moiety can be joined together using chemical reactions. The chemical reactions can involve any active group as long as it does not affect the biological activity of the molecule. Examples of suitable chemical reactions for conjugating two molecules include reactions involving maleimides or iodoacetyl groups.

[0090] Alternatively, when no appropriate chemical conjugation can be used, or chemical conjugation is not desired, a linker can be used. A linker can be attached to the N- or C-terminus of a peptide moiety. A linker can be a covalent bond such as a peptide bond or a disulfide bond. One skilled in the art will recognize that a variety of other linkers with appropriate functional groups such as carbon linkers {e.g., straight or branched-chain carbon linkers, heterocyclic carbon linkers) or polyether linkers may also be useful to practice the present invention. These linkers may be joined to a peptide's constituent amino acids through their side groups (for example, through a disulfide linkage to cysteine). The linkers may also be joined to the a-carbon amino or carboxyl groups of the peptide's terminal amino acids. [0091] In some embodiments, the linker comprises a reactive cysteine. In one embodiment, the chimeric molecule has a linker comprising a cysteine (Cys or C) positioned C-terminal of the di-glycine motif (GG) located at the C-terminal end of ubiquitin. In one embodiment, the linker comprises the amino acid sequence AGGGGSC (SEQ ID NO: 18) to provide a spacer between the ubiquitin and the effector moiety.

[0092] When peptides of the present invention are obtained through recombinant means, a nucleotide sequence encoding a peptide linker can be included during the subcloning process and the resulting recombinant polypeptide will thus already have a proper linker attached. When the peptides of the present invention are obtained through synthetic means, heterologous peptides serving as linkers may be included at the time of peptide synthesis.

V. Therapeutic Methods

A. Targeted Toxin approach

[0093] The present invention provides methods for targeting and killing tumor cells using a chimeric molecule referred to as a targeted toxin. The method comprises contacting a tumor cell with a chimeric molecule comprising ubiquitin, a toxin as the effector moiety and a cytosol- targeting moiety that binds to tumor cells. In some embodiments, the tumor cell is contacted with a chimeric molecule comprising the anthrax LF cytosol-targeting moiety. In these embodiments, the method further comprises contacting the tumor cell with PA or a modified PA. PA is activated by cell surface proteases, which cleave off the 20 kDa fragment (PA20) from the 83 kDa form, leaving the 63 kDa form (PA63) bound to the cell surface receptor. The protease- activated PA assembles into an oligomeric protein-conducting channel that efficiently delivers the anthrax toxin catalytic effector proteins to endosomes and then translocates them to the cytosol. Replacing the site naturally cleaved by furin and related proteases with sequences recognized by matrix metal lopro teases or urokinase plasminogen activator has yielded potent agents having high specificity and efficacy in mouse tumor models. Thus, in some

embodiments, the modified PA comprises sequences recognized by a protease expressed by tumor cells, including but not limited to matrix metal loproteases or urokinase plasminogen activator. The modified PA provides tumor specificity, such that cleavage of PA by the tumor cell protease results in binding of PA to receptors on the tumor cell surface, which allows binding of the LFn domain of the chimeric molecule to the modified PA on the tumor cell. Thus, modification of PA such that it is cleaved and activated by a tumor specific protease allows internalization of the chimeric molecule by tumor cells and not healthy cells.

[0094] For targeting of tumors, the native anthrax effector proteins lethal factor (LF) and edema factor can be replaced with a fusion containing the N-terminal 254 amino acids of anthrax toxin lethal factor (LFn) and the Pseudomonas aeruginosa exotoxin A (PE) catalytic domain (PEIII). Once in the cytosol, PEIII will transfer ADP-ribose to eEF2, resulting in protein synthesis inhibition and cell death. This system is highly effective in terms of cytosolic delivery and tumor-specific activation. It has been tested successfully on a number of tumor types (Abi- Habib,R.J. et al. Mol. Cancer Ther. 5, 2556-2562 (2006)), and is expected to be active on nearly all types of solid tumors.

[0095] In some embodiments of the present invention, a ubiquitin molecule (wild type or modified) is inserted between the LFn domain and the PEIII domain. The present methods demonstrate the surprising and unpredictable result that insertion of a ubiquitin molecule having one or more lysines substituted with another amino acid can increase the cytotoxicity of the chimeric molecule compared to a chimeric molecule that does not comprise a ubiquitin domain, or compared to a chimeric molecule having a wild-type ubiquitin. Insertion of a ubiquitin domain into a chimeric molecule provides the advantage of allowing release of the effector domain from the cytosol-targeting domain, thereby preventing or slowing degradation of the effector domain. Another advantage of the chimeric ubiquitin molecules described herein is the ability to release an effector domain having a specific, desired N-terminal residue, e.g., an N- terminal residue that is an N-end rule stabilizing residue.

[0096] As described herein, the ubiquitin molecule can be a wild-type ubiquitin or a modified ubiquitin. In some embodiments, the modified ubiquitin has one or more lysine residues substituted with another amino acid. In some embodiments, the modified ubiquitin has amino acid substitutions at all lysine residues except for the lysine at residue 48. In some embodiments, the modified ubiquitin has amino acid substitutions at all lysine residues except for the lysine at residue 63. In some embodiments, the modified ubiquitin has amino acid substitutions at all lysine residues. In some embodiments, the one or more lysine residues are substituted with arginine. Thus, in some embodiments, the method of targeting a tumor cell comprises contacting the tumor cell with a construct having the structure LFn-ubiquitin -PEIII, where ubiquitin can be Ut>K48, Ub>K₆3, or Ubjcnuii- Examples of chimeric fusion proteins that function as targeted toxins are shown in Figure 1.

[0097] In some embodiments, the method comprises the steps of 1 ) contacting the tumor cell with a chimeric molecule comprising LFn, ubiquitin, and an effector toxin and 2) contacting a tumor cell with PA or a modified PA. Steps 1 ) and 2) may be performed simultaneously or sequentially. The methods can be performed on tumor cells in vitro or in vivo. When the methods are performed in vitro, the tumor cells are cultured with PA or a modified PA and a ubiquitin containing chimeric fusion protein described herein. To target tumor cells in vivo, the PA or modified PA proteins and the ubiquitin containing chimeric molecules are co-administered to the subject in need of treatment. The PA or modified PA proteins circulate in the blood or other bodily fluids until the PA or modified PA binds to the target tumor ceil. The PA is cleaved and activated by the protease expressed by the tumor cell, and the chimeric molecule binds to the activated PA. The chimeric molecule with the effector toxin is then co-internalized with PA into the cytosol of the tumor cell, resulting in cleavage and release of the effector toxin by DUB. [0098] The PA or modified PA and the ubiquitin containing chimeric fusion proteins can be administered to a subject by various methods well known in the art, for example, intravenously, parenterally, intramuscularly, or intraperitoneally.

B. Targeting HIV infected cells

[0099] The present invention further provides methods for targeting and killing cells infected with human immunodeficiency virus (HIV). In some embodiments, the method comprises directly contacting an HIV-infected cell with a chimeric molecule described herein. In some embodiments, the.method comprises indirectly contacting an HIV-infected cell with a chimeric molecule described herein. For example, in some embodiments, the method comprises the step of contacting a cell infected with HIV with a modified PA that binds to a protein expressed on the surface of an HIV-infected cell. In some embodiments, the modified PA comprises the translocation domain and LF binding domain of native PA and a ligand domain that specifically binds to a protein expressed on the surface of an HIV-infected cell. In some embodiments, the C-terminal domain of PA is replaced with the extracellular domains of CD4 that are capable of binding to viral gpl20 envelope proteins displayed on HIV-infected cells. In order to kill the HIV infected cell, the method further comprises the step of contacting a cell infected with HIV with a chimeric ubiquitin fusion protein described herein, where the chimeric fusion protein binds to the modified PA. The modified PA protein and the ubiquitin chimeric molecule can be co-administered to a subject that is infected with HIV.

