WO2014134187A1 - Compositions and methods for the treatment of cancer - Google Patents

Abstract

A mutant form of the protective antigen (PA) subunit of anthrax lethal toxin (LT) is described. Methods and compositions for treating a subject with cancer using a mutant form of PA or LT or fragment thereof are also described. Methods and compositions for the use of imaging agents comprised of a mutant form of PA or LT or fragment thereof are also described. Methods and compositions for the delivery of therapeutic delivery agents comprised of a mutant form of PA or LT or fragment are also described.

Description

TITLE OF THE INVENTION

Compositions and Methods for the Treatment of Cancer

STATEMENT REGARDING FEDERALLYSPONSORED RESEARCH OR

DEVELOPMENT

This invention was made with government support under Grant No.

CA086306 awarded by the National Institutes of Health (NIH). The government therefore has certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application Serial No. 61/769,447 filed February 26, 2013, the entire disclosure of which is incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

All solid tumors require the formation of new blood vessels, a process known as angiogenesis, and are dependent on blood supply for growth and metastasis.

Endothelial cells (ECs) that make-up tumor vasculature are derived from non-cancerous tissues; therefore, the tumor vasculature represents a single therapeutic target for a number of distinct cancers. Anti-angiogenic drugs and vascular targeting agents (VTAs) are important classes of therapeutics that treat solid tumors. These agents comprise small molecules that mainly target VEGF signaling and microtubule dynamics, or they are ligand-based biologies including antibodies and immunotoxins. Antibody-based therapies are becoming a significant and important clinical tool for treatment of various diseases including cancer. However, antibody-based VTAs are rarely curative in unmodified form

(Chaudhary et al, 2012, Cancer Cell 21 :212-216; Chaudhary et al, 2012, Cell Cycle

11 :2253-2259; Gerber et al, 2009, Proc. Natl. Acad. Sci. USA 106: 12424-12429). To overcome this hurdle, antibody drug conjugates (ADCs) are currently under development, but they often require complicated chemical modification (Gerber et al., 2009, Proc. Natl. Acad. Sci. USA 106: 12424-12429). Immunotoxins, made by fusing antibodies or other tumor-targeting moieties to bacterial or plant toxins, show clinical efficacy but are often limited as to the types of therapeutic payload that can be delivered. In addition, these latter agents require use of selective and efficient antibodies that are against tumor- specific antigens. Indeed, numerous anti-angiogenic drugs and VTAs, including small molecules, antibodies, antibody drug conjugates, and anthrax lethal toxin (LT), are currently in development (Alfano et al, 2008, Cell Cycle 7; Gerber et al, 2009, Proc. Natl. Acad. Sci. USA 106: 12424-12429). Notably, a number of these VTAs target microtubule dynamics, and antiangiogenic effects of these compounds have been linked to alterations of the actin cytoskeleton via Rho GTPases (Bijman et al., 2008, Biochem. Pharmacol. 76:707-716; Bijman et al, 2006, Mol. Cancer Therapeutics 5:2348-2357).

Targeting the development of new blood vessels in growing tumors as well as compromising the integrity of established tumor vasculature are important treatment strategies for a variety of cancers (Chaudhary et al., 2012, Cell Cycle 11 :2253- 2259; Folkman, 1971, N. Eng. J. Med. 285: 1182-1186; Gerber et al, 2009, Proc. Natl. Acad. Sci. USA 106: 12424-12429; Thorpe, 2004, J. Am. Assoc. Cancer Res. 10:415- 427). Indeed, numerous therapeutics that target angiogenesis by blocking VEGF signaling have been implemented clinically. However, these drugs suffer from limited efficacy, due in part to the fact that VEGF-independent angiogenesis pathways also exist (Abbadessa et al, 2007, 20:307-313). A further limitation of these approaches is that VEGF is critical for normal adult physiology, leading to on-target toxicity (Eremina et al, 2006, JASN 17:724-735; Lee et al, 2007, Cell 130:691-703; Sung et al, 2010, Curr. Opin. Hematol. 17:206-212).

Bacterial toxins, such as diphtheria toxin and ricin, have been used to treat cancers in humans by re-engineering target-cell specificity (i.e. "immunotoxins") (Frankel et al., 2002, Protein Pept. Lett. 9:1-14). This typically involves redirecting toxins away from their natural receptors in favor of a heterologous protein upregulated on cancer cells. Bacterial toxins and engineered immunotoxins have a distinct advantage over other targeted therapeutics in that toxins have evolved to efficiently and selectively translocate enzymes, i.e. toxin catalytic domains, into host cells. Further, toxin catalytic domains have evolved to efficiently alter specific host biological pathways including, but not limited to, signal transduction pathways (including Rho, MAPK, cAMP), protein synthesis, cytoskeletal dynamics and DNA replication (Alouf, 2000, Met. Mol. Biol. 145: 1-26).

Tumor endothelial marker 8 (TEM8) is a plasma membrane protein originally found to be upregulated on tumor endothelium associated with human colorectal cancer (St. Croix et al, 2000, Science 289: 1197-1202). Subsequent analysis of xenograft and naturally occurring tumors in mouse and human demonstrated that TEM8 is expressed at high levels in tumor neovasculature and stroma but is undetectable in many normal adult tissues, including new blood vessels associated with wound healing and corpus luteum (Carson- Walter et al, 2001, Cancer Res. 61 :6649-6655; Davies et al, 2006, Int. J. Oncol. 29: 1311-1317; Rmali et al., 2005, World J. Gastroenterol. 11 : 1283- 1286; Rmali et al, 2004, Int. J. Mol. Med. 14:75-80; Felicetti et al, 2007, Cytotherapy 9:23-34; Chaudhary et al, 2012, Cancer Cell 21 :212-226; Chaudhary and St. Croix, 2012, Cell Cycle 11 :2253-2259). Despite the obvious benefit of TEM8 as a tumor marker, it has proven difficult to generate reagents that specifically recognize the conserved extracellular domain of this protein (Chaudhary and St. Croix, 2012, Cell Cycle 11 :2253-2259). Therefore, development of a toxin that binds specifically to TEM8 is of great value for delivery of therapeutics as well as tumor imaging.

Anthrax lethal toxin (LT) is a proteinaceous bacterial toxin produced by

Bacillus anthracis. LT comprises two separate proteins: an 83 kDa cell binding subunit, protective antigen (PA), and a 90 kDa catalytic subunit, lethal factor (LF). PA binds to specific receptors on host cells and must be proteolytically activated by host furin-like proteases in order to oligomerize then bind and translocate LF into the host cell.

Importantly, TEM8 has been identified as an anthrax toxin receptor (Bradley et al., 2001, Nature 414:225-229). Anthrax toxin binds cell-surface TEM8 with nanomolar affinity and oligomerizes to generate high avidity complexes that bind nearly irreversibly to TEM8 on cell surfaces. The identification of TEM8 as a receptor for LT has implications for cancer targeting. Although anthrax toxin is currently being developed as a therapeutic for various forms of cancer (Alfano et al, 2008, Cell Cycle 7:452-461; Cryan and Rogers, 2011, Frontiers in Bioscience 16: 1574-1588; Frankel et al, 2003, Curr. Pharm. Des. 9:2060-2066; Frankel et al, Curr. Protein Pept. Sci. 3:399-407), such approaches are complicated by the existence of a second receptor for this toxin called capillary morphogenesis gene 2 (CMG2) (Scobie et al, 2003, Proc. Natl. Acad. Sci. USA

100:5170-5174). While TEM8 is tumor-specific (Carson- Walter et al, 2001, Cancer Res. 61 :6649-6655; Davies et al, 2006, Int. J. Oncol. 29: 1311-1317; Rmali et al, 2005, World J. Gastroenterol. 11 : 1283-1286; Rmali et al, 2004, Int. J. Mol. Med. 14:75-80; Felicetti et al, 2007, Cytotherapy 9:23-34), CMG2 is expressed ubiquitously on normal adult cells (Scobie et al, 2003, Proc. Natl. Acad. Sci. USA 100:5170-5174). CMG2 is required for toxin-induced pathology and is the physiologically relevant anthrax toxin receptor in normal (i.e. non-cancerous) adult tissues (Liu et al, 2009, Proc. Natl. Acad. Sci. USA 106: 12424-12429). Expression of CMG2 on most adult cell types has limited the use of LT as an anti-cancer therapeutic agent. Indeed, the therapeutic index for LT in mice with experimentally induced melanoma is 1.3 (Abi-Habib et al., 2006, Clin. Cancer Res.

12:7437-7443), indicating that the dose required to achieve maximal tumor regression is very close to the toxic dose. Despite a decade of research to develop anthrax toxin as a therapeutic to treat various cancers, the cellular targets responsible for anti-tumor activity are not known.

Wild type anthrax toxin was tested in mouse models more than a decade ago as a treatment for melanoma, based on the finding that the majority of melanoma cells require MAPK signaling, which is blocked by LF (Koo et al., 2002, Proc. Natl. Acad. Sci. 99:3052-3057). However, other observations indicated that LT could reduce xenograft tumor burden from cancer cells that are resistant to LT (Duesbery et al., 2001, Proc. Natl. Acad. Sci. USA 98:4089-4094). Further, LT displays anti-tumor activity against a wide range of cancers including fibrosarcoma, neuroblastoma, and non-small cell lung and renal cell carcinoma models in mice (Frankel et al., 2002, Protein Pept. Lett. 9: 1-14; Huang et al, 2008, Cancer Res. 68:81-88; Rouleau et al, Int. J. Oncol. 32:739- 748; Su et al, 2007, Cancer Res. 67:3329-3336; Abi-Habib et al, 2006, Mol. Cancer Ther. 5:2556-2562; Liu et al, 2008, J. Biol. Chem. 529-540). These findings led to the hypothesis that LT may elicit anti-tumor effects by targeting tumor vasculature (Alfano et al, 2008, Cell Cycle 7:452-461; Bradley and Young, 2003, Biochem. Pharmacol. 65:309- 314; Liu et al, Exp. Opin. Biol. Ther. 3:843-853). However, this hypothesis has not been tested to date. Many cell types, both normal and cancerous, express CMG2 and are therefore intoxicated by LT (Scobie et al, 2003, Proc. Natl. Acad. Sci. USA 100:5170- 5174; Rouleau et al, 2006, Int. J. Oncol. 32:739-748).

