WO2013066471A2

WO2013066471A2 - Methods and compositions for preventing and treating an anthrax toxin mediated condition in a subject

Info

Publication number: WO2013066471A2
Application number: PCT/US2012/050770
Authority: WO
Inventors: Mikhail MARTCHENKO; Sun Young Jeong; Stanley N. Cohen
Original assignee: The Board Of Trustees Of The Leland Stanford Junior University
Priority date: 2011-08-15
Filing date: 2012-08-14
Publication date: 2013-05-10
Also published as: WO2013066471A3

Abstract

Methods and compositions for preventing or treating an anthrax toxin mediated disease condition in an individual are provided. Aspects of the subject methods include administering to a subject an effective amount of an agent that inhibits cellular entry of an anthrax toxin mediated by complexes comprising a β1 integrin subunit. Also provided are active agents suitable for use in the subject methods, as well as pharmaceutical preparations thereof and kits thereof. Methods of screening candidate agents for anti-toxin activity are also provided.

Description

METHODS AND COMPOSITIONS FOR PREVENTING AND TREATING AN ANTHRAX TOXIN MEDIATED CONDITION IN A SUBJECT

GOVERNMENT RIGHTS

This invention was made with Government support under contract HDTRA1 -06-C- 0039 awarded by the Department of Defense/Defense Threat Reduction Agency. The Govt has certain rights in this invention..

CROSS REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. § 1 19 (e), this application claims priority to the filing date of the United States Provisional Patent Application Serial No. 61/523,693 filed August 15, 201 1 ; the disclosure of which is herein incorporated by reference.

BACKGROUND

Anthrax is a lethal disease of humans and other animals caused by the gram- positive spore-forming eubacterium, Bacillus anthracis. While this disease has long been endemic to most countries, its incidence in human populations largely has been controlled. However, the worldwide prevalence of B. anthracis and the ease of culturing this lethal microbe have focused much recent attention on anthrax as an agent of bioterrorism.

The virulence of B. anthracis infection is due to the cellular effects of separate complexes formed by interaction of a carrier protein, protective antigen (PA, 83 kDa) with lethal factor (LF, 90 kDa) or edema factor (EF, 89 kDa). During infection by B.anthracis, PA binds to receptors on the surface of macrophages and is cleaved by the protease furin, releasing a 20kDa fragment and enabling the residual receptor-bound C-terminal 63kDa PA peptide fragment (PA63) to form heptamers and interact with LF or EF. After entering target cells by endocytosis, PA LF and PA/EF complexes translocate across the endosomal membrane into the cytosol where they dissociate, allowing LF and EF to exert their toxic effects.

LF is a Zn++ protease that cleaves the amino terminus of all MAPK kinases and consequently is responsible for anthrax lethality. EF is a calmodulin-dependent adenylate cyclase that elevates intracellular levels of cAMP, producing profound edema as a typical clinical symptom and impairing the immune response to infection.

SUMMARY

Methods and compositions for modulating entry of an anthrax toxin into a cell are provided. These methods and compositions find a number of uses, including reducing or preventing anthrax susceptibility in a cell; treating anthrax toxicity in a cell; preventing an anthrax toxin mediated disease condition in a host; and treating an anthrax toxin mediated disease condition in a host. Aspects of the subject methods include administering to a subject an effective amount of an agent that inhibits cellular entry of an anthrax toxin mediated by complexes comprising a β1 integrin subunit. Also provided are active agents suitable for use in the subject methods, as well as pharmaceutical preparations thereof and kits thereof. Methods of screening candidate agents for anti-toxin activity are also provided.

Aspects of the invention include methods of inhibiting entry of an anthrax toxin into a cell. In some embodiments, the methods comprise contacting a cell with an effective amount of an agent, i.e., an anti-anthrax agent, which inhibits cellular entry of an anthrax toxin mediated by complexes comprising a β1 integrin subunit. In some embodiments, the anthrax toxin is selected from Lethal Factor (LF) and Edema Factor (EF).

In some embodiments, the agent that inhibits cellular entry of an anthrax toxin mediated by a β1 integrin subunit is an agent that inhibits binding of protective antigen (PA) to a complex comprising a β1 integrin subunit. In some embodiments, the complex comprising a β1 integrin subunit is α4β1 or α5β1 . In some embodiments, the agent binds to PA or to the complex comprising the β1 integrin subunit. In certain embodiments, the agent is an antibody or fragment thereof, e.g. Volociximab, or PF-5412. In certain embodiments, the agent is a small molecule, e.g. BI0121 1 , BI05192, ELND002, a

piperazinylphenylalanine derivative, JSM6427, JSM8757, SJ749, or SJ755. In certain embodiments, the agent is a peptide, e.g. LLP2A, ATN-161 , or a cyclotetrapeptide mimetic comprising a PMRI Arg-Gly-Asp sequence. In some embodiments, the agent that inhibits binding of PA to a complex comprising a β1 integrin subunit is an agent that inhibits the activation of the complex comprising β1 integrin subunit, e.g. activation which promotes binding between the PA and the complex. In some such embodiments, the anti-anthrax agent inhibits the activation of the complex comprising the β1 integrin subunit by inhibiting an activator of the complex, e.g. CD44. In some such embodiments, the agent inhibits CD44 activation, e.g. by inhibiting the binding of a CD44 ligand, e.g. the OPN ligand, to CD44. In some such embodiments, the inhibitor is an antibody or fragment thereof, e.g. a CD44-specific antibody, e.g. ARH460-16-2, or an OPN-specific antibody, e.g. 23C3.