[0100] In some embodiments, the HIV infected cell is contacted with a chimeric molecule comprising the cytosol-targeting moiety LFn. In some embodiments, the HIV infected cell is contacted with a chimeric molecule comprising the effector moiety PEIII.

[0101] In some embodiments, the chimeric molecule comprises a sequence recognized by an HIV protease. In one embodiment, sequence recognized by an HIV protease is GSGIF.LETSL (SEQ ID NO: 14), where the period indicates the site of proteolytic cleavage. In some embodiments, the sequence recognized by the HIV protease is located between the ubiquitin domain and the effector domain. This allows cleavage and release of the effector domain by HIV protease in virus-infected cells, providing an HIV-specific targeting molecule that minimizes non-specific damage to healthy cells and tissue. Thus, in some embodiments, the chimeric molecule is a fusion protein and has the structure, from N-terminus to C-terminus, LFn- ubiquitin-TS-PEIII, where TS represents the target sequence recognized by an HIV protease. [0102] In some embodiments, the effector moiety is the effector domain of the cytolethal distending toxin ("CdtB") (SEQ ID NO:22) produced by various gram-negative pathogens. CdtB contains a natural nuclear localization signal, which promotes its entry into the cell nucleus, where it can destroy cellular genomic DNA and lead to cell death of the HIV infected cell. In some embodiments, the chimeric molecule is a fusion protein and has the structure, from N-terminus to C-terminus, LFn-ubiquitin-TS-CdtB. In some embodiments, the effector moiety is substantially identical to SEQ ID NO:22.

[0103] In some embodiments, the method comprises contacting an HIV infected cell with a chimeric molecule comprising the HIV viral protein R (Vpr), which provides a means for targeting newly formed virus particles. In some embodiments, the Vpr is located between the ubiquitin moiety and the effector moiety. Cleavage of the C-terminal GG motif of ubiquitin by DUBs in the cytosol of HIV-infected cells results in the release of the Vpr-effector domain fusion protein, which is then packaged with the viral genetic information into newly formed viral particles. In some embodiments, the HIV- 1 protease cleavage site described above can be introduced between Vpr and the effector domain to separate the effector domain from Vpr once the HIV-1 protease becomes active in the viral particle. Any infection from this modified virus would immediately release the effector domain into the cytosol of the infected cell. If the effector domain is a toxin, the toxin results in cell death preventing further spreading of HIV. In some embodiments, the effector domain is PEIII. Thus, in some embodiments, the chimeric molecule is a fusion protein and has the structure, from the N-terminus to the C-terminus, LFn- ubiquitin-Vpr-PEIII. In some embodiments, the effector domain is CdtB. In some

embodiments, the chimeric molecule is a fusion protein and has the structure, from the N- terminus to the C-terminus, LFn-ubiquitin-Vpr-CdtB. In some embodiments, the fusion proteins further comprise an HIV protease recognition sequence described above located between the Vpr domain and the effector moiety.

[0104] In some embodiments, the effector moiety is an RNAse that degrades RNA viruses such as HIV. Any RNAse can be used, e.g., human angiogenin. The same delivery mechanism as described above for PEIII would result in the packaging of Vpr-RNAse in nascent virions and RNase activity would destroy viral particles. In some embodiments, the chimeric molecule has the structure LFn-ubiquitin-TS-RNase. In some embodiments, the fusion proteins further comprise an HIV protease recognition sequence described above located between the Vpr domain and the RNAse domain.

C. Methods of delivering PNA into cells

[0105] The invention also provides a method of delivering a PNA into a cell by contacting a cell with a chimeric molecule comprising the PNA. PNAs are nucleic acid mimics that can have similar functions as antisense nucleic acids and can be used for altering gene expression, pre- mRNA splicing, etc. It will be understood that whereas PNAs are not strictly defined as toxins, they can have functional activities that result in death of a target cell. PNAs closely resemble natural polypeptides, and thus can potentially be translocated into cells using the chimeric molecules described herein. In some embodiments, the PNA is conjugated to a reactive cysteine that is in turn attached to a ubiquitin moiety. In some embodiments, the reactive cysteine is chemically conjugated to a PNA. In some embodiments, the reactive cysteine is attached to the C-terminal end of the ubiquitin moiety. For example, the reactive cysteine can be attached to the C-terminal end of the ubiquitin moiety- by a linker as described herein. In one embodiment, the method comprises contacting a cell with a chimeric molecule having the structure LFn-ubiquitin -SH-PNA, where SH represents a reactive cysteine. VI. Functional Assays

[0106] The biological activity of the chimeric molecules described herein can be determined using standard tests of cytotoxicity known in the art as well as those described in this application. For example, cells of the targeted type can be cultured with different concentrations of a chimeric molecule and PA, and dose response curves generated. Further, cell viability can be quantified to determine relative cell survival and generate a survival index (SI). The

concentration at which 50% of the cells survive can be determined and compared between different chimeric molecules to ascertain which chimeric molecule has the greatest activity (i.e., cytotoxicity). Examples of representative functional assays using tumor cells are provided in the Examples. The dose response curve and/or survival index can be used to determine the effective amount of a chimeric molecule to be administered to a subject in need of treatment.

VII. Pharmaceutical compositions

[0107] The chimeric molecules, fusion proteins, and PA or modified PA proteins of the present invention can be administered in various pharmaceutical compositions. The pharmaceutical compositions can include an effective amount of the chimeric molecule in a physiologically acceptable carrier or excipient. A physiologically acceptable carrier or excipient can include, for example, carbohydrates (such as glucose, sucrose or dextrans), antioxidants (such as ascorbic acid or glutathione), chelating agents, low molecular weight proteins or other stabilizers or excipients. Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives, which are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. Suitable formulations for use in the present invention are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985). Proteins of the invention can also be mixed with physiologically acceptable fluids such as saline solutions and phosphate buffered solutions. Pharmaceutical compositions of the invention are suitable for use in a variety of drug delivery systems. For a brief review of methods for drug delivery, see, Langer, Science 249: 1527-1533 (1990).

[0108] The pharmaceutical compositions of the present invention can be administered by various routes, e.g., oral, subcutaneous, intramuscular, intravenous, or intraperitoneal. The effective dose can be determined by one skilled in the art. An effective amount of the pharmaceutical compositions can be administered according to various dosing amounts and schedules. For example, the pharmaceutical compositions can be administered in a dose range of about 2 ug/kg/day to 2 mg/kg/day. In some embodiments, daily doses of about 0.01 - 5000 mg, or 5-500 mg, of a chimeric molecule for a 70 kg adult human may be used. The appropriate dose may be administered in a single daily dose or as divided doses presented at appropriate intervals, for example as two, three, four, or more subdoses per day.

[0109] The pharmaceutical compositions containing chimeric molecules of the invention can be administered for prophylactic and/or therapeutic treatments. In therapeutic applications, compositions are administered to a patient already suffering from a condition that may be treated by the chimeric molecules described herein. An amount adequate to accomplish this is defined as a "therapeutically effective dose." Amounts effective for this use will depend on the severity of the disease or condition and the weight and general state of the patient, but generally range from about 0.1 mg to about 2,000 mg of the chimeric molecule per day for a 70 kg patient, with dosages of from about 5 mg to about 500 mg of the chimeric molecule per day for a 70 kg patient being more commonly used.