ECs are highly dependent on MAPK signaling during angiogenesis (Depeille et al., 2007, Oncogene 26: 1290-1296), and the combination of elevated TEM8 expression and increased dependence on MAPKK signaling is predicted to make these cells especially sensitive to LT (Alfano et al, 2008, Cell Cycle 7:452-461). In some instances, other catalytic subunits with more potent cytotoxicity display increased efficacy (Rono et al., 2006, Mol. Cancer Ther. 5:89-96). A hybrid toxin based on urokinase plasminogen activator-activated PA combined with a fusion of LFn and Pseudomonas exotoxin A (FP-59) showed anti-tumor activity, but still targeted CMG2 and was limited by off-target toxicity (Rono et al., 2006, Mol. Cancer Ther. 5:89-96).

One critical barrier associated with LT as a therapeutic is that PA binds to both TEM8 and CMG2, and CMG2 is expressed on a wide variety of normal cell types (Scobie et al, 2003, Proc. Natl. Acad. Sci. USA 100:5170-5174; Deshpande et al, 2006, FEBSLett. 580:4172-4175). In contrast, TEM8 is a validated marker of tumor

endothelium that is not expressed in normal adult angiogenic processes such as wound healing, menstruation, or ovulation (Carson- Walter et al., 2001, Cancer Res. 61 :6649- 6655; Felicetti et al, 2007, Cytotherapy 9:23-34; Chaudhary et al, 2012, Cancer Cell 21 :212-226; Chaudhary and St. Croix, 2012, Cell Cycle 11 :2253-2259; Cullen et al, 2009, Cancer Res. 69:6021-6026; Duan et al, 2007, J. Natl. Cancer Inst. 99: 1551-1555; Fernando and Fletcher, 2009, Cancer Res. 69:5126-5132; Nanda and St. Croix, 2004, Curr. Opin. Oncol. 16:44-49). Antibodies targeting TEM8 exhibit anti-tumor activity in mouse syngeneic and human xenograft models of melanoma, colon and lung cancers, likely by directly blocking TEM8 function (Chaudhary et al., 2012, Cancer Cell 21 :212- 226; Chaudhary and St. Croix, 2012, Cell Cycle 11 :2253-2259). Fusing truncated tissue factor to an anti-TEM8 antibody induced thrombosis in tumor vasculature in a mouse model of colorectal cancer, resulting in 55% inhibition of tumor growth and a 15% cure rate (Fernando and Fletcher, 2009, Cancer Res. 69:5126-5132).

The use of LT as a potential imaging agent has also been complicated by the nonselective binding of PA between TEM8 and CMG2. A peptide derived from PA conjugated to the positron-emitting radionuclide ¹⁸F was recently reported as a novel PET imaging agent for TEM8-expressing tumors (Quan et al., 2011, Eur. J. Nuc. Med. Mol. Imag. 38: 1806-1815). However, this peptide is predicted to bind CMG2 as well as TEM8. Despite this limitation, the ¹⁸F-peptide demonstrated higher tumor labeling in mice with UM-SCC1 xenograft tumors, which express high levels of TEM8, compared with MD- MB-435 xenografts, which express low levels of TEM8 (Quan et al., 2011, Eur. J. Nuc. Med. Mol. Imag. 38: 1806-1815), indicating TEM8 is a viable target for PET- based imaging of tumors.

LT has been used to treat multiple cancers in animal models (Felicetti et al, 2007, Cytotherapy 9:23-34; Frankel et al, 2003, Curr. Pharm. Des. 9:2060-2066; Cullen et al, 2009, Cancer Res. 69:6021-6026; Koo et al, 2002, Proc. Natl. Acad. Sci. 99:3052-3057; Rouleau et al, 2008, Int. J. Oncol. 32:739-748; Liu et al, 2008, J. Biol. Chem. 283:529-540; Liu et al, 2003, Exp. Opin. Biol. Ther. 3:843-853; Abi-Habib et al, 2005, Mol. Cancer Ther. 4: 1303-1310; Liu et al, 2000, Cancer Res. 60:6061-6067; Rono et al., 2006, Mol. Cancer Ther. 5:89-96; Venanzi et al., 2010, Cancer Immunol.

Immunother. 59:27-34; Wang et al, 2007, Curr. Top Med. Chem. 7: 1364-1378).

However, existing approaches are limited by toxicity in normal tissues. In order to increase tumor targeting by LT and reduce side effects, Leppla and colleagues engineered versions of PA in which the furin cleavage site, which is required for oligomerization and LF binding, is replaced with consensus cleavage sites for proteases upregulated on the surface of tumors (i.e. MMP-2/MMP-9, and urokinase plasminogen activator) (Liu et al., 2003, Exp. Opin. Biol. Ther. 3:843-853; Liu et al, 2000, Cancer Res. 60:6061-6067; Liu et al, 2001, J. Biol. Chem. 276: 17976-17984; Liu et al, 2007, J. Biol. Chem. 283:529- 540; Alfano et al, 2009, Mol. Cancer Res. 7:452-461). Although these PA mutants induce tumor regression with reduced toxicity, this targeting method is imperfect and dose-limiting toxicity was still observed (Liu et al, 2008, J. Biol. Chem. 283:529-540; Rono et al, 2006, Mol. Cancer Ther. 5:89-96). Further, these mutants bound

preferentially to CMG2 excluding their use as imaging/detection agents. The toxicity and binding affinity of additional PA mutants have also been examined, however they have yet to be developed into a therapeutic (Rosovitz et al, 2003, J. Biol. Chem. 278:30936- 30944; Chen et al, 2007, J. Biol. Chem. 282:9834-9845; Mourez et al, 2003, PNAS 100: 13803-13808; Lacy et al, 2004, Proc. Natl. Acad. Sci. USA 101 :13147-13151).

There is a need in the art for compositions and methods that specifically and effectively target tumor but not normal angiogenesis and/or vascular integrity. The present invention addresses this unmet need.

SUMMARY OF THE INVENTION

The present invention relates to an isolated nucleic acid encoding a mutant form of anthrax protective antigen (PA) or fragment thereof. The mutant form of PA or fragment thereof preferentially binds to TEM8 as compared with binding of the mutant form of PA or fragment thereof to CMG2, and the mutant form of PA or fragment thereof includes a mutation at Y688.

The present invention also relates to an isolated peptide. The isolated polypeptide includes a mutant form of anthrax protective antigen (PA) or fragment thereof, wherein the mutant form of PA or fragment thereof preferentially binds to TEM8 as compared with binding of the mutant form of PA or fragment thereof to CMG2, and further wherein the mutant form of PA or fragment thereof comprises a mutation at Y688.

The present invention also relates to a method of treating a subject with cancer. The method includes the steps of administering to the subject a therapeutically effective amount of a mutant form of PA or LT or fragment thereof, wherein the mutant form of PA or LT or fragment thereof preferentially binds to TEM8 as compared with binding of the mutant form of PA or LT or fragment thereof to CMG2, and further wherein the mutant form of PA or LT or fragment thereof comprises at least a mutation at Y688 within the PA subunit or fragment thereof. The present invention also relates to an imaging agent. The imaging agent includes a mutant form of PA or LT or fragment thereof, wherein the mutant form of PA or LT or fragment thereof preferentially binds to TEM8 as compared with binding of the mutant form of PA or LT or fragment thereof to CMG2, and further wherein the mutant form of PA or LT or fragment thereof comprises a mutation at Y688 within the PA subunit or fragment thereof.

The present invention also relates to a method of imaging a tumor. The method includes the steps of contacting a tumor tissue with an imaging agent, and detecting the presence of the imaging agent so contacted, wherein the imaging agent comprises a mutant form of PA or LT or fragment thereof, wherein the mutant form of PA or LT or fragment thereof preferentially binds to TEM8 as compared with binding of the mutant form of PA or LT or fragment thereof to CMG2, and further wherein the mutant form of PA or LT or fragment thereof comprises a mutation at Y688 within in the PA subunit or fragment thereof.

The present invention also relates to a therapeutic delivery agent. The agent includes a mutant form of PA or LT or fragment thereof, wherein the mutant form of PA or LT or fragment thereof preferentially binds to TEM8 as compared with binding of the mutant form of PA or LT or fragment thereof to CMG2, and further wherein the mutant form of PA or LT or fragment thereof comprises a mutation at Y688 within the PA subunit or fragment thereof.

The present invention also relates to a delivery system for delivering a therapeutic agent. The system includes a therapeutic delivery agent, wherein the therapeutic delivery agent specifically binds to a tumor for targeted delivery of a therapeutic agent to the tumor, further wherein the therapeutic delivery agent comprises a mutant form of PA or LT or fragment thereof, wherein the mutant form of PA or LT or fragment thereof preferentially binds to TEM8 as compared with binding of the mutant form of PA or LT or fragment thereof to CMG2, and further wherein the mutant form of PA or LT or fragment thereof comprises a mutation at Y688 within the PA subunit or fragment thereof. In some embodiments, the Y688 mutation is selected from the group consisting of Y688G, Y688A, Y688T and Y688V. In some embodiments, the mutant form of PA or fragment thereof comprises at least three mutations. In some

embodiments, the at least three mutations are R659S, M662R, and Y688G. In some embodiments, the mutant form of PA or fragment thereof comprises a cysteine. In some embodiments, the mutant form of PA or fragment thereof additionally comprises the mutation K563C. In some embodiments, the imaging agent includes a detectable label. In some embodiments, the detectable label is a radionuclide. In some embodiments, the radionuclide is chelated by DOTA-maleimide. In some embodiments, the imaging method is positron emission tomography.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

Figure 1, comprised of Figures 1A-1B, depicts TEM8-specific PA. Figure 1A is a graph depicting cells expressing TEM8. Figure IB is a graph depicting cells expressing CMG2. For Figures 1A-1B, cells were challenged with an increasing amount of indicated PA mutant and 5 ng/mL of a modified catalytic subunit (LFnDTA). Forty- eight hours later, cell viability was measured using ATPlite (PerkinElmer). Relative luminescence units (RLU) directly correlate with viability.