In some embodiments, the agent that inhibits cellular entry of an anthrax toxin by a complex comprising a β1 integrin subunit is an agent that inhibits toxin endocytosis mediated by the complex comprising a β1 integrin subunit. In some embodiments, the complex comprising a β1 integrin subunit is α4β1 or α5β1 . In certain embodiments, the anti-anthrax agent that inhibits endocytosis inhibits calpain. In some such embodiments, the agent inhibits calpain by inhibiting calpain-promoted cleavage of a polypeptide that links complexes comprising the β1 integrin subunit to the cytoskeleton, e.g. Talin, FAK, paxillin, alpha-actinin, vinculin. In some such embodiments, the inhibitor of calpain is DL28170. In some embodiments, the agent is the polypeptide calpasiatin. In some embodiments, the agent is a nucleic acid encoding calpasiatin. In certain embodiments, the anti-anthrax agent that inhibits endocytosis inhibits clathrin.

In some embodiments, the agent that inhibits cellular entry of an anthrax toxin mediated by complexes comprising a β1 integrin subunit is an agent that inhibits mediators of signaling by complexes comprising the β1 integrin subunit. In some embodiments, the complex comprising the β1 integrin subunit is α4β1 or α5β1 . In some embodiments, the mediator is an anthrax toxin. In some embodiments, the mediator is a focal adhesion protein, e.g. vinculin, paxillin, talin, etc. In some embodiments, the mediator is a kinase, e.g. Fak, Fyn, Src, etc. In some embodiments, the mediator is a cell surface receptor, e.g. an integrin, a growth factor receptor, CMG2, etc.

In some embodiments, the agent that inhibits cellular entry of an anthrax toxin is an agent that inhibits expression of the β1 integrin subunit. In some such embodiments, the agent is an siRNA, e.g. a β1 integrin subunit-specific siRNA or shRNA.

In some embodiments, the method is performed ex vivo. In some embodiments, the method is performed in vivo. In some embodiments, the method further comprises the step of contacting the cell with an effective amount of a second anti-anthrax agent. In some embodiments, the second anti-anthrax agent is an agent that inhibits entry of B. anthracis spores into a cell. In some embodiments, the second anti-anthrax agent is an agent that prevents B. anthracis from multiplying. In some embodiments, the second anti-anthrax agent is an agent that inhibits entry of an anthrax toxin into a cell. In some embodiments, the second anti-anthrax agent is an agent that inhibits anthrax toxin toxicity in a cell.

In some aspects of the invention, the methods reduce anthrax susceptibility in a cell.

In some aspects of the invention, the methods treat anthrax toxicity in a cell. In some aspects of the invention, the methods inhibit an anthrax toxin-mediated condition in a host. In some such embodiments, the methods comprise administering to a host an effective amount of an anti-anthrax agent that inhibits cellular entry of an anthrax toxin mediated by complexes comprising a β1 integrin subunit In some such embodiments, the method is a method of prophylactically conferring an anthrax toxin-resistant phenotype on the subject. In some such embodiments, the method is a method of treating a subject suffering from an anthrax-toxin mediated disease condition.

Aspects of the invention also include anti-anthrax compositions that confer protection from the toxicity of an anthrax toxin to a subject upon administration to the subject, or that treat anthrax toxin toxicity to a subject upon administration to the subject. In some such compositions, the composition comprises a first anti-anthrax agent that inhibits cellular entry of an anthrax toxin mediated by complexes comprising a β1 integrin subunit; and a second anti-anthrax agent. In some embodiments, the second anti-anthrax agent is an agent that inhibits entry of B. anthracis spores into a cell, e.g. by inhibiting a protein, e.g. α2β1 , αηιβ2, etc., that mediates spore entry into the cell, In some embodiments, the second anti-anthrax agent is an agent that prevents B. anthracis from multiplying, e.g. an antibiotic. In some embodiments, the second anti-anthrax agent is an agent that inhibits entry of an anthrax toxin into a cell e.g. by inhibiting a polypeptide, e.g. CMG2, LRP6, TEM8, etc. that mediates anthrax toxin entry into a cell. In some embodiments, the second anti-anthrax agent is an agent that inhibits anthrax toxin toxicity in a cell, e.g. by inhibiting the anthrax toxin. In some embodiments, the second anti-anthrax agent is an antibody or binding fragment thereof. In some embodiments, the second anti-anthrax agent is a dominant negative peptide. In some embodiments, the second anti-anthrax agent is a small molecule. In some embodiments, the second anti-anthrax agent is a nucleic acid. In some embodiments, the composition comprises a pharmaceutically acceptable vehicle.

Aspects of the invention also include methods for screening candidate agents for anti-toxin activity. In some embodiments, the methods comprise contacting a cell expressing a β1 integrin or other toxin receptor with a toxin and a candidate agent; and comparing the viability of the cell to the viability of a cell expressing a β1 integrin /other toxin receptor that was contacted with the toxin not contacted with the candidate agent; wherein enhanced viability of the cell contacted with the candidate agent indicates that the candidate agent has anti-toxin activity. In some embodiments, the toxin is an anthrax toxin, e.g. a PA- bound anthrax toxin.

Aspects of the invention also include kits that may be used in the methods disclosed herein. In some embodiments, the kit comprises an anti-anthrax agent that inhibits cellular entry of an anthrax toxin mediated by complexes comprising the β1 integrin subunit. In some embodiments, the kit further comprises a second anti-anthrax agent.