[0110] In prophylactic applications, pharmaceutical compositions containing chimeric molecules of the invention are administered to a patient susceptible to or otherwise at risk of developing a disease or condition that may be treated by the chimeric molecules described herein, in an amount sufficient to delay or prevent the onset of the symptoms. Such an amount is defined to be a "prophylactically effective dose." In this use, the precise amounts of the chimeric molecule again depend on the patient's state of health and weight, but generally range from about 0.1 mg to about 2,000 mg of the polypeptide for a 70 kg patient per day, more commonly from about 5 mg to about 500 mg for a 70 kg patient per day.

[0111] Single or multiple administrations of the compositions can be carried out with dose levels and pattern being selected by the treating physician. In any event, the pharmaceutical formulations should provide a quantity of a chimeric molecule sufficient to effectively treat the disease or condition, either therapeutically or prophylatically.

VIII. Diseases Targeted

[0112] The chimeric molecules described herein can be used to target cells associated with or having the characteristics of different diseases or medical conditions. For example, a target cell associated with a disease state can be targeted by PA molecules that have been modified to bind to cell surface receptors that are specific for the cells being targeted. PA can also be modified to be activated by a target cell-specific protease to confer cell-type specificity. Further, PA can be modified using a combination of the above modifications to confer cell-type specificity. The PA or modified PA can be co-administered with the chimeric molecules provided herein.

[0113] In some embodiments, tumor cells can be targeted and killed using the chimeric molecules provided herein. The chimeric ubiquitin molecules can be used to target cells infected with viruses such as HIV. The chimeric ubiquitin molecules can also be used to deliver antigens to cells that can be presented by the MHC class I pathway and thus induce cellular immunity. EXAMPLES

[0114] The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially the same or similar results.

EXAMPLE 1

[0115] This example demonstrates that ubiquitin fusion proteins provide improved cytotoxicity compared to fusion proteins without ubiquitin.

Introduction

[0116] The therapeutic benefit of drugs depends on achieving high potency for the target (an enzyme, cell, bacterium, parasite, virus, etc.) while avoiding damage to the host organism. One approach to killing of tumor cells has been to use highly potent bacterial and plant toxins that act catalytically in the cytosol of the targeted cells. Most commonly, specificity has been sought by linking these toxins chemically or genetically to antibodies that bind to cell surface materials enriched on tumor cells. These proteins, initially termed "immunotoxins" and now more generally described as "targeted toxins" (TT), have been under development for decades, but few have reached clinical use (Weldon, J.E. & Pastan, I., FEBS J., 278, 4683-4700 (201 1 ); Alfano, R.W. et al., Cell Cycle, 7, 745-749 (2008); Hetzel, C. et al., Curr. Pharm. Des., 15, 2700-271 1 (2009)). This appears to be due to inadequate specificity for tumor vs. host (i.e., a low therapeutic index), low efficiency of delivery to the cytosol, along with additional factors.

Several approaches have been explored for improving the therapeutic indices of TTs (reviewed in (Hetzel, C. et al., Curr. Pharm. Des. , 15, 2700-271 1 (2009)) or to increase the uptake of TTs into the cytosol of tumor cells (Bachran, C. et al., J. Immunother. , 32, 713-725 (2009)). [0117] In this laboratory, efforts have emphasized an alternative approach to achieving tumor cell specificity. This exploits the fact that anthrax toxin activity depends on proteolytic activation of the receptor-bound protective antigen protein (PA) by cell surface proteases (Liu, S. et al., Cancer Res., 60, 6061-6067 (2000); Liu, S. et al., Nat. Biotechnol., 23, 725-730 (2005); Liu, S. et al., J. Biol. Chem., 283, 529-540 (2008)). Replacing the site normally cleaved by furin and related proteases with sequences recognized by matrix metalloproteases or urokinase

plasminogen activator has yielded potent agents having high specificity and efficacy in mouse tumor models. The protease-activated PA assembles into an oligomeric protein-conducting channel that efficiently delivers the anthrax toxin catalytic effector proteins to endosomes and then translocates them to the cytosol. For targeting of tumors, the native anthrax effector proteins lethal factor (LF) and edema factor can be replaced with a fusion containing the N-terminal 254 amino acids of anthrax toxin lethal factor (LFn) and the Pseudomonas aeruginosa exotoxin A (PE) catalytic domain (PEIII). Once in the cytosol, PEIII will transfer ADP-ribose to eEF2, resulting in protein synthesis inhibition and cell death. This system is highly effective in terms of cytosolic delivery and tumor-specific activation. It has been tested successfully on a number of tumor types (Abi-Habib, R.J. et al., Mol. Cancer Ther. , 5, 2556-2562 (2006)), and is expected to be active on nearly all types of solid tumors.

[0118] One factor that affects the potency of all TT, but that has received limited attention, is the issue of the stability of the effector proteins once they have reached the cytosol. It was noted in 1989 that many protein toxins have a strong bias against the presence of lysine residues in their catalytic domains (London, E. & Luongo, C.L., Biochem. Biophys. Res. Commun., 160, 333-339 (1989)). In retrospect, it is now evident that this feature limits the attachment of ubiquitin and the resulting proteasomal degradation of toxins (Falnes, P.O. & Olsnes, S., EMBO J, 17, 615-625 (1998)). The cytosolic stability and resulting potencies of several toxins has been shown to depend on the N-end rule, which specifies that the N-terminal amino acid of a polypeptide determines the efficiency with which side chain lysine residues are ubiquitinated for proteasomal targeting (Varshavsky, A., Protein Sci. , 20, 1298-1345 (201 1 )). The N-end rule applies to LF and LFn-based fusion proteins (Gupta, P.K. et al., PLoS. ONE, 3, e3 130 (2008); Wesche, J. et al., Biochemistry 37, 15737-15746 ( 1998)), indicating that it will impact the efficacy of anthrax toxin-based TTs.

[0119] Ubiquitin is a small eukaryotic protein that plays a major role in signal transduction and many other processes in addition to its role in protein degradation. Ubiquitin consists of a di- glycine motif at its C-terminus that is conjugated to the epsilon amine of a lysine within the target protein. According to the N-end rule noted above, ubiquitination occurs on proteins with specific destabilizing N-terminal residues (Varshavsky, A., Protein Sci. , 20, 1298-1345 (201 1 ); Tasaki, T. & Kwon, Y.T., Trends Biochem. Sci. , 32, 520-528 (2007)) and ubiquitination may occur on several sites within one protein. Ubiquitinated proteins are targeted for degradation by the 26S proteasome system (Thrower, J.S., EMBO J., 19, 94-102 (2000)). Ubiquitin itself may be ubiquitinated after its conjugation to a target protein, leading to creation of polyubiquitin chains. These chains may be built upon any of the seven lysine residues within ubiquitin by ubiquitin ligases, although Lys48 is most often used (Kim, H.T. et al., J. Biol. Chem., 282, 17375-17386 (2007)). Furthermore, polyubiquitination of Lys63 instead of Lys48 seems to result in less protein degradation (Jacobson, A.D. et al., J. Biol. Chem., 284, 35485-35494 (2009)).

Ubiquitination is a balanced process with deubiquitinating enzymes (DUBs) counteracting the ubiquitination. The DUBs recognize the C-terminal di-glycine motif of ubiquitin and release ubiquitin from labeled proteins. This specific release of cargo from ubiquitin fusions is described by Varshavsky as the ubiquitin fusion technique (Varshavsky, A., Methods Enzymol., 399, 777- 799 (2005)), which can be used to conditionally stabilize or destabilize a protein.

[0120] In this study, we examined how the insertion of ubiquitin variants within the TT LFn- ΡΕΠΙ altered enzymatic activity, cytotoxicity, and stability of the TTs. We used ubiquitin variants that allowed us to study the accessibility of the TTs to DUBs and polyubiquitination. The results obtained indicate that intracellular release of the catalytic PE1II domain is achievable and that ubiquitination of the TTs controls their persistence in the cytosol and thus determines their potency.