Figure 2 is a graph depicting C57BL/6 mice (n=10/group) which were challenged with anthrax toxin. Toxin was delivered in 8 doses, delivered i.p. every other day at a PA:LF ratio of 5: 1. Key: 24, 48 and 96 indicate total mass of LF delivered, in μg per mouse; wt = wild type PA; 2M = PA(R659S/M662R); 3M =

PA(R659S/M662R/Y688G). Figure 3 is a graph depicting how TEM8-specific PA+LF reduces B16F10 tumor burden similar to a sub-lethal dose of wt PA+LF. C57BL/6 mice were injected s.c. with 7xl0⁶ B16 cells on day 0. Anthrax toxin or saline was injected on days 1, 3, 5, and 7, and tumor volumes were measured daily. Animals received a total of 48 μg LF with PA-3M or 24 μg LF with WT-PA. The experiment was halted at Day 11 as tumors in animals receiving saline reached the maximum allowed size and were euthanized. * p < 0.05 compared to saline control.

Figure 4, comprised of Figures 4A-4E, depicts sequences of anthrax protective antigen (pagA) of Bacillus anthracis. Figure 4A depicts the nucleic acid sequence of anthrax protective antigen (SEQ ID NO: 1). Start/stop codons are in bold text. Figure 4B depicts the amino acid sequence of anthrax protective antigen (SEQ ID NO: 2). Figure 4C depicts the signal peptide and four domain regions within the amino acid sequence of anthrax protective antigen (SEQ ID NO:2). Figure 4D depicts the amino acid sequence of anthrax protective antigen domains 3 and 4 (SEQ ID NO: 3). Figure 4E depicts the amino acid sequence of anthrax protective antigen domain 4 (SEQ ID NO: 4).

Figure 5, comprising Figures 5A and 5B, are graphs depicting an in vitro study demonstrating that mutant PA does not target melanoma cells directly, and instead supports the role of targeting vasculature in anti-tumor activity.

Figure 6 is a set of graphs demonstrating the ability to deliver alternative payloads to tumor cells in vivo.

DETAILED DESCRIPTION

The present invention relates to the discovery that a mutant subunit of anthrax lethal toxin (LT), the PA subunit, selectively targets cellular receptor TEM8 as compared with CMG2. TEM8 is expressed at high levels in tumor neovasculature and stroma, but is undetectable in many normal adult tissues. CMG2 is expressed

ubiquitously on normal adult cells, and is the physiologically relevant anthrax toxin receptor in normal, non-cancerous adult tissues. Therefore, the present invention provides compositions and methods of treating or preventing a disease in a subject by administering a composition comprising a mutant form of the protective antigen (PA) subunit of anthrax lethal toxin (LT), where the mutant PA subunit preferentially binds to TEM8 as compared with CMG2. In some embodiments, the mutant form of PA has at least three mutations located within it. In other embodiments, the mutant form of PA comprises the mutations R659S, M662R, and Y688G of the mature peptide lacking the signal peptide (or alternatively numbered R688S, M691R and Y717G, respectively, when including the leading 29 amino acids of the signal peptide).

The present invention also relates to compositions and methods for imaging applications using the mutated forms of PA described herein. In some embodiments, the present invention provides imaging agents and methods for their use in positron emission topography (PET). The present invention also provides compositions and methods related to the delivery of therapeutic agents using the mutated forms of PA described herein.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

As used herein, each of the following terms has the meaning associated with it in this section.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

"About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. The term "abnormal" when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cells or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from those organisms, tissues, cells or components thereof that display the "normal" (expected) respective characteristic. Characteristics which are normal or expected for one cell or tissue type, might be abnormal for a different cell or tissue type.

A "disease" is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.

In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.

A disease or disorder is "alleviated" if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a patient, or both, is reduced.

An "effective amount" or "therapeutically effective amount" of a compound is that amount of compound which is sufficient to provide a beneficial effect to the subject to which the compound is administered. An "effective amount" of a delivery vehicle is that amount sufficient to effectively bind or deliver a compound.

"Allogeneic" refers to a graft derived from a different animal of the same species.

"Xenogeneic" refers to a graft derived from an animal of a different species.

The term "xenograft," as used herein, is synonymous with the term

"heterograft" and refers to a graft transferred from an animal of one species to one of another species.

The term "cancer" as used herein is defined as disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like.

The terms "patient," "subject," "individual," and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.

The term "therapeutic" as used herein means a treatment and/or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state or disorder.

The term "therapeutically effective amount" refers to the amount of the subject compound that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term "therapeutically effective amount" includes that amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.

To "treat" a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

The term "delivery vehicle," as used herein, refers to a molecule or composition useful for binding or carrying another molecule, such as a nucleic acid or drug, and delivering it to a target site, such as a cell. "Delivery vehicle" is used interchangeably with terms such as "drug delivery vehicle" "nucleic acid delivery vehicle," or "delivery vector."

As used herein, an "instructional material" includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the compositions and methods of the invention. The instructional material of the kit of the invention may, for example, be affixed to a container which contains the nucleic acid, peptide, and/or composition of the invention or be shipped together with a container which contains the nucleic acid, peptide, and/or composition. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively by the recipient.

"Isolated" means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified

polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

As used herein, the term "substantially the same" amino acid sequence is defined as a sequence with at least 70%, preferably at least about 80%>, more preferably at least about 85%, more preferably at least about 90%, even more preferably at least about 95%, and most preferably at least 99% homology with another amino acid sequence, as determined by the FASTA search method in accordance with Pearson & Lipman, 1988, Proc. Natl. Inst. Acad. Sci. USA 85:2444-48.

The term "amino acid sequence variant" refers to polypeptides having amino acid sequences that differ to some extent from a native sequence polypeptide. Ordinarily, amino acid sequence variants will possess at least about 70% homology, or at least about 80%, or at least about 90% homology to the native polypeptide. The amino acid sequence variants possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence of the native amino acid sequence.

A "variant" as the term is used herein, is a nucleic acid sequence or a peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence respectively, but retains essential properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of peptide variants are typically limited or conservative, so that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical. A variant and reference peptide can differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A variant of a nucleic acid or peptide can be a naturally occurring such as an allelic variant, or can be a variant that is not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides may be made by mutagenesis techniques or by direct synthesis.

As used herein, the term "fragment," as applied to a nucleic acid, refers to a subsequence of a larger nucleic acid. As used herein, the term "fragment," as applied to a protein or peptide, refers to a subsequence of a larger protein or peptide. For example, with regard to a fragment of the PA subunit of LT, the fragment may have any subsequence that is less than the entire sequence of the wild type or full length PA subunit. In some instances and without limitation, the PA subunit fragment may include at least domain 4, or at least domains 3 and 4.

The term "preferentially binds" and grammatical variations thereof, as used herein, refers to the ability of the mutant form of PA to bind TEM8 with at least a 10-fold higher affinity than it binds CMG2 and/or displays at least 10-fold more binding or functional activity under equivalent experimental conditions.

The term "intoxication," as used herein, refers to the mechanism by which anthrax lethal toxin penetrates a cell, disrupting various cell functions, such as cellular signaling and cell migration, and possibly inducing cell lysis. First, PA binds to a receptor on the host cell, followed by proteolytic cleavage of PA to remove a 20-kDa fragment of PA. The remaining 63-kDa fragment of PA then oligomerizes, forming a complex comprised of a heptameric prepore and bound molecules of LF and/or edema factor (EF). Alternatively, proteolytic cleavage of PA, oligormerization and binding of LF and/or EF may occur prior to cell binding. Regardless, the cell-bound toxin complex is internalized into an endosome by receptor-mediated endocytosis, resulting in increasing acidity and inducing a conformation change in the prepore. The

conformational change permits penetration of the prepore into the endosomal membrane and formation of a pore through which LF or EF may translocate into the cytosol of the host cell.

The term "DOTA-maleimide" as used herein refers to 2,2',2"-(10-(2-((2- (2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)ethyl)amino)-2-oxoethyl)-l,4,7,10- tetraazacyclododecane- 1 ,4,7-triyl)triacetic acid.

By "mutant form of protective antigen (PA)" as used herein means a PA subunit of LT that comprises a mutation.

By "mutant form of anthrax lethal toxin (LT)" as used herein means an LT protein that comprises a mutant form of PA.

As described herein, amino acid mutant "K563" and "K592" correspond to the same amino acid location of the PA subunit of LT. As numbered, K563 corresponds to the amino acid location of a mature PA that lacks the 29 amino acid signal peptide region, while K592 corresponds to the amino acid location of PA that includes the 29 amino acid signal peptide region.

As described herein, amino acid mutant "R659" and "R688" correspond to the same amino acid location of the PA subunit of LT. As numbered, R659 corresponds to the amino acid location of a mature PA that lacks the 29 amino acid signal peptide region, while R688 corresponds to the amino acid location of PA that includes the 29 amino acid signal peptide region.

As described herein, amino acid mutant "M662" and "M691" correspond to the same amino acid location of the PA subunit of LT. As numbered, M662 corresponds to the amino acid location of a mature PA that lacks the 29 amino acid signal peptide region, while M691 corresponds to the amino acid location of PA that includes the 29 amino acid signal peptide region.

As described herein, amino acid mutant "Y688" and "Y717" correspond to the same amino acid location of the PA subunit of LT. As numbered, Y688 corresponds to the amino acid location of a mature PA that lacks the 29 amino acid signal peptide region, while Y717 corresponds to the amino acid location of PA that includes the 29 amino acid signal peptide region.

Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to

6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

Description

The present invention relates to the discovery that a mutant form of the PA subunit of anthrax lethal toxin (LT) selectively targets the cellular receptor TEM8 as compared with CMG2, and preferentially binds TEM8 as compared with binding to CMG2. TEM8 is expressed at high levels in tumor neovasculature and stroma, but is undetectable in many normal tissues. CMG2 is expressed ubiquitously on normal cells, and is the physiologically relevant anthrax toxin receptor in normal, non-cancerous tissues. Therefore, the present invention provides compositions and methods of treating cancer in a subject by administering a composition comprising a mutant form of the PA subunit of LT, where the mutant PA subunit preferentially binds to TEM8 as compared with CMG2. In some embodiments, the mutant form of PA has at least three mutations located within the PA subunit of LT. In other embodiments, the mutant form of PA comprises the mutations R659S, M662R, and Y688G of the mature peptide lacking the signal peptide (or alternatively numbered R688S, M691R and Y717G, respectively, when including the leading 29 amino acids of the signal peptide).

The present invention also relates to compositions and methods of detecting tumors using the mutated forms of PA described herein. In some

embodiments, the present invention provides imaging agents and methods for their use in positron emission topography (PET). The present invention also provides compositions and methods related to the delivery of therapeutic agents using the mutated forms of PA described herein.

The invention includes methods for treatment of cancer by administering to a subject having cancer a mutant form of PA or LT, which mutant form preferentially binds to cellular receptor TEM8 as compared with CMG2. TEM8 is a validated marker of tumor endothelium as well as a known anthrax toxin receptor. Endothelial cells (ECs) that make-up tumor vasculature are derived from non-cancerous tissues. As a result, tumor vasculature represents a single therapeutic target for a variety of cancers.

Advantages of targeting the tumor vasculature rather than the cells that comprise the tumor include: 1) vasculature represents a common target for many solid tumors based on common requirement for blood flow; 2) destruction of vasculature leads to death of many more tumor cells due to nutrient deprivation, providing an amplification of therapeutic effect; 3) ECs are derived from non-cancerous or normal host tissue, and are less likely to undergo somatic mutation, resulting in resistance to therapy; and, 4) the potential for synergy with standard chemotherapies. However, a major challenge associated with the use of LT as a therapeutic is that the PA subunit of LT binds to both TEM8 and CMG2, which is expressed on a wide variety of normal cell types. Thus, the present invention provides compositions and methods for treating cancer in a subject which are selective for TEM8 as compared with CMG2.

Mutant Forms of PA

The mutant forms of PA described herein may be produced by any method known in the art. PA mutants that target TEM8 specifically may be even more potent than other TEM8 targeting agents such as monoclonal antibodies as these toxins mutants not only block TEM8 function, but also provide targeted delivery of enzymatic payloads into tumor ECs. TEM8-preferential binding by the mutant form of PA of the present invention combines the advantages of using a monoclonal antibody along with tumor- specific delivery of bacterial enzymes to increase disruption of endothelium and tumors. As would be understood by the skilled artisan, methods for assessing binding of the mutant form of PA to either TEM8 or CMG2 may be accomplished using any method known in the art. In some embodiments, the mutant form of PA exhibits a 10-fold, or 100-fold, or 1000-fold, or 10,000-fold, or 100,000-fold, and any and all ranges therebetween, increase in binding specificity for TEM8 when compared with binding to CMG2.

In one embodiment, the mutant form of PA comprises at least one mutation in the PA subunit of LT. In another embodiment, the mutant form of PA comprises at least two mutations in the PA subunit of LT. In yet another embodiment, the mutant form of PA comprises at least three mutations in the PA subunit of LT. In a further embodiment, the mutant form of PA comprises at least four mutations in the PA subunit of LT. In yet another embodiment, the mutant form of PA comprises at least five mutations in the PA subunit of LT. Referring to the sequence of wild type (WT) PA (Figure 4A: nucleic acid sequence: SEQ ID NO 1; Figure 4B amino acid sequence: SEQ ID NO 2; Table 2), in one embodiment, the mutant form of PA comprises a mutation at Y688. While the preferred mutation is Y688G, the invention should be construed to encompass any other mutation at this site that results in preferential binding to TEM8 over CMG2. Non-limiting examples of such mutations include, for example, Y688A, Y688T, and Y688V. In some embodiments, the mutant form of PA comprises a mutation in Y688 in combination with at least one other mutation. In one embodiment, the mutant form of PA comprises the mutations Y688G and R659S. In another embodiment, the mutant form of PA comprises the mutations Y688G and M662R. Table 1 : Preferred Mutations of Amino Acids in Mutants of WT-PA

Mutations are noted in reference to WT-PA including the signal peptide (SEQ ID NO 2)

As described elsewhere herein, the present invention is based in part on the unexpected discovery that a mutant form of PA comprising the mutations R688S, M692R, and Y717G results in a significant loss of binding of PA and therefore LT to CMG2. This loss of binding results in an increased preferential binding of PA and therefore LT to TEM8. In some embodiments, the mutant form of PA comprises the mutation Y717G in combination with at least two other mutations. In a preferred embodiment, the mutant form of PA comprises the mutations R688S, M691R, and

Y717G. The skilled artisan will understand that the present invention contemplates any mutant form of PA which exhibits preferential binding of TEM8 as compared with CMG2 as defined elsewhere herein.

In other embodiments, the mutant form of PA may include any of the aforementioned mutations, and further comprise a shortened or truncated form of PA. For example, the mutant form of PA may comprise domains 3 and 4 of PA (amino acids 517-764 or SEQ ID NO: 3) or the mutant form of PA may comprise domain 4 of PA (amino acids 625-764 or SEQ ID NO:4). Such shortened or truncated forms comprising domains 3 and 4 have been shown to bind cell surface receptors (Cirino et al (1999)), and it was previously reported that domain 4 of PA fused with glutathione S-transferase (GST) is functional for binding to TEM8 (Bradley et al (2001)). Further, it has been reported that cysteine scanning mutagenesis of PA identified residues in domains 3 and 4 that would potentially tolerate site-specific labeling with PET probes or other cargo (Mourez et al (2003)). The present invention also contemplates mutant forms of PA of the present invention optionally comprising at least one mutation for the purpose of attaching a detectable label, as would be understood by a skilled artisan. By way of example, certain detectable labels require a cysteine residue for attachment. Because PA does not contain any cysteine residues, the mutant form of PA may contain an optional mutation in the PA subunit substituting at least one amino acid therein for a cysteine. While there are many sites in the PA subunit where a cysteine can be added using technology readily available to the skilled artisan, in a preferred embodiment, the mutant form of PA comprises the mutation K592C, although the invention should not be construed to be limited solely to this specific mutation.

As contemplated herein, one embodiment of the invention includes a peptide comprising the sequence of SEQ ID NO: 2 having at least one of the mutations described herein (e.g., mutations at at least one of K592, R688, M691, Y717, and any combinations thereof), so long as the peptides preferentially bind to TEM8 as compared to CMG2. In another embodiment, the invention includes a peptide comprising the sequence of SEQ ID NO: 3 (corresponding to domains 3 and 4 of the PA subunit) and having at least one of the mutations described herein, so long as the peptide preferentially binds to TEM8 as compared to CMG2. In yet another embodiment, the invention includes a peptide comprising the sequence of SEQ ID NO: 4 (corresponding to domain 4 of the PA subunit) and having at least one of the mutations described herein, so long as the peptide preferentially binds to TEM8 as compared to CMG2. It should be appreciated that the invention includes variants of SEQ ID NOs:2, 3 or 4, provided that the peptide still preferentially binds to TEM8 as compared to CMG2. Variants may have additional mutations in addition to those described herein, comprising insertions, deletions, or substitutions of amino acids. Variants preferably comprise conservative amino acid substitutions.

For example, in one embodiment, the peptide of the invention comprises a peptide having at least one of the mutations described herein (e.g., mutations at at least one of K592, R688, M691, Y717, and any combinations thereof), and having at least 75% homology with SEQ ID NOs: 2, 3 or 4. In one embodiment, the peptide of the invention comprises a peptide having at least one of the mutations described herein and having at least 80% homology with SEQ ID NOs: 2, 3 or 4. In one embodiment, the peptide of the invention comprises a peptide having at least one of the mutations described herein and having at least 85% homology with SEQ ID NOs: 2, 3 or 4. In one embodiment, the peptide of the invention comprises a peptide having at least one of the mutations described herein and having at least 90% homology with SEQ ID NOs: 2, 3 or 4. In one embodiment, the peptide of the invention comprises a peptide having at least one of the mutations described herein and having at least 95% homology with SEQ ID NOs: 2, 3 or 4. In one embodiment, the peptide of the invention comprises a peptide having at least one of the mutations described herein and having at least 99% homology with SEQ ID NOs: 2, 3 or 4. Included in the invention are nucleic acid sequences that encode the peptides of the invention. In one embodiment, the invention includes nucleic acid sequences corresponding to the amino acid sequence of SEQ ID NOs: 2, 3 or 4.

Accordingly, subclones of a nucleic acid sequence encoding a peptide mimetic of the invention can be produced using conventional molecular genetic manipulation for subcloning gene fragments, such as described by Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, New York (2012), and Ausubel et al. (ed.)

In addition, chemical synthesis can also be employed using techniques well known in the chemistry of proteins such as solid phase synthesis or synthesis in homogenous solution.

Variants of suitable peptides of the invention can also be expressed.

Variants may be made by, for example, the deletion, addition, or alteration of amino acids that have either (i) minimal influence on certain properties, secondary structure, and hydropathic nature of the polypeptide or (ii) substantial effect on one or more properties of the peptide of the invention.

Variants of the peptides of the invention can also be fragments of the peptides of the invention having at least one of the mutations described herein (e.g., mutations at at least one of K592, R688, M691, Y717, and any combinations thereof) and that include one or more deletion, addition, or alteration of amino acids of SEQ ID NOs: 2, 3 or 4. The substituted or additional amino acids can be either L-amino acids, D-amino acids, or modified amino acids. Whether a substitution, addition, or deletion results in modification of the activity of the peptide may depend, at least in part, on whether the altered amino acid is conserved. Conserved amino acids can be grouped either by molecular weight or charge and/or polarity of R groups, acidity, basicity, and presence of phenyl groups, as is known in the art.