BRIEF DESCRIPTION OF THE FIGURES

The file of this patent contains at least one drawing executed in color. Copies of this patent with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.

Figure 1 depicts the localization of β1 , α4, and α5 integrins during PA binding and endocytosis in RAW264.7 cells. Fluorescence microscopy images showing the cellular localization of fluorescently labeled PA (green) and fluorescently labeled antiintegrin antibodies (red). {A} β1 -Integrin, (B) a4-integrin, and (C) a5-integrin localization during PA binding (4 °C) and PA internalization (37 °C). Integrin internalization was also measured in the absence of PA.

Figure 2 demonstrates that Integrin and PA interact. (A) Fluorescence microscopy analysis of the effects of inhibitory monoclonal an ts— β 1 -integrin and anii— β3 antibodies on PA internalization. RAW284.7 cells were incubated with Alexa Fluor 488-!abeied PA (1 Mg/mL) in the presence or in the absence of anti-βΐ or antH33 integrin antibody for 20 min at 37 °C followed by fluorescence microscopy. Western blot analysis of the influence of anti- pi and -β3 integrin inhibitory antibody on monomeric PA binding to the cell surface (6), and on the internalization of heptameric PA in the cytoplasm (C). Numbers represent the average of relative intensities of PA ± SD obtained from three independent experiments.

Figure 3 illustrates the effect of anti-integrin inhibitory monoclonal antibodies on anthrax LF-PA~-mediated lethality in RAW284.7 macrophages. (A) Effect of inhibition of antibody to integrin subunits cr4, ct5, αν, β1 , and β3 or to the integrin-activating protein CD44. RAW264.7 cells were incubated for 24 h with 3 ng/mL PA and 500 ng/mL LF and in the presence of increasing amounts of purified monoclonal antibodies (0-10 Mg/mL). (6) Soluble α5β1 -integrin protein inhibits the LF-PA intoxication of RAW264.7 cells. RAW264.7 ceils that express endogenous anthrax toxin receptors were incubated with 3 ng/mL PA and 500 ng/mL LF and in the presence of increasing amounts of purified α5β1 -integrin protein (0-31 .75 Mg/mL) for 24 h.

Figure 4 illustrates the effects of CD44 and HA on the sensitivity of RAW264.7 macrophages to LF-PA. (A) Fluorescence microscopy shows effects of HA on PA internalization. Western blot analysis of the influence of HA on the internalization of monomeric PA in the cytoplasm (S), and on heptameric PA binding to the cell surface (C). Numbers represent the average of relative intensities of PA ± SD obtained from three independent experiments. (D) Effect of HA on colocalization between β1 -integrin and PA. Fluorescence microscopy images showing the colocalization of fluorescent!y labeled anii— β1 -integrin antibodies (red) and PA oligomers (green). (E) Effects of exogenously added HA on susceptibility of RAW264.7 cells to LF-PA toxin. RAW264.7 ceils were plated in the presence or in the absence of HA (100 pg/mL), anti-βΐ -integrin inhibitory antibody, or both in 98-well plates and were treated with serially diluted LF in the presence of constant of PA (200 ng/mL) for 24 h. Numbers represent 1C₅₀ values.

Figure 5 demonstrates the pathways involved in LF-PA entry. Effects of

exogenously added HA on susceptibility of WT RAW284.7 ceils to 24 h exposure to LF-PA toxin were assessed on ARAP3 knockdown (A), LRP8 knockdown (S), or C G2

knockdown ( D) cells. (C) Effects of exogenously added HA on susceptibility of WT RAW264.7 cells or C G2 knockdown cells to 4 h exposure to LF-PA toxin. Numbers represent iC₅₀ values. The analysis was done as in Figure 4E.

Figure 8 demonstrates the association between expression of selected genes and LF-PA sensitivity in differentially sensitive human cancer cell lines. GABRIEL identification of genes that are more highly expressed in LF-PA-sensitive cell lines and also shows reduced expression in LF-PA-resistant cell lines. Red shades represent an increase and green shades represent a decrease in hybridizing cDNA. Black indicates no detectable change in transcript level. Sensitive ceil lines were defined as the ones whose growth was inhibited down to 50% by less than 60 ng/mL of toxin. Resistant ceil lines were defined as the ones whose growth was not inhibited down to 50% by at least 830 ng/mL of toxin. Gene expression profiles were previously reported.

Figure 7 illustrates the binding kinetics of PA to immobilized 0.2 μΜ α5β1 -iniegrin as measured by SPR. Protein concentrations of PA ranged from 148 nmol/L to 12 moi/L in PBS buffer with or without 1 mmoi/L nGl2. Equilibrium dissociation constants were calculated on the basis of the kinetic measurements of the association and dissociation rate constants according to the formula Kd = kd/ka. It is known that all three of Mn, g, and Ca are physiologically relevant cations bound to integrin MIDAS domains. According to manufacturer's recommendations, the purified α5β1 -integrin is active (i.e., binds ligands) in PBS buffer. Both Mg and Ca form visible precipitates in PBS, precluding the additional use of Mg or Ca in these assays.

Figure 8 demonstrates the effect of inhibition of antibody to integrin subunits cr4, a5, av, β1 , and β3 or to the integrin-activating protein CD44 on growth of RAW264.7

macrophages. RAW264.7 cells were incubated in the absence of toxin and in the presence of increasing amounts of purified monoclonal antibodies (0-10 pg/mL). Cell viability was determined by MTT assay (Materials and Methods) and is shown as the percent of survivors obtained relative to treatment without antibodies (100%).