Materials and Methods

Chemicals [0121] The ubiquitin El ligase inhibitor PYR-41 (Yang, Y. et al., Cancer Res. 67, 9472-9481 (2007)) and the DUB inhibitor ubiquitin aldehyde (Jacobson, A.D. et al., J. Biol. Chem., 284, 35485-35494 (2009)) were obtained from Calbiochem, San Diego, CA. The oxidative indicator 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Sigma Aldrich, St. Louis, MO. Cloning of ubiquitin-containing targeted toxins [0122] All TTs used for this study are based on the anthrax fusion toxin FP59, consisting of LFn-PEIII (Liu, S. et al., Cancer Res., 60, 6061-6067 (2000)). LFn consists of the N-terminal 254 residues of anthrax toxin lethal factor (see Fig. 1) (but in FP59 includes an additional N- terminal HM added due to cloning). PEIII consists of the C-terminal 216-residue catalytic domain of Pseudomonas exotoxin A (SEQ ID NO: 13). For cloning, ubiquitin and LFn were amplified by fusion PCR and cloned into pET19b via the Ndel and BamHI restriction sites. PEIII was inserted into this plasmid 3' of ubiquitin. Finally, ubiquitin was substituted by other variants of ubiquitin (ubiquitin with an uncleavable sequence at the C-terminus of ubiquitin, ubiquitin with all lysine residues except Lys48 or Lys63 changed to arginine residues, and ubiquitin with all lysine residues changed to arginine residues) after amplification by PCR. The ubiquitin variants containing mutations were amplified from plasmids purchased from Addgene,

Cambridge, MA (see the internet at www.addgene.org). All constructs encoded an N-terminal 10 x histidine (SEQ ID NO: 19) tag, followed by the enterokinase cleavage site DDDDK (SEQ ID NO:20) for histidine tag removal. After histidine tag removal, all constructs obtained an N- terminal HMAGG (SEQ ID NO: 16) sequence. All ubiquitin constructs contained in addition a GGGS (SEQ ID NO:21 ) linker at the C-terminus of LFn and a GS or AS linker preceding PEIII (Fig. 1 ).

Expression and purification of ubiquitin-containing targeted toxins

[0123] PA and the mutant PAAFF (PA deleted of Phe₃ i₄-Phe₃i₅) were expressed as described earlier (Liu, S. et al., Cell. Microbiol, 9, 977-987 (2007); Singh, Y. et al., J. Biol. Chem., 269, 29039-29046 (1994)). All targeted toxins were expressed in 3 1 LB medium supplemented with 100 μg/ml ampicillin for 5 h at 37 °C in E. coli strain BL21 (DE3) Gold. Expression was induced by 1 mM isopropyl-P-D-thiogalactopyranoside after reaching an optical density (600 nm) of 0.8. Cells (6 g wet weight) were harvested by centrifugation, culture pellets resuspended in 120 ml sonication buffer (50 mM Tris, pH 8.0, 0.3 M KC1, 5 mM EDTA, 1 tablet protease inhibitor (Roche, Indianapolis, IN) / 50 ml) supplemented with 1 mg/ml lysozyme, sonicated for 2 min and incubated 20 min on ice. After a subsequent centrifugation, the pellet was resuspended (in 35 ml 1 .5% N-lauroyl sarcosine, 1 % Triton-X 100, 50 mM Tris, pH 8.0, 0.3 M KC1), sonicated for 2 min, recentrifuged and 4 ml Ni-NTA-agarose (Qiagen, Valencia, CA) was added. The proteins were eluted by 0.5 M imidazole (in 50 mM Tris, pH 8.0, 10 % glycerol, 0.3 M KC1) after washing with 1 M and 0.3 M KC1 (in 50 mM Tris, pH 8.0, 10 % glycerol, 20 mM imidazole). Fractions containing the TTs were pooled, dialyzed against 5 mM HEPES, pH 7.5, 0.5 mM EDTA overnight and loaded on a Q-Sepharose Fast Flow column for purification with an Akta chromatography system (GE Healthcare, Waukesha, WI) and eluted using a linear gradient of 0- 0.5 M NaCl in 20 mM Tris, pH 8.0, 0.5 M EDTA. Fractions containing the TTs were again dialyzed as described above, concentrated (Amicon Ultrafiltration devices, 30 kDa molecular weight cutoff, Millipore, Billerica, MA), filter sterilized, and stored in aliquots at -80 °C. All proteins were analyzed by electrospray ionization mass spectrometry to confirm that the masses matched those calculated from their sequences.

[0124] For cytotoxicity studies, enterokinase cleavage was performed to remove the N- terminal 10 x histidine (SEQ ID NO: 19) tag, exposing an N-terminal HMAGG (SEQ ID NO: 16) sequence. The TTs (at 0.3 mg/ml) were incubated 24 h with 0.5 U/ml enterokinase (Stratagene, La Jolla, CA) in 50 mM Tris, pH 8.0, 50 mM NaCl, 2 mM CaCl₂, 0.1 % Triton X-100). The cleavage of the samples was confirmed by SDS-PAGE and the TTs were used in cytotoxicity assays without further purification. Enzymatic activity of TTs

[0125] The enzymatic activities of PEIII within all TTs were measured as described (Bachran, C. et al., Clin. Chem., 53, 1676-1683 (2007)). Incubation of the TTs with purified elongation factor 2 (eEF2) and biotinylated NAD⁺ allows the detection of ADP-ribosylated eEF2 by using streptavidin to detect biotin on eEF2 after SDS PAGE and Western blotting. Cell culture

[0126] Cell culture experiments were performed on HN6 cells (human head and neck cancer cell line) (Yeudall, W.A. et al., Carcinogenesis, 15, 2683-2686 (1994)), CHO TEM 8 T4 cells (Chinese hamster ovary cells stably transfected with anthrax receptor tumor endothelial marker 8) (Liu, S. et al., Cell. Microbiol, 9, 977-987 (2007); Liu, S. & Leppla, S.H. J. Biol. Chem., 278, 5227-5234 (2003)), and RAW264.7 cells (murine leukemic monocyte / macrophages). HN6 and RAW264.7 cells were maintained in Dulbecco's modified Eagle's medium with Glutamax™-1 (Gibco, Life Technologies, Grand Island, NY), CHO TEM8 T4 cells were maintained in modified Eagle's medium alpha with Glutamax™-1 (Gibco, Life Technologies, Grand Island, NY). All media were supplemented with 10% fetal bovine serum (Gibco, Life Technologies, Grand Island, NY) and 50 g/ml gentamicin (Quality Biological, Gaithersburg, MD). CHO TEM 8 T4 cells were additionally grown in the presence of 500 μg/ml hygromycin (Invitrogen, Life Technologies, Grand Island, NY).

Cleavage by deubiquitinating enzymes

[0127] Cell lysates of HN6 cells were obtained after growing 6 x 10⁶ HN6 cells overnight, washing them twice with phosphate-buffered saline (PBS; 150 mM NaCl, 8.3 mM Na₂HP0₄, 1.7 mM KH₂P0₄, pH 7.4) and incubation with 300 μΐ of PBS supplemented with 1% Triton X-100. After 30 min at 4 °C on a rotary shaker, cells were resuspended and centrifuged (30 min, 4 °C, 16,000 x g). The supernatant was used for in vitro cleavage of ubiquitin fusion toxins by incubation of 75 ng of the TTs for up to 120 min at 37 °C with 8 μΐ of the cell lysate

supplemented with 2 mM DTT in a total volume of 9 μΐ. Additional experiments were performed with ubiquitin aldehyde to inhibit deubiquitinating activity. Ubiquitin aldehyde (0.5 μΐ, final concentration 2.5 μΜ) was incubated with the lysate 5 min at 37 °C prior to TT addition.