The variants of the peptides according to the present invention may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, (ii) one in which there are one or more modified amino acid residues, e.g., residues that are modified by the attachment of substituent groups, (iii) one in which the peptide is an alternative splice variant of the peptide of the present invention, (iv) fragments of the peptides and/or (v) one in which the peptide is fused with another peptide. Variants may be post-translationally, or chemically modified. Such variants are deemed to be within the scope of those skilled in the art from the teaching herein.

As known in the art the "similarity" between two peptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one peptide to a sequence of a second peptide. Variants are defined to include peptide sequences different from the original sequence, preferably different from the original sequence in less than 40% of residues per segment of interest, more preferably different from the original sequence in less than 25% of residues per segment of interest, more preferably different by less than 10% of residues per segment of interest, most preferably different from the original protein sequence in just a few residues per segment of interest and at the same time sufficiently homologous to the original sequence to preserve the functionality of the original sequence. The present invention includes amino acid sequences that are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90%, or 95% similar or identical to the original amino acid sequence. The degree of identity between two peptides is determined using computer algorithms and methods that are widely known for the persons skilled in the art.

The peptides of the invention can be post-translationally modified. For example, post-translational modifications that fall within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding and proteolytic processing, etc. Some modifications or processing events require introduction of additional biological machinery.

The peptides of the invention may include unnatural amino acids formed by post-translational modification or by introducing unnatural amino acids during translation as would be understood by those skilled in the art. A peptide of the invention may be conjugated with other molecules, such as proteins, to prepare fusion proteins. This may be accomplished, for example, by the synthesis of N-terminal or C-terminal fusion proteins provided that the resulting fusion protein retains functionality as would be understood by those skilled in the art. As contemplated herein, any of the aforementioned mutant forms of PA or LT, or fragments thereof, may be used to deliver any toxin that can be fused or otherwise conjugated to the mutant form of PA or LT or fragment thereof. For example, the edema factor and/or lethal factor of the anthrax toxin may be conjugated to the mutant PA or LT or fragment thereof, or a lethal factor-diphtheria toxin conjugate (LFnDTA) may be delivered via the mutant form of PA or LT or fragment thereof. In a further embodiment, cytolethal distending toxin (Cdt) or a component thereof may be conjugated to the mutant PA or LT or fragment thereof. It should be appreciated that the present invention is not limited to any particular toxin, and therefore any toxin or other payload may be used with the present invention, provided such toxin or payload can be suitably conjugated, fused or otherwise deliverable to a TEM8 expressing cell when the mutant form of PA or LT or fragment thereof component binds to the targeted cell.

Methods

As described elsewhere herein, the present invention provides mutant forms of PA or LT that preferentially bind cells associated with a tumor, for example, non-cancerous cells that form the tumor vasculature, and actual tumor cells that express TEM8 as compared with binding to cells that express CMG2. Any of the herein- described mutants of PA is useful in methods for treating cancer in a subject alone or in combination with other methods for treating cancer and/or in conjunction with other therapeutic agents. As such, the present invention includes methods for the treatment of any cancer having tumor-associated cells expressing TEM8. Cancers that may be treated include tumors that are not vascularized, or not yet substantially vascularized, as well as vascularized tumors. The cancers may comprise non-solid tumors (such as hematological tumors, for example, leukemias and lymphomas) or may comprise solid tumors. Types of cancers to be treated with the mutated form of PA or LT of the invention include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas. Adult tumors/cancers and pediatric tumors/cancers may also be treated using the compositions of the invention.

Hematologic cancers to be treated include cancers of the blood or bone marrow. Non-limiting examples of hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non- Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

Non- limiting examples of solid tumors that can be treated using the compositions of the present invention include sarcomas and carcinomas, including fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,

rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors (such as a glioma (such as brainstem glioma and mixed gliomas), glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma

craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brain metastases). Generally, the mutated forms of PA or LT described herein may be used in the treatment and prevention of diseases that arise in individuals who are

immunocompromised. In certain embodiments, the mutated forms of PA or LT of the invention are used in the treatment of patients at risk for developing cancer. Thus, the present invention provides methods for the treatment or prevention of cancer comprising administering to a subject in need thereof, a therapeutically effective amount of a mutated form of PA or LT of the invention.

In one embodiment, a mutated form of PA or LT of the present invention is administered as monotherapy. However, in another embodiment, a mutated form of PA or LT of the present invention is administered in a combination therapy, as would be understood by a skilled artisan.

Pharmaceutical Compositions

The mutated forms of PA or LT of the present invention may be administered either alone, or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2 or other cytokines or cell populations. Briefly, pharmaceutical compositions of the present invention may comprise a mutant form of PA or LT, as described herein, in combination with one or more

pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. In addition, the mutant form of PA or LT can be conjugated with polyethylene glycol or other compound that serves to improve serum half-life of the mutant form of PA or LT.

Compositions of the present invention are preferably formulated for intravenous administration.

Pharmaceutical compositions comprising the mutant forms of PA or LT of the present invention may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials. A

pharmaceutical composition of the present invention may comprise any of the mutant forms of PA or LT described herein.

When "an immunologically effective amount," "an anti-tumor effective amount," "an tumor-inhibiting effective amount," or "therapeutic amount" is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising a mutated form of PA or LT described herein may be administered at a dosage of 10⁴ to 10⁹ cells/kg body weight, preferably 10⁵ to 10⁶ cells/kg body weight, including all integer values within those ranges. A mutant form of PA or LT may also be administered multiple times at these dosages. The mutant form of PA or LT can be administered using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al, New Eng. J. of Med. 319: 1676, 1988). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

The administration of the subject compositions may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (z^'.v.) injection, or intraperitoneally. In one embodiment, the mutated form of PA or LT of the present invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the mutated form of PA or LT of the present invention is preferably administered by i.v. injection. The mutated form of PA or LT may be injected directly into a tumor, lymph node, or site of infection.

In certain embodiments of the present invention, a mutated form of PA or LT of the present invention is administered to a patient in conjunction with {e.g., before, simultaneously or following) any number of relevant treatment modalities, including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, Cytarabine (also known as ARA-C) or natalizumab treatment for MS patients or efalizumab treatment for psoriasis patients or other treatments for PML patients. In further embodiments, the mutated form of PA or LT of the invention may be used in combination with chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAM PATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. These drugs inhibit either the calcium dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase that is important for growth factor induced signaling (rapamycin) (Liu et al., Cell 66:807- 815, 1991; Henderson et al, Immun. 73:316-321, 1991; Bierer et al, Curr. Opin. Immun. 5:763-773, 1993). In a further embodiment, the mutated form of PA or LT of the present invention of the present invention is administered to a patient in conjunction with (e.g., before, simultaneously or following) bone marrow transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another embodiment, the mutated form of PA or LT of the present invention is administered following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan. For example, in one embodiment, subjects may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, following the transplant, subjects receive an infusion of the mutated form of PA or LT of the present invention. In an additional embodiment, a mutated form of PA or LT is administered before or following surgery.

The dosage of the above treatments to be administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The scaling of dosages for human administration can be performed according to art-accepted practices. Imaging Agents

The invention also includes use of the mutant form of PA or LT of the invention as a diagnostic agent, e.g., as a tumor imaging agent. Any of the herein- described mutants of PA is useful as a diagnostic imaging agent alone or in combination with other diagnostic imaging agents and/or in conjunction with other therapeutic agents. The preferential binding to TEM8 as compared with CMG2 of the mutant forms of PA or LT of the present invention provides tumor-specific targeting for diagnostic or theragnostic purposes. The imaging agent can be used in vivo in human patients known to have or have had a TEM8-associated disorder. Optionally, the disorder includes TEM8-positive cells that are localized, e.g., in a solid tumor.

There are any number of methods available in the art that can be used to convert the mutant form of PA or LT of the present invention into an imaging agent. In one such method, a detectable label is added, either directly or indirectly to the mutant PA or LT. Non-limiting examples of such labels include a fluorophore or a positron emitting radionuclide, which when added to mutated PA, generates a mutated PA- or LT- based imaging agent. For example, a positron emitting radionuclide may be attached site-specifically to the mutated PA. In one embodiment, the positron emitting

radionuclide is ⁶⁴Cu²⁺ or ⁶⁸Ga³⁺ chelated by DOTA-maleimide. DOTA-maleimide is a metal chelating agent which attaches to thiol side chains of cysteine.

A target cell obtained from, or in a subject may be contacted with the mutated PA- or LT -based imaging agent. For example, a tumor sample may be contacted with the imaging agent that will bind any TEM8 expressed therein. The presence and/or level of TEM8 expressed in the sample is then assessed as a measure of for example, tumor burden, efficacy of a treatment regime, and the like. Methods of detection of binding of the imaging agent to TEM8 include imaging techniques such as PET (positron emission tomography), SPECT (single photon emission computed tomography), or computerized tomography. In one embodiment, the imaging technique is positron emission tomography. In another embodiment, the imaging technique is computerized tomography. In some embodiments, a combination of imaging techniques is used, as would be understood by one skilled in the art. In one embodiment, the imaging techniques are positron emission tomography and computerized tomography in combination.

Therapeutic Delivery Agents

Also contemplated in the present invention is the use of a mutant form of

PA or LT as a delivery vehicle for delivery of additional therapeutic agents to cells and tissues (e.g., a tumor). Any of the herein-described mutants of PA is useful as a therapeutic delivery agent alone or in combination with other therapeutic delivery agents and/or in conjunction with other therapeutic agents. The preferential binding to TEM8 as compared with CMG2 of the mutant forms of PA or LT of the present invention provides a therapeutic delivery agent with greatly reduced off-target liability. The therapeutic delivery agent may be used in vitro or in vivo to deliver a therapeutic agent directly to cells expressing TEM8. These TEM8-positive cells may be localized, e.g., in a solid tumor.