Figure 9 illustrates CD44 localization during PA binding (4 °C), PA internalization (37 °C), or in the absence of PA. Fluorescence microscopy images showing the

coiocalization of fiuorescently labeled anti-CD44 antibodies (red) and PA oligomers (green). For PA binding assay, ceils were incubated with fiuorescently Iabeied PA and anti-CD44 antibody for 1 h at 4 °C before being examined by fluorescence microscopy following the protocol described in Materials and Methods in the main text. For PA internalization assays, cells were incubated with fiuorescently labeled PA and anti-CD44 antibody for 20 min at 37 °C followed by fluorescence microscopy. CD44 internalization was also measured in the absence of PA. Figure 10 illustrates the quantification of cytoplasmic β 1 -integrin in the presence and in the absence of HA as determined by histogram analysis using Adobe Photoshop software. The intensities of fluorescently labeled anti-βΐ -integrin antibodies signal were quantified by histogram analysis: pixel-intensity value from identical areas of the

cytoplasmic regions were analyzed in at least 30 ceils per condition. Average intensity values and SD values were calculated.

Figure 11 depicts how the minimal amount of HA needed to cause maximal reduction of sensitivity of RAW284.7 cells to LF-PA killing was determined. RAW264.7 cells were plated in the absence (0 Mg/mL) or presence of various amounts of HA (18-150 Mg/mL) in 98-weil plates and were treated with serially diluted LF in the presence of constant PA (200 ng/mL) for 24 h. MTT ceil viability assays were performed, and data were plotted as shown.

Figure 12 illustrates the characteristics of expression of CAST EST. (A) Cells were pre-incubated in the presence or absence of 1 μg/ml of Dox for 2 days, and then seeded in a 96-weli plate at a concentration of 2x104 cells/mi with or without 1 μg/ml of Dox. in the presence of absence of 1 g/ml of Dox, ceils were treated with serial diluted PA plus fixed 250 ng/ml LF. After 2 days, MTT assay was performed as described in Experimental Procedures. The data represents the mean and standard deviation of four independent replicates. (B-D) Phenotype reconstitution by introducing CAST EST. CAST EST identified in clone 3-12 was reintroduced into pLEST vector, and then RAW284.7tTA cells were infected with ientivirus containing pLEST vector alone or ientivirus expressing CAST EST and selected by 800 μg/ml G418. (B) Effect of expression of CAST EST on cytotoxicity of PA-LF. Cells were treated with serial diluted LF combined with 200 ng/ml PA. MTT assay was performed 1 day after toxin treatment. The values represent the mean and standard deviation of three independent replicates. (C) The rnRNA expression level of CAST was examined by quantitative RT-PCR and normalized to β-actin. The data represents the mean and standard deviation of three independent replicates. (D) Abundance of CAST protein was determined by Western blot analysis using anti-CAST antibody, β-actin was used as a loading control. (E) Caipain activity was determined using synthetic fiuorogenic substrates from total ceil iysates as described in Example 2 Materials and Methods.

Figure 13 illustrates the effect of inhibition of caipain activity on cellular susceptibility to PA-LF. (A) Effect of MDL28170 on killing of RAW264.7 cells by PA-LF. Ceils were plated in a 96-welI plate at a density of 1 x 105 cells/mi. 20 or 40 μΜ MDL28170 was added to ceils 1 h prior to toxin treatment. The final concentration of DMSO in ail samples was adjusted to 0.1 % since MDL28170 was dissolved in DMSO. After cells were incubated with 0.5 μg/mi PA-LF for 4 h, ceil viability was assessed by MTT assay as described in Experimental Procedures. (B) Effect of addition of MDL28170 at different time points on PA- LF induced cytotoxicity. RAW284.7 ceils were treated with 40 μΜ MDL28170 for 80 min prior to PA-LF addition, at the same time as the toxin addition, or 75 min after toxin addition. A final concentration of 0.1 % DMSO was added in sample wells without MDL28170. After incubation with serial diluted LF in the presence of 200 ng/ml PA for 4 h, TT assay was performed. The values represent the mean and standard deviation of three replicates.

Figure 14 illustrates the anthrax toxin uptake assay. (A) Effect of caipain inhibition on PA-LF- mediated cleavage of MEK-2. RAW264.7 cells were incubated in the absence or the presence of 80 μΜ DL28170 for 1 h, and then exposed to 0.5 ig/m\ PA-LF for 90 min. Cell extracts were analyzed by Western blot with anti-N-term MEK-2 antibody to detect intact MEK-2. β-actin was used as a loading control. PA binding (B) and internalization (C) assays. RAW264.7 cells were incubated with 80 μΜ MDL28170 for 1 h prior to PA addition. The ceils were exposed to 0.5 μg/mi PA at 4^QC for 1 h for binding assay (B) or to 0.5 μg/m\ PA at 37^eC for the indicated time for internalization assay (C). Cell iysates were analyzed by Western blot with anti-PA antibody as described in Example 2 Materials and Methods, β- actin was used as a loading control.