Subsequently, samples were separated by SDS PAGE and Western blotted using the iBlot system (Invitrogen, Life Technologies, Grand Island, NY). TTs were detected by a polyclonal rabbit anti-LF serum and infrared dye-conjugated secondary antibodies on the Odyssey Imager infrared detection system (LI-COR, Lincoln, NE).

[0128] Detection of intracellular cleavage was performed on CHO TEM8 T4 cells (1 x 10⁶ cells overnight in 12-well plates). Cells were incubated with 1 μg/ml PA or PAAFF (PA deleted of Phe314 and Phe315, a mutant that fails to deliver LF to the cytosol (Singh, Y. et al., J. Biol. Chem., 269, 29039-29046 (1994)), and 1 μ^πιΐ of the TTs in 0.5 ml medium for 1 h or washed off immediately (0 h). Further samples were pre-incubated with 50 μΜ PYR-41 for 1 h at 37 °C before adding PA and the TTs at the indicated concentrations for 1 h. All cells were washed twice with PBS, incubated with trypsin/EDTA at 37 °C until all cells could be transferred into new tubes for a centrifugation (5 min, 4 °C, 1000 x g). For cytosol isolation, cells were resuspended in 125 μg/ml saponin (Sigma Aldrich, St. Louis, MO) in PBS, supplemented with protease inhibitor cocktail (Roche) and incubated 10 min on ice (Newman, Z.L. et al., Infect. Immun., 77, 4327-4336 (2009)). The complete supernatants after centrifugation (30 min, 4 °C, 16,000 x g) were separated by SDS-PAGE and Western blotted using the iBlot system and the Western Blot Signal Enhancer kit (Thermo, Waltham, MA) for signal enhancement. TTs were detected by polyclonal rabbit anti-LF serum, polyclonal rabbit anti-PE serum (Sigma Aldrich, St. Louis, MO), and infrared dye-conjugated secondary antibodies (Rockland Immunochemicals, Gilbertsville, PA) on the Odyssey Imager infrared detection system (LI-COR, Lincoln, NE).

Cytotoxicity of TTs

[0129] Dose-response curves for the TTs were obtained by incubation on HN6 cells for 2 or 48 h in the presence of 0.25 μg/ml PA. The cells (10,000 cells per well in 100 μΐ medium) were seeded in 96-well plates and incubated at 37 °C for 6 h before addition of PA and the TTs. TTs were added in 100 μΐ to achieve final concentrations ranging from 0.3 pM-1 nM and the cells were incubated a further 48 h. For the experiments with 2 h TT exposure, the TT-containing medium was removed after 2 h and the cells were incubated a further 46 h in fresh medium. Cell survival was determined in an MTT assay by adding 30 μΐ MTT solution (5 mg/ml in medium without fetal bovine serum) to each well and incubation for 1 h at 37 °C. All medium was removed and the cells were treated with 50 μΐ solubilizer (90 % isopropanol, 1 % sodium dodecyl sulfate, 80 mM HCl). Plates were shaken for 1 min and absorbance was measured at 570 nm and 630 nm (for background subtraction for each sample) in a microplate reader (Spectra Max Gemini, Molecular Devices, Sunnyvale, CA). The relative cell survival, designated as survival index (SI), was calculated after blank subtraction (wells without cells) as the percentage of living cells in treated wells in relation to untreated cells (cells without toxin). For experiments using the inhibitor PYR-41 , RAW264.7 cells (1 5,000 cells per well in 100 μΐ medium) were seeded in 96-well plates and incubated at 37 °C for 12 h. Cells were treated with 50 μΐ medium containing the inhibitor diluted so as to achieve the desired final 15.8 μΜ PYR-41 in 200 μΐ and incubated for 1 h at 37 °C. Subsequently, 50 μΐ medium supplemented with the TTs was added and incubated for a further 18 h at 37 °C before measurement of cell survival as described above.

Data analysis

[0130] The statistical significance of different 50 % survival indices (SI₅o, 50 % cell survival in comparison to untreated controls) values for cytotoxicity analyses was determined by a paired t- test using GraphPad Prism 5.02 and data obtained from a nonlinear regression curve fit. The method used for the nonlinear regression curve fit was "Iog(inhibitor) versus normalized response" (by using a least square fit). A two-tailed significance ofp < 0.05 was interpreted as being statistically significant. Results Design of TTs

[0131] Six different TTs were constructed and analyzed in this study (Fig. 1A). The five ubiquitin-containing TTs are based on the TT FP59 that contains LFn at the N-terminus and PEIII at its C-terminus. An alignment of the amino acid sequences shows the differences between the ubiquitin fusions (Fig. I B). The TT designated "Ub" (Fig. 1 A) contains the human wildtype ubiquitin with the C-terminal di-glycine motif that is specifically recognized by DUBs. The TT Ubu_N has the same sequence but with replacement of three glycines (including the di- glycine motif) by a sequence of three alanines, which renders the sequence uncleavable by DUBs. The mutants UbK.48 and Ubj<;63 have all lysine residues of ubiquitin replaced by arginine residues except for one single lysine residue, either Lys48 or Lys63, respectively. Ubi nuii contains ubiquitin with all seven lysines replaced by arginine. Ubi s, Ubi<63, and Ubi nuii all retain the intact C-terminal di-glycine motif.

[0132] All of the ubiquitin-containing TTs are expected to follow the uptake pathway described for FP59, by binding to the PA heptamer and translocating from endosomes through the PA channel into the cytosol. Cytosolic DUBs are expected to cleave the TTs at the di-glycine motif located at the C-terminus of the ubiquitin moiety to release PEIII as a free polypeptide in the cytosol where it can produce its toxic effects. Ubu lacks this cleavage site and is predicted to be less toxic due to its more rapid degradation. Furthermore, it is hypothesized that ubiquitin variants with few or no lysines (Ub_K48, Ub_K63 and UbKnuii) will be stabilized compared to the TT Ub, which will be polyubiquitinated and targeted for proteasomal degradation. FP59 contains no ubiquitin, but is expected to be degraded as well by the ubiquitin / proteasomal degradation system due to the high lysine content of LFn.

Purification of TTs and in vitro ADP-ribosylation by TTs

[0133] All proteins were successfully purified in yields of at least 1.5 mg per liter of culture, and the analyses of the final products showed that all proteins were > 90% pure (Fig. 2A). The molecular masses of all six proteins were confirmed by electrospray ionization mass

spectrometry. Since the accessibility of PEIII within a larger polypeptide or toxin may limit its activity (Leppla, S.H. et al., Biochem. Biophys. Res. Commun. , 81 , 532-538 (1978)), we measured the catalytic activity of all the fusion proteins. The five TTs containing ubiquitin or variants of ubiquitin showed the same ADP-ribosylation activity as the fusion protein FP59 (Fig. 2B). All samples show a band for biotin-containing ADP-ribosylated eEF2 at a molecular mass of 100 kDa and an additional band of the same intensity at around 40 kDa. The latter is a degradation product of eEF2, an artifact of the purification of eEF2 from yeast. This fragment contains the site of ADP-ribosylation of eEF2 and is thus likewise ADP-ribosylated and detected (Bar, C. et al., Mol. Microbiol, 69, 1221-1233 (2008)). In vitro deubiquitination of TTs

[0134] In order to analyze and compare the cleavability of the ubiquitin-containing TTs, the fusion proteins were incubated with HN6 cell lysates. The lysates contain active DUBs that are expected to cleave at the C-terminal di-glycine motif of the ubiquitin (Cronican, J.J. et al., ACS Chem. Biol, 5, 747-752 (2010)). All TTs containing ubiquitin with the natural C-terminal di- glycine sequence were cleaved within the 2 h reaction time (Fig. 3A, B, and C). The cleavage product, LFn-ubiquitin, has an expected molecular mass of 41 kDa and is detected as a band migrating between the 50 kDa and 36 kDa marker bands. FP59, which lacks ubiquitin, showed no cleavage within 2 h. Furthermore, UBU_N, containing three alanines instead of the di-glycine motif, remained intact. The other proteins differed in their susceptibility. Ubi¾₃ showed reduced cleavage compared to the Ub and ΙΛκ₄₈ constructs, for which cleavage was almost complete. UbKnuii was also less efficiently cleaved by DUBs. These differences may reflect decreased recognition of the modified ubiquitin domains by the DUBs that are active in the lysates used. All TTs were also exposed to lysates preincubated with the DUB inhibitor ubiquitin aldehyde. Pretreatment of the lysates with the inhibitor prevented cleavage of the fusion proteins, demonstrating the specificity of the cleavage.