Any method known in the art to convert the mutant form of PA or LT of the present invention into a therapeutic delivery agent can be used, as would be understood by a skilled artisan. In one such method, one or more mutant forms of PA or LT are directly conjugated to a therapeutic agent for delivery to cells or tissues expressing TEM8. Such an approach is analogous to gemtuzumab (MYLOTARG ®; Wyeth), which consists of a humanized mAb targeted to a cell surface marker (CD33) linked to the cytotoxic antibiotic ozogamicin (N-acetyl-γ calicheamicin). Suitable therapeutic agents include, but are not limited to, radioactive isotopes, antifolates, vinca alkaloids, taxanes, cytokines, nucleic acids such as antisense RNA, ribozymes, short interfering RNA (siRNA), apoptosis-inducing ligands such as TRAIL, and

anthracyc lines. The mutant forms of PA or LT may also be conjugated to various nanoparticles that have desired properties, i.e. paramagnetic cores for magnetic resonance imaging or loaded with therapeutics including but not limited to anti-tumor drugs or siRNA. Therapeutic agents may be provided encapsulated in particles such as liposomes, which are in turn conjugated to one or more mutant forms of PA or LT of the present invention. In these embodiments, peptide linkers that are stable in serum but which can be readily degraded in intracellular compartments (e.g., disulfide linkers) may be superior to non-cleavable linkers. The mutated forms of PA or LT of the present invention may be conjugated to various catalytic subunits that block well-defined cellular processes without the need for chemical modifications, as would be understood by the skilled artisan. Examples of these cellular processes include, but are not limited to,

ERK/p38/JNK signaling, protein synthesis, and cytoskeletal dynamics.

In alternative aspects, a mutant form of PA or LT may be useful as a therapeutic delivery agent for directed enzyme prodrug therapy approach (analogous to ADEPT), which specifically aims at causing bystander effects by targeting enzymes to a targeted cell and delivering a prodrug that is locally converted to a chemotherapeutic by the targeted enzyme.

A target cell obtained from, or in, a subject may be contacted with the mutated LT -based therapeutic delivery agent. By way of example, a tumor may be contacted with the therapeutic delivery agent, which will bind to any TEM8 receptor expressed on the tumor. Thus, the therapeutic agent, which is a component of the therapeutic delivery agent, is delivered directly to the tumor.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure. Example 1 : Targeting Tumor Vasculature with Anthrax Toxin

Described herein is a novel mutant of anthrax toxin for use as a therapeutic agent in its own right and as a therapeutic-delivery agent and/or an imaging agent. Targeting the development of new blood vessels in growing tumors, as well as compromising the integrity of established tumor vasculature, is an important treatment strategy for a variety of cancers. The success of such approaches relies on specific cell- surface markers that enable delivery of therapeutics to tumor vasculature while sparing non-cancerous blood vessels and tissues.

The materials and methods used and the results of these experiments are now described.

Construction of TEM8-Specific Mutant of PA

A mutated form of the PA subunit of LT that preferentially binds TEM8 as compared with binding to CMG2 has been generated. Amino acid position Y688 of the PA subunit of LT was mutated to alanine or glycine to generate Y688A or Y688G, respectively. Without wishing to be bound by any particular theory, it was hypothesized that the Y688 residue of PA would pose a steric clash with residues in TEM8 but not with residues in CMG2. Therefore, it was predicted that the Y688G or Y688A mutants would have a higher affinity for TEM8 without changing their affinity for CMG2. However, unexpectedly, no difference was observed in either mutant's ability to intoxicate cells expressing either TEM8 or CMG2. A double mutation in PA comprising R659S/M662R was generated and the Y688 mutations were tested in this background for preferential binding to TEM8 or CMG2. It was hypothesized that binding of this mutated form of the PA subunit of LT to TEM8 would improve, but that binding to CMG2 would not be affected.

Unexpectedly, the triple mutant [PA(R659S/M662R/Y688G); termed "PA-RMY" or "PA-3M" for 3 mutations, Table 1] resulted in intoxication of TEM8- expressing cells, but did not result in intoxication of CMG2 expressing cells at concentrations up to ~2 μ§/ηιΙ, (Figure 1). In addition, a significant decrease in binding of this triple mutant in the PA subunit of LT to CMG2 was observed.

The toxicity of PA(R659S/M662R) and PA-3M in mice was assessed in vivo when combined with the catalytic subunit of anthrax toxin, lethal factor (LF). The previously published TEM8-specific PA(R659S/M662R) (Table 1) exhibited a two-fold reduction in toxicity in mice when co-injected into the mice with LF. In contrast, injection of PA-3M + LF was non-toxic even when administered to the mice at the highest dose used (Figure 2).

PA-3M was next tested for its ability to treat melanoma using a B16 melanoma model in immunocompetent C57BL/6 mice. Administration of PA-3M to these mice resulted in reduced tumor growth that was similar to the situation when the mice were administered WT-PA in combination with LF (Figure 3). Importantly, one out of six animals receiving toxin comprised of WT-PA + LF (24 μg) became moribund as a result of the toxin treatment prior to the experimental endpoint. In contrast, none of the animals receiving the mutant PA-3M + LF (48 μg) presented toxin-mediated clinical signs. Therefore, PA-3M provides protection from toxicity associated with

administration of anthrax toxin as a treatment for melanoma in an animal model.

TEM8-Specific PA-3M Preferentially Targets Tumors In Vivo

In this set of experiments, anthrax toxin, and specifically PA-3M, is examined for whether it manifests anti-tumor activity by preferentially targeting tumors in which TEM8 expression is high. First, the distribution and localization of WT-PA and PA-3M in tumor-free and tumor-bearing mice is determined. PA localization is monitored in real time using micro Positron Emission Tomography (PET) and

Computerized Tomography (CT) imaging. Importantly, these studies provide a novel imaging reagent to detect tumor endothelium based on radiolabeled PA-3M. It was hypothesized that tumor targeting by PA or LT may be more efficient if both the vasculature and tumor cells express TEM8. Thus, a determination of whether PA-3M preferentially localizes to tumors, and whether targeting is influenced by TEM8 expression on tumor cells as well as tumor endothelium, is assessed. In order to develop PA or LT for PET imaging, a radiolabeling protocol was established that enables site-specific attachment of positron emitting radionuclides. The PA subunit of LT contains no cysteine residues. Prior cysteine-scanning

mutagenesis of the entire PA protein identified several amino acid positions where cysteine mutations did not affect receptor binding, oligomerization, or toxin activity (Mourez et al, 2003, Proc. Natl. Acad. Sci. USA 100:13803-13808). One such mutant named K563C was used in the experiments now described (Table 1). This mutation was introduced into WT-PA and PA-3M. A metal-chelating agent developed for attachment to the thiol side chain of cysteine (DOTA-maleimide; Macrocyclics, Texas, U.S.) was then conjugated to each molecule. DOTA-maleimide conjugation to K563C was highly efficient and no unlabeled protein was detected by non-reducing SDS-PAGE. DOTA- PA(K563C) displayed no defect in binding to TEM8 or intoxication of cultured cells when compared with WT-PA or unlabeled PA(K563C). Tumor Targeting of PA-3M for In Vivo Imaging Based on PET/CT

To target PA-3M to a tumor, DOTA-conjugated PA or LT mutants are labeled with the positron emitter ⁶⁴Cu²⁺. Because of slow on-rates for metal chelation by DOTA at room temperature, ⁶⁴Cu/DOTA-maleimide is pre-chelated at 90°C for 1 h, then conjugated to PA(K563C) mutants, which can be performed in as little as 10 min

(Wangler et al, 2009, Bio. Med. Chem. Lett. 19: 1926-1929). Conjugation and labeling are validated by instant thin layer chromatography (ITLC). In pilot experiments, receptor- binding activity is retained using a cell-binding assay followed by scintillation counting. After quality control, labeled PA or LT mutants are injected via tail vein into nude mice bearing subcutaneous xenograft tumors or tumor- free control animals and are detected using PET/CT at the UCLA Crump Institute.

To establish cell lines having defined levels of TEM8 expression, a TEM8-negative melanoma cell line (WM-115) (Rouleau et al, 2008, Int. J. Oncol.

32:739-748) is engineered to express high levels of TEM8 using lentiviral vectors.

Comparisons are made between mice bearing WM-115 (no expression of TEM8) and WM-115-TEM8 (expressing TEM8) xenografts. In addition to the fact that TEM8 expression is lacking in the WM-115 cell line, this cell line was chosen because WM-115 xenografts are resistant to the vascular targeting agent bevacizumab/Avastin (anti-VEGF antibody) (Adamcic et al., 2012, Neoplasia 14:612-623).

An initial pilot experiment is comprised of two animals per group, which are injected with ⁶⁴Cu/DOTA labeled PA or LT mutants followed immediately by microPET imaging at t= 1, 2, 3, 4, 5, 10, 15, 20, 30, 45, and 60 minutes. This time course is expected to be sufficient to monitor kinetics and final distribution of PA (serum half life of 6 min) (Liu et al, 2008, J. Biol. Chem. 283:529-540; Quan et al, 2011, Eur. J. Nuc. Med. Mol. Imag. 38: 1806-1815; Dadachova et al, 2008, Nucl. Med. Biol. 755-761), though additional time points are implemented as necessary (Quan et al., 2011, Eur. J. Nuc. Med. Mol. Imag. 38: 1806-1815). To determine whether tumor size influences targeting, mice are imaged at multiple stages of tumor development: early (before tumors are measurable by calipers), mid-phase (when tumor size is -500 mm³), and late [when tumors reach maximal size allowed (-2,000 mm³)]. Initial PA or LT dosage is based on concentrations used to treat tumors, but can easily be adjusted according to the PET signal detected (i.e. increased or decreased depending on signal strength). Based on ⁶⁴Cu labeling efficiency of DOTA-conjugated diabodies (Olafsen et al., 2004, Protein Eng. Des. Sel. 17:21-27), the initial dose is expected to correspond to -50-100 μθ of ⁶⁴Cu.