Figure 15 illustrates the effect of caipain inhibition on cellular localization of PA and β1 integrin during PA-internaiization process. RAW264.7 cells were preincubated without (A-D) or with (E-G) 80 μΜ MDL28170 for 1 h, followed by exposure to Alexa 488-labeied PA (B-G) in the presence of 0.5 μg/ml anti-βΐ integrin-APC (HMB1 -1 ) antibody at 37^eC for 20 min. After fixation and permeabilization, the cellular localization of PA and β1 integrin was monitored by using a fluorescence microscope. Fluorescence microscopy images showing the localization of PA (green; B and E) and β1 integrin (red; A, C, and F).

Figure 16 illustrates the effect of caipain inhibition on cellular localization of CMG2 during PA-internalization process. RAW264.7 cells expressing EGFP-CMG2 were preincubated without (A and C) or with (B and D) 80 μΜ DL28170 for 1 h, followed by exposure to 1 g/ml PA (C and D) at 37^eC for 20 min. After washing and fixation, the cellular localization of CMG2 was monitored by using a fluorescence microscope.

Figure 17 illustrates the effect of talin cleavage by caipain on PA-LF lethality.

RAVV264.7 cells were transfected with an empty vector pEGFP-C1 or containing wild type or mutant form of talin (TLN1 or TLN 1 -L432G, respectively). (A) The mRNA expression of EGFP-tagged talin was assessed in parental cells (Ctrl) and the transfected cells by quantitative RT-PCR and normalized to β-actin. The data represents the mean and standard deviation of three independent replicates. (B) PA-internaiization kinetics. The cells were treated with 1 ug/m! of PA at 4-C for 1 h, and then shifted to 37^eC for the indicated time. Cell extracts were analyzed by Western blot with anti-PA antibody. (C) Cellular susceptibility to PA-LF. The cells were exposed to serial diluted PA in the presence of 200 ng/ml LF for 4 h, and then MTT assay was performed. The values represent the mean and standard deviation of three replicates.

Figure 18 illustrates the effect of MDL28170 on calpain activity. RAW284.7 cells were treated with 40 μΜ MDL281 70 or 0.1 %(v/v) DMSO for 2 h. Caipain activity was determined using synthetic fluorogenic substrates from total ceil lysates as described in

Example 2 Materials and Methods.

Figure 19 illustrates the effect of calpain inhibition on cellular susceptibility to PA-

FP59 (a hybrid toxin comprising the PA binding site of LF plus a toxin domain derived from Pseudomonas aeruginosa exotoxin A) or PE toxin (Pseudomonas aeruginosa exotoxin) .

RAW264.7 cells were pretreated with 40 μΜ MDL281 70 for 1 h prior to exposure to serial diluted FP59 in the presence of 200 ng/ml PA (A) or serial diluted PE alone (B). After 1 d of toxin treatment, ceil viability was determined by MTT assay as described in Example 2

Materials and Methods. (C) PA uptake in the presence of LF or FP59. Cells were incubated with 80 MDL281 70 for 1 h, followed by exposure to 0.5 \\g/m\ PA in the presence of 0.25 μ9/ηι! LF or FP59 at 4^?C for 1 h, and then shifted to 37^SC. After 15 rnin, the ceils were collected and analyzed by Western blot with anti-PA antibody.

Figure 20 demonstrates the protective effect of MDL281 70 against anthrax lethal toxin challenge in CAST/EiJ mice.

Figure 21 documents the behavior of mice after lethal toxin challenge. Active: moving around, and climbing the walls of the cage. Sick: less moving and no climbing.

Very sick: no moving and half-closed eyes.

DETAILED DESCRIPTION

Methods and compositions for preventing or treating an anthrax toxin mediated disease condition in an individual are provided. Aspects of the subject methods include administering to a subject an effective amount of an agent that inhibits cellular entry of an anthrax toxin mediated by complexes comprising a β1 integrin subunit. Also provided are active agents suitable for use in the subject methods, pharmaceutical preparations thereof and kits thereof. Methods of screening candidate agents for anti-toxin activity are also provided.

Before the present invention is described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

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 also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. In further describing various aspects of the subject invention, a more detailed review of embodiments of the methods is first provided, followed by a review of embodiments of utilities in which the methods find use and embodiments of compositions that find use in the methods.

METHODS

Aspects of the invention include methods of inhibiting entry of an anthrax toxin, or simply "anthrax toxin", into a cell. By inhibiting entry of an anthrax toxin into a cell it is meant that the amount of the anthrax toxin that enters a cell, as measured using any convenient protocol, e.g. as described in the examples below, is decreased by an amount of 2-fold or more, usually by 5-fold or more and including by 10-, 25-, 50-, 100-fold or more, as compared to a control, i.e., a cell that is not subjected to the methods of the present invention. In certain embodiments, the methods are methods of inhibiting entry of anthrax toxin into a cell, such that the cell does not die upon exposure of the cell to anthrax toxin.

The anthrax toxin whose entry into a cell is inhibited via the subject methods is one that is produced by B.anthracis. In certain embodiments, the anthrax toxin of interest is one whose entry is mediated by protective antigen (PA) of B.anthracis, e.g. lethal factor (LF) or edema factor (EF). In certain embodiments, the methods result in a decreased presence, e.g., of free cytosolic LF and/or EF in the cell cytosol following exposure of the cell to B.anthracis, as compared to a control (e.g., by an amount of 2-fold or more, usually by 5- fold or more and including by 10-, 25-, 50-, 100-fold or more).

Aspects of the invention include contacting a cell with an effective amount of an anti- anthrax agent, or simply "agent", that inhibits entry of the toxin into the cell. By "anti-anthrax agent" it is meant an agent which prevents or treats anthrax toxicity. In some embodiments, the anti-anthrax agent is one that inhibits toxin entry mediated by a cell surface protein. In some embodiments, the cell surface protein is an integrin.