Deubiquitination of TTs

[0135] Studies on deubiquitination of TTs within cells were done using CHO TEM8 T4 cells, which overexpress the tumor endothelial marker 8 anthrax toxin receptor and internalize more TT, thereby facilitating its detection in the cytosolic fractions. All full length TTs were detectable in the cytosol after 1 h toxin exposure and Western blotting with anti-LF and anti-PE combined (upper filled arrowhead, Fig. 4A-C). Mutant PAAFF binds and delivers LFn and TTs to endosomes but cannot support their translocation to the cytosol (Singh, Y. et al., J. Biol.

Chem., 269, 29039-29046 (1994)). Thus, replacing PA by PAAFF prevented the delivery of the TTs into the cytosol, as seen by the weaker bands for the full length TTs in the cytosolic extracts. The presence of small amounts of full length TTs is probably an indication that the cytosolic extracts contain some endosomal contents as a contamination. The bigger cleavage product, LFn- ubiquitin, with an apparent molecular mass of 41 kDa, was detected as a very weak band in all fractions (open arrowhead), even in the PAAFF samples and in the uncleavable UBUN fractions. However, PEIII, the released catalytic domain of the TTs, is clearly detectable in the cytosol of cells incubated with Ub] ₄₈, Ub]<₆₃, and Ubi nui! (arrow with dashed line at an apparent molecular mass of 24 kDa, Fig. 4B and 4C). The corresponding band for the Ub TT is very weak in comparison and the UbuN sample has no band for PEIII (Fig. 4A). After incubation with the mutant PAAFF, no PEIII is detectable in any of the samples. The data demonstrate that DUB processing of the susceptible TT occurs in the cytosol to release the free PEIII domain. The ubiquitination inhibitor PYR-41 did not increase the amounts of any of the detected proteins, neither the uncleaved TTs nor the cleaved LFn-ubiquitin or PEIII. FP59 showed only the expected band for full length FP59.

Cytotoxicity of TTs

[0136] Cytotoxicity analyses employed HN6 cells. This human cell line was chosen as a model cell line for proof of principle, since this cell line is a suitable model for human head and neck cancer when transplanted into nude mice. A 48-h toxin exposure resulted in dose-dependent cytotoxicity with cytotoxicity in the order Ubj nuii > Ub«6₃ > FP59 > ΙΛ 48 » Ub > UbuN (Fig. 5A). This result indicates that the TTs efficiently reach the cytosol, since they induce

cytotoxicity at very low concentrations, comparable to that of FP59, which was described in earlier studies (Liu, S. et al., Cancer Res., 60, 6061 -6067 (2000)). The SI₅o values determined for the TTs are shown in Table 1. The observed SI₅₀ values are in the range of 615 pM (Ubu ) to 3.7 pM (Ubi nuii)- These values are 0.02-fold and 3-fold changes of the SI50 of the ubiquitin-free FP59 (SI50 FP59 1 1 pM), respectively. A shorter (2 h) period of toxin exposure reduced the observed cytotoxicities for all TTs (Fig. 5B). However, the reductions in potency were about the same for all TTs, since the relative SI50 values compared to FP59 remained similar for all TTs (Table 1). A 2-h exposure of UbuN on the HN6 cells showed no cytotoxicity at the doses up to 10 nM.

Table 1 : SI50 values, jP-values and factors of enhancement for the cytotoxicity of the TTs on HN6 cells, obtained from Fig. 5.

toxin 48 h exposure 2 h toxin exposure

Targeted SIso p- Factor of SI50 p- Factor of

toxin (pM) value" enhancement¹⁵ (pM) value^a enhancement^b Ubu 615 0.052 0.02 nd^d 0.066 nd

Ub 100 0.045^c 0.1 1 773.0 0.053 0.03

Ut>K48 014 0.093 0.79 79 0.012 0.34

Ub_K63 006.2 0.051 1.8 018 0.496 1.5

Ub n li 003.7 0.038 3 ί Oi l 0.1 14 2.5

a The p-value was calculated for FP59 versus all TTs. b The factor of enhancement was calculated by comparison of the SI₅₀ values of FP59 to all other TTs. c Bold jo-values are below 0.05, which is defined to be a significant difference. d Not determined

Effects of inhibitors on TT cytotoxicity

[0137] In order to demonstrate the effects of the ubiquitin variants within the TTs, FP59- sensitive RAW264.7 cells were pre-incubated with the El ubiquitin activating enzyme inhibitor PYR-41 (Yang, Y. et al., Cancer Res. 67, 9472-9481 (2007)) and subsequently with the different TTs for a further 18 h before cell survival was measured. RAW264.7 cells were used since they die within 18 h of continuous toxin exposure, while other cell lines need up to 48 h. This shorter time course was chosen because incubation of several cell lines with PYR-41 for longer times led to toxicity (data not shown). Longer incubations of PYR-41 on cells were possible with lower concentrations (5 μΜ), but this concentration would not effectively prevent ubiquitination. In the cell culture model used, all TTs induced cytotoxicity on RAW264.7 and PYR-41 increased the cytotoxicity of all TTs (Fig. 6A-F). However, certain differences were observed. PYR-41 increased the cytotoxicities of Ub_K63, Ubxnuii, and FP59 only slightly (Fig. 6D-F), while increasing the toxicities of Ubx₄8, and especially Ub and UBUN, to larger, if still modest extents (Fig. 6A-C), consistent with the expectation that the latter TTs are more susceptible to ubiquitination and inactivation.

Discussion

[0138] Current anti-cancer drugs are often associated with strong off-target effects that limit the doses that can be administered. Thus, our aim has been the development of improved drugs which can be administered in doses that eliminate the tumor cells without induction of side effects. TTs containing bacterial toxin catalytic domains possess high potency, at least in theory, but this high potency requires that the TT be targeted with high specificity to avoid damage to non-target cells. A high inherent potency allows lower doses to be used, with can help to limit the inevitable damage that comes from clearance of administered proteins to, and accumulation in, the liver and kidneys (Soler-Rodriguez, A.M. et al., Int. J. Immunopharmacol, 14, 281-291 (1992)). However, for low protein doses to be effective, the protein must be efficiently bound and internalized, translocated to the cytosol, and once there it must have sufficient stability to inactivate its target. The studies described here focus on the latter steps in TT action. The modified anthrax toxin used in our study utilizes a highly efficient delivery system which has been extensively studied as a protein-conducting channel (Thoren, K.L. & Krantz, B.A., Mol. Microbiol, 80, 588-595 (201 1 ); Pentelute, B.L. et a\., Angew. Chem. Int. Ed. Engl, 50, 2294- 2296 (201 1 )). In addition, high tumor cell specificity has been achieved by making these TTs dependent on activation by cell-surface proteases that are expressed only on tumor cells (Liu, S. et al., Cancer Res. , 60, 6061 -6067 (2000); Abi-Habib, R.J. et al., Mol Cancer Ther., 5, 2556- 2562 (2006); Liu, S. et al., J. Biol Chem., 276, 17976-17984 (2001); Liu, S. et al., Proc. Natl. Acad. Sci. U. S. A., 100, 657-662 (2003)). The system has been modified and optimized in the last decade, and shown to be effective in limiting tumor growth in mouse models of anaplastic thyroid carcinoma (Alfano, R.W. et al., Mol. Cancer Ther., 9, 190-201 (2010)), non-small cell lung cancer (Su, Y. et al., Cancer Res. , 67, 3329-3336 (2007)), and others. In one modification of the system, the TTs have been engineered to require activation by two separate tumor cell proteases, thereby further increasing specificity (Liu, S. et al., Nat. Biotechnol , 23, 725-730