Following PET scanning, animals are subjected to microcomputed tomography (microCT) to detect anatomical features including subcutaneous tumors.

Based on the results obtained from these experiments, the studies are expanded to include 5-10 animals/group, focusing on the most informative PA-3M concentrations and time points. These studies will determine whether PA-3M preferentially localizes to tumors, whether tumor targeting is influenced by TEM8 expression in tumor cells, and whether there is a difference in targeting between early, mid, or late stage tumors. This

information will guide the use of PA-3M in additional preclinical studies aimed at developing therapeutic and imaging applications.

In other instances, tumor imaging may be inefficient due to pharmacokinetics (PK) and/or pharmacodynamics (PD) of full-length PA-3M (83 kDa monomer which can oligomerize to -500 kDa in blood). While PA displays similar PK as the recently reported ¹⁸F-peptide [serum half-life on the order of minutes with renal clearance (Huang et al, 2008, Cancer Res. 68:81-88; Quan et al, 2011, Eur. J. Nuc. Med. Mol. Imag. 38: 1806-1815; Dadachova et al.,2008, Nucl. Med. Biol. 35:755-761)], PK/PD of PA in tumor-bearing animals is unknown. Without wishing to be bound to any particular theory, it is possible that only a small percentage of injected PA is biologically active. Indeed, it has previously been reported that the majority of PA is rapidly cleared and only a small fraction associates with cells. However, this small fraction retained the full ability to bind and translocate LF (Moayeri et al., 2007, Infection and Immunity 75:5175-5184). The ability of PA-3M to specifically target tumors/tumor endothelium is tested using a functional toxin-translocation assay described elsewhere herein for the examination of PA-3M specificity using TEM8 expressing and non-expressing tumor models. In some instances, development of a tumor imaging agent involves truncated mutants of PA or LT that retain receptor binding and TEM8 specificity but more closely match the size of the published ¹⁸F-peptide. Indeed, N-terminally truncated mutants of PA with molecular weight of 32 or 16 kDa can be expressed as functional receptor binding proteins and these fragments retain the residues mutated in PA-3M

(Krishnanchettiar et al., 2003, Protein Exp. Pur. 27:325-330; Cirino et al., 1999, Infection and Immunity 67:2954-2963). Determination of Cell Types Targeted By TEM8-Specific PA-3M

To determine whether PA-3M manifests anti-tumor activity by specifically intoxicating tumor endothelium, the following experiments can be performed. While PET/CT imaging reveals whether WT-PA and/or PA-3M display tumor targeting in mice, the level of resolution provided by PET cannot reveal target cell-type specificity. Even if no preferential localization of PA-3M to tumors is found, functional targeting as seen by reduced tumor growth in the absence of toxicity (Figures 2 and 3) suggests that translocation of LF/LFnBLAM may still exhibit specificity for tumor endothelium. In addition to ECs, pericytes associated with tumor endothelium also express TEM8 and therefore may be targeted by PA-3M (Chaudhary et al., 2012, Cancer Cell 21 :212-226). Further, TEM8 is expressed in multiple cancer cell lines (Rouleau et al, 2008, Int. J. Oncol. 32:739-748), indicating that PA-3M may serve to target not only endothelium, but tumors themselves. To determine cell-type targeting by PA-3M, a powerful technology is employed, which is based on a genetically fused hybrid protein LFnBLAM, which consists of the N-terminal 254 amino acids of lethal factor (LFn) fused to beta-lactamase (Hu and Leppla, 2009, PLoS ONE 4:e7946). PA+LFnBLAM has been used to study cell type specificity of intoxication in murine spleens (Hu and Leppla, 2009, PLoS ONE 4:e7946). A similar approach is used to determine which cells are intoxicated using PA- 3M. A finding that PA-3M + LFnBLAM intoxicates only ECs and/or endothelium would support the hypothesis that toxin mediated tumor regression (Figure 3) is due primarily to alterations in tumor vasculature. If PA-3M + LFnBLAM intoxicates tumor cells, then alternative toxin subunits that directly kill cancer cells may prove to be more effective, as described elsewhere herein.

Examination of PA-3M Specificity Using TEM8 Expressing and Non-Expressing Tumor Models

PA-3M specificity is examined using TEM8 expressing and non- expressing tumor models as described elsewhere herein for tumor targeting of PA-3M using in vivo imaging based on PET/CT. Athymic nude mice bearing WM-115 and WM- 115-TEM8 xenografts, or tumor- free mice, are challenged with 200 μg WT-PA or PA- 3M combined with 200 μg LFnBLAM (based on LD90 dose of PA + LF). One hour post- challenge, mice are euthanized, tumors are extracted and tumor cells are dissociated as described (Chaudhary et al., 2012, Cancer Cell 21 :212-226). Cells are stained for TEM8, markers of endothelium (i.e. CD31) and the tumor itself [i.e. melanoma chondroitin sulfate proteoglycan (Richards et al, 2012, J. Int. Soc. Anal. Cytol. 81 :374-381)]. All samples are incubated with the fluorescent BLAM substrate CCF2-AM and analyzed by flow cytometry to identify cell types into which PA-3M delivered LFnBLAM in vivo. Known targets of WT-PA+LF, including lung epithelium and endothelium (Abi-Habib et al., 2005, Mol. Cancer Ther. 4: 1303-1310; Langer et al., 2012, Infection and Immunity) and splenocytes (Hu and Leppla, 2009, PLoS ONE 4:e7946) are harvested, stained with cell-type specific markers (ECs = CD31; lung epithelium = P2X7 and GABAA^; Splenocytes = CD1 lb, F4/80, CD1 lc, B220, CD4, and CD8) and analyzed by CCF2-AM to monitor tumor specificity. Although it is not expected that PA-3M targets lung or spleen, these are important controls to further test off-target effects and safety. Determination of Optimal Therapeutic Payload Delivered By PA-3M

PA-3M may bind and deliver toxin catalytic subunits directly into TEM8- expressing tumor cells, some of which may not depend on MAPKK signaling. To examine whether alternative catalytic subunits have increased efficacy in two tumor models, the following experiments are performed. First, the catalytic subunit of diphtheria toxin (DTA) is delivered in a PA-dependent manner using an LFnDTA fusion protein (Milne et al., 1995, Mol. Microbio. 15:661-666). DTA blocks protein translation and induces apoptosis in all cell types in which it gains entry. Second, a fusion of LFn and the catalytic domain of toxin B from Clostridium difficile (LFnTcdB) is tested (Spyres et al, 2001, Infection and Immunity 69:599-601). TcdB disrupts host-cell cytoskeletal dynamics by inactivating Rho family GTPases. It is predicted that direct inactivation of Rho family members by LFnTcdB disrupts tumor endothelium.

Two major factors are predicted to influence susceptibility of melanoma cells to PA-3M + LF: BRAF mutational status, dictating dependence on MAPKK signaling (Abi-Habib et al., 2005, Mol. Cancer Ther. 5:89-96), and TEM8 expression. To test the functional consequence of targeting tumor vasculature versus tumor cells, these factors are independently tested. Mouse xenograft models are compared using melanoma cells with different dependencies on BRAF/MAPK and thus susceptibility to LT. SK- MEL28 melanoma cells express the V600E mutant of BRAF, and are highly sensitive to LT in vitro with an IC50 value of 32 pM (Abi-Habib et al, 2005, Mol. Cancer Ther. 5:89-96). In contrast, a second human melanoma line, SK-MEL-5, is BRAF -independent and resistant to LT at the maximum dose tested (10,000 pM) (Abi-Habib et al., 2005, Mol. Cancer Ther. 5:89-96). Importantly, both cell lines express similar levels of TEM8. To determine the role of TEM8 expression in tumor cells versus tumor endothelium, WM-115/WM-l 15-TEM8 xenograft models are compared to other models described elsewhere herein. Nude mice are injected subcutaneously with the human melanoma cells described elsewhere herein, and tumors are allowed to grow until they are -500 mm³, though this size may be adjusted if tumor size influences targeting, as described elsewhere herein. Mice are injected every other day with saline (control) or PA-3M in combination with LF, LFnDTA, or LFnTcdB for a total of 4-8 treatments. The number of treatments and dosing are based on maximum tolerated doses in non-tumor bearing mice (Rouleau et al, 2008, Int. J. Oncol. 32:739-748; Abi-Habib et al, 2006, Clin. Cancer Res. 12:7437-7443). Tumor volume is measured for 60 days or until tumors reach 2,000 mm³, at which point animals are euthanized. Data is analyzed for differences in response to PA-3M + LF as a function of BRAF mutational status, i.e. whether BRAF-dependent tumors are more sensitive to LF, or whether targeting endothelium is sufficient.

Differences in response are compared between WM-115 and WM-115-TEM8 xenografts to determine if targeting vasculature with various catalytic subunits is sufficient for maximal anti-tumor effect, and if so, which catalytic subunit is optimal. These data will reveal whether PA-3M functions solely by targeting tumor vasculature or whether additional mechanism associated with targeting tumor cells are important, guiding efforts to optimize anthrax toxin-based therapeutics for cancer.

In some instances, overexpression of TEM8 in WM-115 cells may lead to changes in cell physiology and tumor behavior (Kim et al, 2011, PLoS ONE 6:e27425), thereby compromising the ability to directly compare tumors +/- TEM8. A TEM8 mutant that lacks the C-terminal cytosolic tail and therefore no longer contributes to signaling while maintaining the ability to take-up toxin may then be used (Liu and Leppla, 2003, J. Biol. Chem. 278:5227-5234). In some instances, comparison of BRAF dependence using two distinct cell lines, SK-MEL-5 and SK-MEL-28, may be complicated due to myriad differences between cell lines. If gross differences are noted in vivo, a paired set of cell lines may be used. BRAF-dependent cell lines have been selected in vitro for resistance to the BRAF inhibitor PLX4032/Vemurafenib (Nazarian et al, 2010, Nature 468:973- 977). The matched parental and BRAF- inhibitor-resistant lines are compared.