Integrins are heterodimers comprising an a polypeptide subunit and a β polypeptide subunit. In some embodiments, the integrin comprises an a subunit, i.e. any a subunit, and a β1 subunit. A β1 subunit of integrin, or "β1 integrin subunit" or "integrin β1 subunit" refers to a polypeptide encoded by the gene ITGB1 the sequence for which can be found at Genbank Accession Nos. NM_00221 1 .3 (isoform 1 A), NM_033668.2 (isoform 1 D), and NM 133376.2 (isoform 1 E). There are 18 known a integrins to which β1 integrin subunit can bind: a1 , a2, a3, a4, a5, a6, a7, a8, a9, a10, a1 1 , ad, aE, al_, aM, aV, aW, and aX. An integrin comprising an a subunit, i.e. any a subunit, and a β1 subunit is referred to herein as a "β1 integrin". In some embodiments, the anti-anthrax agent is one that inhibits cellular entry of toxin that is mediated a complex comprising the β1 integrin subunit. As used herein, a "complex comprising the β1 integrin subunit" includes β1 integrin, i.e. a heterodimer comprising an a subunit and β1 subunit, and protein complexes that comprise a β1 integrin, e.g. a β1 integrin/"XXX" complex, where "XXX" refers to a non-integrin protein, e.g. a FGF receptor, a VEGF receptor, a CMG2 receptor, a focal adhesion protein, etc,

Integrins, including β1 integrins, are membrane receptors that are involved in cell adhesion and recognition in a variety of processes including embryogenesis, hemostasis, tissue repair, immune response and metastatic diffusion of tumor cells. Without being bound by theory, it is believed that activation of integrins by cellular proteins, e,g. cell surface proteins such as CD44, intracellular proteins such as focal adhesion proteins, etc., activates integrin binding to ligands. Ligand binding activates intracellular signaling by the integrin that is mediated by a host of cellular proteins, e.g., focal adhesion proteins, kinases, receptors, etc. This intracellular signaling modulates a number of cellular responses, including cytoskeletal reorganization, endocytosis of the ligand by the complex comprising β1 integrin and other receptors, cell motility, cell survival, cell proliferation, gene

transcription. Thus, for example, the anti-anthrax agent that inhibits cellular entry of an anthrax toxin may be an agent that inhibits the binding of PA to a complex comprising a β1 integrin subunit, e.g. by inhibiting proteins that activate the complex to bind to PA, or by physically interfering with the binding of the complex to PA. As a second example, an anti- anthrax agent that inhibits cellular entry of an anthax toxin may be an agent that inhibits toxin endocytosis mediated by a complex comprising a β1 integrin subunit. As a third example, an anti-anthrax agent that inhibits cellular entry of an anthrax toxin may be an agent that inhibits mediators of signaling by a complex comprising a β1 integrin subunit. As a fourth example, an anti-anthrax agent that inhibits cellular entry of an anthrax toxin may be an agent that inhibits β1 integrin subunit expression.

Inhibitors of PA binding to complexes comprising β 1 integrin subunits

In some embodiments, the anti-anthrax agent that inhibits cellular entry of an anthrax toxin is an agent that inhibits binding of PA to complexes comprising a β1 integrin subunit. To inhibit binding between PA and complexes comprising a β1 integrin subunit, an agent that acts as member of a specific binding pair with complexes comprising a β1 integrin subunit, e.g. that binds to a complex comprising a β1 integrin subunit or binds to PA and thereby inhibits binding of PA to the complexes comprising a β1 integrin subunit, may be employed. The term "specific binding member" or "binding member" as used herein refers to a member of a specific binding complex of molecules, e.g., two or more molecules, where the molecules may be the same or different molecules, where one of the molecules (i.e., first specific binding member) through chemical or physical means specifically binds to the other molecule(s) (e.g., second specific binding member). The complementary specific binding members are sometimes referred to as a ligand and receptor; or receptor and counter- receptor. For the purposes of the present invention, the binding members may be known to associate with each other, for example where an assay is directed at detecting compounds that interfere with the association of a known binding pair. Alternatively, candidate compounds suspected of being a binding partner to a compound of interest may be used. The specific binding pairs may include analogs, derivatives and fragments of the original specific binding member. For example, a receptor and ligand pair may include peptide fragments, chemically synthesized peptidomimetics, labeled protein, derivatized protein, etc.

In certain embodiments, the specific binding member is an antibody. The term

"antibody" or "antibody moiety" is intended to include any polypeptide chain-containing molecular structure with a specific shape that fits to and recognizes an epitope, where one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope. Antibodies that bind specifically to a complex comprising a β1 integrin subunit are referred to as anti-βΐ integrin specific antibodies, or more particularly, by the particular β1 integrin complex to which they bind, e.g. anti-o^1 specific antibody, α5β1 specific antibody, etc. In some instances, the antibody binds to the a subunit or the β1 subunit of the β1 integrin. In some instances, the antibody binds to both the a and β1 subunits, e.g., it binds to a face on the β1 integrin that is created by the dimerization of the a and β1 subunits. The specific or selective fit of a given structure and its specific epitope is sometimes referred to as a "lock and key" fit. The archetypal antibody molecule is the immunoglobulin, and all types of immunoglobulins, IgG, IgM, IgA, IgE, IgD, etc., from all sources, e.g. human, rodent, rabbit, cow, sheep, pig, dog, other mammal, chicken, other avians, etc., are considered to be "antibodies." Antibodies utilized in the present invention may be polyclonal or monoclonal antibodies.