(2005) ). The system can deliver a number of different "payloads" in addition to the natural toxin lethal factor, as it was shown that the system is able to deliver Pseudomonas exotoxin A catalytic domain (Arora, N. & Leppla, S.H., J. Biol. Chem., 268, 3334-3341 (1993)), diphtheria toxin A chain, shiga toxin catalytic domain (Arora, N. & Leppla, S.H., Infect. Immun., 62, 4955-4961 (1994)), as well as the reporter beta- lactamase (Hobson, J.P. et al., Nat. Methods, 3, 259-261

(2006) ; Hu, H. & Leppla, S.H., PLoS. ONE, 4, e7946 (2009)). Here, it was shown that ubiquitin can be used to successfully release a cargo, e.g. PEIII, from the PA-binding delivery peptide, LFn.

[0139] A key feature of the TTs described here is the insertion of a ubiquitin domain between the targeting domain (LFn) and the catalytic payload (PEIII). A prerequisite for success of this design is that the ubiquitin domain not limit the efficiency of translocation of the enlarged, 3- domain polypeptides to the cytosol. Previous studies of the translocation process show that polypeptides must completely unfold to pass through the narrow lumen of the oligomeric PA channel (Wesche, J. et al., Biochemistry 37, 15737-15746 (1998); Thoren, K.L. & Krantz, B.A., Mol. Microbiol , 80, 588-595 (201 1 )). Proteins that are tightly folded, either naturally or due to ligand binding, will not be translocated, although it is possible that chaperones may facilitate translocation of certain proteins (Tamayo, A.G. et al., Mol. Microbiol, 81 , 1390-1401 (201 1)). Ubiquitin is a tightly folded protein (Ralat, L.A. et al., J. Mol. Biol, 406, 454-466 (201 1)), and it was not certain that it would readily translocate. However, the fact that several of the proteins characterized here had potencies very similar to that of the FP59 protein indicate that ubiquitin unfolding occurred readily, at least in the context of this LFn fusion proteins and in the HN6 cells.

[0140] A second prerequisite for success of the strategy described here is that cytosolic DUBs be able to cleave the fusion proteins at an adequate rate. Here, the strategy is aided by the great diversity of DUBs present in cells (Reyes-Turcu, F.E. et a\., Annu. Rev. Biochem. , 78, 363-397 (2009)). It was evident from the cleavages we observed (Fig. 3 and 4) and the pattern of toxicities (Fig. 5 and Table 1 ) that at least a few of the DUBs recognized the ubiquitin and the modified ubiquitins in the context of the 3-domain fusions. Even replacement of all lysines with arginine (in the Ubi nuii protein) did not greatly diminish the ability of the DUBs to cleave the fusion proteins.

[0141] The release of a free catalytic domain (e.g., PEIII) in the cytosol has a number of potential and real advantages, including the opportunity to increase its stability and potency. Typically, it can be expected that any polypeptide used in the delivery of the catalytic domain will contain a number of lysines that provide targets for ubiquitination. This is clearly the case with LFn fusion proteins, since 32 of the 254 residues of LFn are lysine. Thus, an LFn-PEIlI fusion (e.g., FP59) might be expected to be rapidly degraded. In fact, we and others have shown that LF and LFn are subject to the N-end rule (Gupta, P.K. et al., PLoS. ONE, 3, e3130 (2008); Wesche, J. et al., Biochemistry 37, 15737-15746 (1998)), and therefore that the side chain lysines are ubiquitinated, provided the N-terminal has a destabilizing residue under the N-end rule. In retrospect, it is rather surprising that LF and LFn fusion protein possess a high potency. Thus, the introduction of a ubiquitin domain into a TT provides a way to cause release of the catalytic domain, freeing it from other (usually N-terminally located) domains that would promote its degradation. In the case of the PEIII fusions described here, the catalytic domain contains only 2 lysines, and therefore is inherently resistant to proteasomal degradation.

[0142] Another advantage of the strategy described here is the ability to release the TT catalytic domain having a specific, desirable N-terminal residue. This objective was one impetus to the original development of the ubiquitin fusion method (Varshavsky, A., Methods EnzymoL, 399, 777-799 (2005)). In the case of TTs, it is often desirable to generate a free catalytic domain having an N-terminal residue that is an N-end rule stabilizing residue. Actually, this may be less important for TT catalytic domains than for other materials delivered as LFn-Ub fusions, since catalytic toxin domains are already "protected" from ubiquitination because of their very low lysine content (London, E. & Luongo, C.L., Biochem. Biophys. Res. Commun., 160, 333-339 (1989)). However, there are cases where it is important to control the N-terminal amino acid of the payload polypeptide because it has a specific role as part of the active site.

[0143] Introducing ubiquitin as a linker between the two domains of FP59 to produce the TT designated "Ub" decreased its potency nearly 10-fold (Table 1 ). The introduction of ubiquitin targets the fusion protein to the proteasome after polyubiquitination (Stack, J.H., Nat.

Biotechnol, 18, 1298-1302 (2000)) and prevented efficient accumulation of PEIII in the cytosol of cells (Fig. 4). Even though in vitro cleavage was as efficient as for the other cleavable TTs (Fig. 3), release of PEIII may be too slow to save the majority of the fusion protein from degradation. The uncleavable UbuN showed no cleavage and was apparently degraded too fast for the PEIII to efficiently inhibit protein synthesis (Fig. 4). Only the DUB-cleavable TTs having a reduced number of Lys demonstrated successful accumulation of PEIII in the cytosol and thus much higher cytotoxicities. The release of PEIII from the rest of the molecule helps PEIII to persist longer in the cytosol, which is a requisite for high cytotoxicity (Falnes, P.O., J. Biol. Chem., 275, 4363-4368 (2000)). Ubicnuii, which is resistant to ubiquitination of the inserted ubiquitin domain (Lim, K.L. et al., J. Neurosci., 25, 2002-2009 (2005)), is even more toxic than FP59 and presents the lowest SI₅o of all TTs studied here (Table 1 ). Its high cytotoxicity is apparently due to the release of PEIII at rates well above those at which ubiquitination of the lysines on the LFn domain targets the intact fusion protein to the proteasome. The UbK63 mutant is slightly more toxic than the Ub_K48 mutant (Table 1 ), implicating a role in their individual targeting mechanism when they become ubiquitinated in the cytosol. Jacobson et al. described differences for the proteasomal processing of ubiquitin chains either on lysine 48 or lysine 63, resulting in faster deubiquitination of lysine 63-linked polyubiquitin chains and less proteasomal accessibility (Jacobson, A.D. et al, J. Biol. Chem., 284, 35485-35494 (2009)). This would explain the lower SI₅o value for Ubi 63 compared to Ub_K4₈. Ub_K6₃ would persist longer in the cytosol before it is degraded and counteract the lower release of PEIII detected (Fig. 3C). Thus, the different ubiquitin variants allow the modulation of PEIII release and protein stability so as to achieve different levels of cytotoxicity.