PA-RMY (or PA-3M Does Not Target B16 Melanoma Cells Directly Mouse melanoma B16-F10 cells were seeded on 384-well plates and treated with a constant amount of either LF or LFnDTA (a heterologous fusion protein consisting of the non-toxic N-terminus of LF genetically fused to the catalytic domain from diphtheria toxin (DTA)) in the presence of an increasing concentration of WT PA or PA-RMY as indicated on the x-axis of Figure 5 A. After 48 hours, cell viability was measured using ATP-lite reagent relative light units (RLU, y-axis of Figure 5B), directly correlate with cell viability).

As depicted in the results shown in Figure 5, it is demonstrated that LF (catalytic domain of LT) does not kill B16-F10 cells and that PA-RMY cannot deliver toxic LFnDTA into B16-F10 cells. These data support the conclusion that tumor regression in vivo is a result of targeting host cells (i.e. endothelial and/or stromal cells that make up tumor vasculature) rather than targeting the tumor directly.

PA-RMY (or PA-3M) in vivo Delivery of Alternative Payload

To demonstrate the ability to delivery alternative payloads to TEM8 expressing cells in vivo, C57BL/6 mice were implanted with B16-F10 melanoma tumor cells followed by injection of either saline or PA-RMY + LFnDTA at days 3, 5, 7, and 9 as indicated in Figure 6. Body weight was measured to monitor dose-limiting toxicity of toxin treatment (Figure 6, top). As shown in Figure 6, it is demonstrated that the toxin was tolerated. Tumor volume was measured daily and graphed (Figure 6, bottom). At day 11 , mice receiving PA-RMY + LFnDTA had tumors that were significantly smaller than those mice receiving saline (p<0.05 by Student's t test). N=10/group.

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

1. An isolated nucleic acid encoding a mutant form of anthrax protective antigen (PA) or fragment thereof, wherein the mutant form of PA or fragment thereof preferentially binds to TEM8 as compared with binding of the mutant form of PA or fragment thereof to CMG2, and further wherein the mutant form of PA or fragment thereof comprises a mutation at Y688.

2. The isolated nucleic acid of claim 1, wherein the Y688 mutation is selected from the group consisting of Y688G, Y688A, Y688T and Y688V.

3. The isolated nucleic acid of claim 2, wherein the Y688 mutation is

Y688G.

4. The isolated nucleic acid of claim 1 , wherein the mutant form of PA or fragment thereof comprises at least three mutations.

5. The isolated nucleic acid of claim 4, wherein the at least three mutations are R659S, M662R, and Y688G.

6. The isolated nucleic acid of claim 1 , wherein the mutant form of PA or fragment thereof comprises a cysteine.

7. The isolated nucleic acid of claim 6, wherein the mutant form of PA or fragment thereof additionally comprises the mutation K563C.

8. An isolated polypeptide comprising a mutant form of anthrax protective antigen (PA) or fragment thereof, wherein the mutant form of PA or fragment thereof preferentially binds to TEM8 as compared with binding of the mutant form of PA or fragment thereof to CMG2, and further wherein the mutant form of PA or fragment thereof comprises a mutation at Y688.

9. The isolated polypeptide of claim 8, wherein the Y688 mutation is selected from the group consisting of Y688G, Y688A, Y688T and Y688V.

10. The isolated polypeptide of claim 9, wherein the Y688 mutation is

Y688G.

11. The isolated polypeptide of claim 8, wherein the mutant form of PA or fragment thereof comprises at least three mutations.

12. The isolated polypeptide of claim 11 , wherein the at least three mutations are R659S, M662R, and Y688G.

13. The isolated polypeptide of claim 8, wherein the mutant form of PA or fragment thereof comprises a cysteine.

14. The isolated polypeptide of claim 13, wherein the mutant form of PA or fragment thereof additionally comprises the mutation K563C.

15. A method of treating a subject with cancer, the method comprising administering to the subject a therapeutically effective amount of a mutant form of PA or LT or fragment thereof, wherein the mutant form of PA or LT or fragment thereof preferentially binds to TEM8 as compared with binding of the mutant form of PA or LT or fragment thereof to CMG2, and further wherein the mutant form of PA or LT or fragment thereof comprises at least a mutation at Y688 within the PA subunit or fragment thereof.

16. The method of claim 15, wherein the Y688 mutation within the PA subunit or fragment thereof is selected from the group consisting of Y688G, Y688A, Y688T and Y688V.

17. The method of claim 16, wherein the Y688 mutation within the PA subunit or fragment thereof is Y688G.

18. The method of claim 15, wherein the mutant form of PA or LT or fragment thereof comprises at least three mutations located within the PA subunit or fragment thereof of LT.

19. The method of claim 18, wherein the at least three mutations located within the PA subunit or fragment thereof of LT comprise R659S, M662R, and Y688G.

20. The method of claim 15, wherein the subject is human.

21. An imaging agent comprising a mutant form of PA or LT or fragment thereof, wherein the mutant form of PA or LT or fragment thereof preferentially binds to TEM8 as compared with binding of the mutant form of PA or LT or fragment thereof to CMG2, and further wherein the mutant form of PA or LT or fragment thereof comprises a mutation at Y688 within the PA subunit or fragment thereof.

22. The imaging agent of claim 21 , wherein the Y688 mutation within the PA subunit or fragment thereof is selected from the group consisting of Y688G, Y688A, Y688T and Y688V.

23. The imaging agent of claim 22, wherein the Y688 mutation within the PA subunit or fragment thereof is Y688G.

24. The imaging agent of claim 21 , wherein the mutant form of PA or LT or fragment thereof comprises at least three mutations located within in the PA subunit or fragment thereof.

25. The imaging agent of claim 24, wherein the at least three mutations located within in the PA subunit or fragment thereof are R659S, M662R, and Y688G.

26. The imaging agent of claim 21 , wherein the mutant form of PA or LT or fragment thereof comprises a cysteine.

27. The imaging agent of claim 26, wherein the mutant form of PA or LT or fragment thereof additionally comprises the mutation K563C.

28. The imaging agent of claim 21, further comprising a detectable label.

29. The imaging agent of claim 28, wherein the detectable label is a radionuclide.

30. The imaging agent of claim 29, wherein the radionuclide is chelated by DOTA-maleimide.

31. A method of imaging a tumor, the method comprising: a) contacting a tumor tissue with an imaging agent; and b) detecting the presence of the imaging agent so contacted, wherein the imaging agent comprises a mutant form of PA or LT or fragment thereof, wherein the mutant form of PA or LT or fragment thereof preferentially binds to TEM8 as compared with binding of the mutant form of PA or LT or fragment thereof to CMG2, and further wherein the mutant form of PA or LT or fragment thereof comprises a mutation at Y688 within in the PA subunit or fragment thereof.

32. The method of claim 31 , wherein the Y688 mutation within the PA subunit or fragment thereof is selected from the group consisting of Y688G, Y688A, Y688T and Y688V.

33. The method of claim 32, wherein the Y688 mutation within the PA subunit or fragment thereof is Y688G.

34. The method of claim 31 , wherein the mutant form of PA or LT or fragment thereof comprises at least three mutations located within in the PA subunit or fragment thereof.

35. The method of claim 34, wherein the at least three mutations located within in the PA subunit or fragment thereof are R659S, M662R, and Y688G.

36. The method of claim 31 , wherein the mutant form of PA or LT or fragment thereof comprises a cysteine.

37. The method of claim 36, wherein the mutant form of PA or LT or fragment thereof additionally comprises the mutation K563C.

38. The method of claim 31 , wherein the imaging agent further comprises a detectable label.

39. The method of claim 38, wherein the detectable label is a radionuclide.

40. The method of claim 39, wherein the radionuclide is chelated by DOTA-maleimide.

41. The method of claim 31 , wherein the imaging method is positron emission tomography.

42. A therapeutic delivery agent comprising a mutant form of PA or LT or fragment thereof, wherein the mutant form of PA or LT or fragment thereof preferentially binds to TEM8 as compared with binding of the mutant form of PA or LT or fragment thereof to CMG2, and further wherein the mutant form of PA or LT or fragment thereof comprises a mutation at Y688 within the PA subunit or fragment thereof.

43. The therapeutic delivery agent of claim 42, wherein the Y688 mutation within the PA subunit or fragment thereof is selected from the group consisting of Y688G, Y688A, Y688T and Y688V.

44. The therapeutic delivery agent of claim 43, wherein the Y688 mutation within the PA subunit or fragment thereof is Y688G.

45. The therapeutic delivery agent of claim 42, wherein the mutant form of PA or LT or fragment thereof comprises at least three mutations located within in the PA subunit or fragment thereof.

46. The therapeutic delivery agent of claim 45, wherein the at least three mutations located within in the PA subunit or fragment thereof are R659S, M662R, and Y688G.

47. A delivery system for delivering a therapeutic agent comprising: a therapeutic delivery agent, wherein the therapeutic delivery agent specifically binds to a tumor for targeted delivery of a therapeutic agent to the tumor, further wherein the therapeutic delivery agent comprises a mutant form of PA or LT or fragment thereof, wherein the mutant form of PA or LT or fragment thereof preferentially binds to TEM8 as compared with binding of the mutant form of PA or LT or fragment thereof to CMG2, and further wherein the mutant form of PA or LT or fragment thereof comprises a mutation at Y688 within the PA subunit or fragment thereof.

48. The delivery system of claim 47, wherein the Y688 mutation within the PA subunit or fragment thereof is selected from the group consisting of Y688G, Y688A, Y688T and Y688V.

49. The delivery system of claim 48, wherein the Y688 mutation within the PA subunit or fragment thereof is Y688G.

50. The delivery system of claim 47, wherein the mutant form of PA or LT or fragment thereof comprises at least three mutations located within in the PA subunit or fragment thereof.

51. The delivery system of claim 50, wherein the at least three mutations located within in the PA subunit or fragment thereof are R659S, M662R, and Y688G.