Polyclonal antibodies can be raised by a standard protocol by injecting a production animal with an antigenic composition, formulated as described above. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In one such technique, an β1 integrin antigen comprising an antigenic portion of the β1 target polypeptide, e.g., found on an extracelluar domain, is initially injected into any of a wide variety of mammals (e.g., mice, rats, rabbits, sheep or goats). When utilizing an entire protein, or a larger section of the protein, antibodies may be raised by immunizing the production animal with the protein and a suitable adjuvant (e.g., Fruend's, Fruend's complete, oil-in-water emulsions, etc.) When a smaller peptide is utilized, it is

advantageous to conjugate the peptide with a larger molecule to make an

immunostimulatory conjugate. Commonly utilized conjugate proteins that are commercially available for such use include bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH). In order to raise antibodies to particular epitopes, peptides derived from the full sequence may be utilized. Alternatively, in order to generate antibodies to relatively short peptide portions of the brain tumor protein target, a superior immune response may be elicited if the polypeptide is joined to a carrier protein, such as ovalbumin, BSA or KLH. The peptide-conjugate is injected into the animal host, preferably according to a predetermined schedule incorporating one or more booster immunizations, and the animals are bled periodically. Polyclonal antibodies specific for the polypeptide may then be purified from such antisera by, for example, affinity chromatography using the polypeptide coupled to a suitable solid support.

Alternatively, for monoclonal antibodies, hybridomas may be formed by isolating the stimulated immune cells, such as those from the spleen of the inoculated animal. These cells are then fused to immortalized cells, such as myeloma cells or transformed cells, which are capable of replicating indefinitely in cell culture, thereby producing an immortal, immunoglobulin-secreting cell line. The immortal cell line utilized is preferably selected to be deficient in enzymes necessary for the utilization of certain nutrients. Many such cell lines (such as myelomas) are known to those skilled in the art, and include, for example: thymidine kinase (TK) or hypoxanthine-guanine phosphoriboxyl transferase (HGPRT). These deficiencies allow selection for fused cells according to their ability to grow on, for example, hypoxanthine aminopterinthymidine medium (HAT).

In certain embodiments, the immortal fusion partners utilized are derived from a line that does not secrete immunoglobulin. The resulting fused cells, or hybridomas, are cultured under conditions that allow for the survival of fused, but not unfused, cells and the resulting colonies screened for the production of the desired monoclonal antibodies. Colonies producing such antibodies are cloned, expanded, and grown so as to produce large quantities of antibody, see Kohler and Milstein, 1975 Nature 256:495 (the disclosures of which are hereby incorporated by reference).

Large quantities of monoclonal antibodies from the secreting hybridomas may then be produced by injecting the clones into the peritoneal cavity of mice and harvesting the ascites fluid therefrom. The mice, which may be primed with pristane, or some other tumor- promoter, and immunosuppressed chemically or by irradiation, may be any of various suitable strains. The ascites fluid is harvested from the mice and the monoclonal antibody purified therefrom, for example, by CM Sepharose column or other chromatographic means. Alternatively, the hybridomas may be cultured in vitro or as suspension cultures. Batch, continuous culture, or other suitable culture processes may be utilized. Monoclonal antibodies are then recovered from the culture medium or supernatant.

In addition, the antibodies or antigen binding fragments may be produced by genetic engineering. In this technique, as with the standard hybridoma procedure, antibody- producing cells are sensitized to the desired antigen or immunogen. The messenger RNA isolated from the immune spleen cells or hybridomas is used as a template to make cDNA using PCR amplification. A library of vectors, each containing one heavy chain gene and one light chain gene retaining the initial antigen specificity, is produced by insertion of appropriate sections of the amplified immunoglobulin cDNA into the expression vectors. A combinatorial library is constructed by combining the heavy chain gene library with the light chain gene library. This results in a library of clones which co-express a heavy and light chain (resembling the Fab fragment or antigen binding fragment of an antibody molecule). The vectors that carry these genes are co-transfected into a host (e.g. bacteria, insect cells, mammalian cells, or other suitable protein production host cell). When antibody gene synthesis is induced in the transfected host, the heavy and light chain proteins self- assemble to produce active antibodies that can be detected by screening with the antigen or immunogen.

In certain embodiments, recombinant antibodies are produced in a recombinant protein production system which correctly glycosylates and processes the immunoglobulin chains, such as insect or mammalian cells. An advantage to using insect cells, which utilize recombinant baculoviruses for the production of antibodies, is that the baculovirus system allows production of mutant antibodies much more rapidly than stably transfected mammalian cell lines. In addition, insect cells have been shown to correctly process and glycosylate eukaryotic proteins, which prokaryotic cells do not. Finally, the baculovirus expression of foreign protein has been shown to constitute as much as 50-75% of the total cellular protein late in viral infection, making this system an excellent means of producing milligram quantities of the recombinant antibodies.