[0144] The effect of the ubiquitin ligase inhibitor PYR-41 was rather weak in the cytotoxicity studies (Fig. 6). Higher concentrations are typically needed to obtain sufficient inhibition of ubiquitin activation (Yang, Y. et al., Cancer Res. 67, 9472-9481 (2007)). However, these concentrations were toxic for the cells lines used here. There was furthermore no increased stability of the TTs observed due to pre-incubation of PYR-41 (Fig. 4). As Fig. 6 shows, the effects of PYR-41 are rather weak, probably due to the instability of PYR-41.

[0145] Cleavable sequences have been introduced in other TTs for separation of the different moieties. Thus, Heisler et al. introduced furin and cytosol ic protease-cleavable peptides sequences in a fusion of epidermal growth factor and the plant toxin saporin to release the toxin in the cytosol (Heisler, I. et al., Int. J. Cancer, 103, 277-282 (2003)). Furin cleavage sites were introduced into TTs by other groups as well to achieve increased cytotoxicity (Goyal, A. & Batra, J.K., Biochem. J, 345 247-254 (2000); Wang, F. et al., Clin. Cancer Res., 16, 2284-2294 (2010)).

[0146] Fusions to ubiquitin have more commonly been used to decrease the stability of proteins, including green fluorescent protein (Deichsel, H. et al., Dev. Genes Evol. , 209, 63-68 (1999)) and beta-lactamase (Stack, J.H., Nat. BiotechnoL, 18, 1298-1302 (2000)). In an approach to enhancing TT potency more like that described here, Tcherniuk et al. used ubiquitin in a fusion protein to saporin, a plant protein toxin (Tcherniuk, S.O. et al., Mol. Ther., 1 1 , 196-204 (2005)). In this study, the DUB cleavage sequence of ubiquitin was exchanged by a prostate- specific antigen cleavage site. Thus, the authors intended to separate ubiquitin and saporin only in the vicinity of tumor cells expressing prostate-specific antigen, following the same idea used for the activation of anthrax toxin PA by urokinase plasminogen activator or matrix

metalloproteases (Liu, S. et al., Nat. BiotechnoL, 23, 725-730 (2005)). In contrast to PA, where the proteolytic cleavage results in activation of PA, prostate-specific antigen cleavage would separate ubiquitin and saporin with the goal of increasing the intracellular stability of saporin. While achieving enrichment of ubiquitin-free saporin in the vicinity of tumor cells, this design did not include any strategy for achieving binding and delivery of the payload to the cytosol, and no gain in potency was achieved. The ubiquitin system for intracellular release of a toxic or therapeutic moiety, efficiently delivered into the cytosol of a target cell as an LFn fusion, provides a sophisticated system for the fine-tuning of toxin uptake, half-life, non-specific toxicity and overall efficacy. Future studies on this system will hopefully provide drugs capable of the safe and efficient elimination of tumor cells in mouse models and, eventually, may warrant clinical trials in humans.

EXAMPLE 2

[0147] This example demonstrates the ubiquitin fusions comprising cytolethal distending toxin B (CdtB) are cytotoxic to four different cell lines.

[0148] Wild-type ubiquitin (Ub, containing 7 lysines, which are targets of polyubiquitination, resulting in protein degradation in the proteasome) or mutated ubiquitin (Ubicnuii, all 7 lysines are mutated to arginine, no polyubiquitination of UbKnuii possible) was inserted between LFn and CdtB to test the hypothesis that Ub or Ub nuii would result in release of CdtB in the cytosol of target cells upon cleavage by cytosol ic deubiquitinating enzymes (DUBs). Targeting to cells was directed by anthrax toxin protective antigen, which binds its receptors on the cell surface and mediates the delivery of the fusion proteins into the cytosol of cells.

[0149] As shown in Figures 7A-7D, fusion proteins in which all seven lysines of Ub were mutated to arginines were cytotoxic to four independent transformed cell lines, including tumor cell lines. In contrast, fusion proteins comprising wild-type Ub were much less toxic. The LFn- CdtB construct was most toxic.

[0150] This example shows that fusion proteins containing ubiquitin as a cleavable linker between the cytosol-targeting domain and the effector domain were cytotoxic for fusions comprising LFn and CdtB. [0151] All patents, patent applications, and other publications, including GenBank Accession Numbers, cited in this application are incorporated by reference in their entirety for all purposes.

Claims

WHAT IS CLAIMED IS: L A chimeric molecule comprising: (1) a cytosol targeting moiety; (2) an effector moiety, and (3) a ubiquitin moiety located between (1 ) and (2), wherein the ubiquitin moiety is a wild-type ubiquitin or a modified ubiquitin that comprises an amino acid substitution at a lysine residue.

2. The chimeric molecule of claim 1 , wherein the cytosol-targeting moiety comprises the anthrax toxin lethal factor (LFn) from Bacillus anthracis.

3. The chimeric molecule of claim 1 , wherein the effector moiety comprises the catalytic domain of Pseudomonas exotoxin A (PEIII), the effector domain of cytolethal distending toxin (CdtB), saporin, a PNA, or an RNase.

4. The chimeric molecule of claim 1 , wherein the ubiquitin moiety is a wild- type human ubiquitin.

5. The chimeric molecule of claim 1 , wherein the modified ubiquitin comprises an amino acid substitution at all lysine residues except for the lysine at residue 48 or 63.

6. The chimeric molecule of claim 1 , wherein the modified ubiquitin comprises an amino acid substitution at all lysine residues.

7. The chimeric molecule of claim 1 , wherein the lysine residue is substituted with arginine.

8. The chimeric molecule of claim 1 , wherein the cytosol-targeting moiety comprises the N-terminal 254 amino acids of anthrax toxin lethal factor (LFn) from Bacillus anthracis, the effector moiety comprises the catalytic domain of Pseudomonas exotoxin A (PEIII), and the ubiquitin moiety comprises an amino acid substitution at a lysine residue.

9. The chimeric molecule of claim 3, further comprising an amino acid sequence recognized by an HIV protease located between the ubiquitin moiety and the effector moiety.

10. The chimeric molecule of any one of claims 1 -9, which is a fusion polypeptide.

1 1. A polynucleotide sequence encoding the chimeric molecule of claim 10.

12. An expression cassette comprising the polynucleotide sequence of claim 1 1.

13. A host cell comprising the expression cassette of claim 12.

14. A method for recombinantly producing the chimeric molecule of any one of claims 1 -9, comprising the steps of:

culturing the host cell of claim 13 under conditions permissible of expression of the chimeric molecule; and

isolating the chimeric molecule.

15. A composition comprising the chimeric molecule of any one of claims 1 -9 and a physiologically acceptable excipient.

16. A method for cytosol delivery of an effector moiety to a target cell, comprising contacting the target cell with the chimeric molecule of any one of claims 1 -9.

17. A method for improving the efficacy of a conjugate intended for cytosol delivery to a target cell, wherein the conjugate comprises a cytosol-targeting moiety and an effector moiety, the method comprising inserting a ubiquitin moiety between the cytosol- targeting moiety and the effector moiety, the ubiquitin moiety being a wild-type ubiquitin or modified ubiquitin that comprises an amino acid substitution at a lysine residue.

1 8. A method for killing tumor cells, comprising contacting a tumor cell with the chimeric molecule of any one of claims 1 -8.

19. A method for killing cells infected with HIV, comprising contacting a cell infected with HIV with the chimeric molecule of claim 9.