Antibodies with a reduced propensity to induce a violent or detrimental immune response in humans (such as anaphylactic shock), and which also exhibit a reduced propensity for priming an immune response which would prevent repeated dosage with the antibody therapeutic or imaging agent are preferred for use in the invention. Thus, humanized, chimeric, or xenogenic human antibodies, which produce less of an immune response when administered to humans, are preferred for use in the present invention. Chimeric antibodies may be made by recombinant means by combining the murine variable light and heavy chain regions (VK and VH), obtained from a murine (or other animal-derived) hybridoma clone, with the human constant light and heavy chain regions, in order to produce an antibody with predominantly human domains. The production of such chimeric antibodies is well known in the art, and may be achieved by standard means (as described, e.g., in U.S. Patent No. 5,624,659, incorporated fully herein by reference).

Humanized antibodies are engineered to contain even more human-like immunoglobulin domains, and incorporate only the complementarity-determining regions of the animal- derived antibody. This is accomplished by carefully examining the sequence of the hyper- variable loops of the variable regions of the monoclonal antibody, and fitting them to the structure of the human antibody chains. Although facially complex, the process is straightforward in practice. See, e.g., U.S. Patent No. 6,187,287, incorporated fully herein by reference.

Alternatively, polyclonal or monoclonal antibodies may be produced from animals that have been genetically altered to produce human immunoglobulins. Techniques for generating such animals, and deriving antibodies therefrom, are described in U.S. Patents No. 6,162,963 and 6,150,584, incorporated fully herein by reference.

Alternatively, single chain antibodies (Fv, as described below) can be produced from phage libraries containing human variable regions. See U.S. Patent No. 6,174,708. In addition to entire immunoglobulins (or their recombinant counterparts), immunoglobulin fragments comprising the epitope binding site (e.g., Fab', F(ab')₂, or other fragments) are useful as antibody moieties in the present invention. Such antibody fragments may be generated from whole immunoglobulins by ficin, pepsin, papain, or other protease cleavage. "Fragment," or minimal immunoglobulins may be designed utilizing recombinant

immunoglobulin techniques. For instance "Fv" immunoglobulins for use in the present invention may be produced by linking a variable light chain region to a variable heavy chain region via a peptide linker (e.g., poly-glycine or another sequence which does not form an alpha helix or beta sheet motif).

Fv fragments are heterodimers of the variable heavy chain domain (V_H) and the variable light chain domain (V_L). The heterodimers of heavy and light chain domains that occur in whole IgG, for example, are connected by a disulfide bond. Recombinant Fvs in which V_H and V_L are connected by a peptide linker are typically stable, see, for example, Huston et al., Proc. Natl. Acad, Sci. USA 85:5879-5883 (1988) and Bird et al., Science 242:423-426 (1988), both fully incorporated herein, by reference.

In addition, derivatized immunoglobulins with added chemical linkers, detectable moieties, such as fluorescent dyes, enzymes, substrates, chemiluminescent moieties and the like, or specific binding moieties, such as streptavidin, avidin, or biotin, and the like may be utilized in the methods and compositions of the present invention. For convenience, the term "antibody" or "antibody moiety" will be used throughout to generally refer to molecules which specifically bind to an epitope of the LPR6 protein targets, although the term will encompass all immunoglobulins, derivatives, fragments, recombinant or engineered immunoglobulins, and modified immunoglobulins, as described above.

Candidate antibodies can be tested for activity by any suitable standard means. As a first screen, the antibodies may be tested for binding against the immunogen, or protein. As a second screen, candidates may be tested for binding to an appropriate cell line. For these screens, the candidate antibody may be labeled for detection. After selective binding to the protein target is established, the candidate antibody agent may be tested for appropriate activity (i.e., the ability to confer anthrax toxin resistance on cells) in an in vitro or in vivo model.

Examples of antibodies that may be used to inhibit binding of PA to complexes comprising a β1 integrin subunit or used to design antibodies or antibody fragments that inhibit binding of PA to a complexes comprising a β1 integrin subunit include Volociximab (an anti-a531 specific chimeric antibody) and PF-04605412 ( "PF-5412"; an anti-a431 specific antibody).

Other agents that disrupt binding of PA to complexes comprising a β1 integrin subunit that find use in the methods described herein include dominant negative mutants of β1 integrin or the β1 integrin subunit. Dominant negative mutants of β1 integrin or the β1 integrin subunit are mutant proteins that exhibit dominant negative β1 integrin activity. As used herein, the term "dominant-negative β1 integrin activity" or "dominant negative activity" refers to the inhibition, negation, or diminution of certain particular activities of β1 integrin or the β1 integrin subunit, e.g. anthrax toxin cellular entry mediated by complexes comprising a β1 integrin subunit. Examples of dominant negative mutants of β1 integrin or the β1 integrin subunit include truncated versions of β1 integrin or the β1 integrin subunit, e.g. peptides comprising the extracellular domain of β1 integrin or the β1 integrin subunit, e.g. soluble peptides or membrane-bound peptides, i.e. peptides comprising β1 integrin extracellular domain(s) plus a transmembrane domain. Such polypeptides can bind to PA and prevent PA binding to complexes comprising a β1 integrin subunit. Other examples include dominant negative mutants comprising point mutations or deletions in the β1 integrin domain that binds PA or intracellular domains of β1 that abrogate binding of β1 integrin to PA or β1 integrin intracellular signaling, respectively. The peptides may be exogenously provided to the cell, i.e. as peptides, or they may be provided to the cell as nucleic acids for expression by the cell, where expression of such mutants in the cell result in a decrease in cellular entry of an anthrax toxin mediated by a complex comprising a β1 integrin subunit.

Dominant negative mutations are readily generated for corresponding proteins. These may act by several different mechanisms, including mutations in a substrate-binding domain; mutations in a catalytic domain; mutations in a protein binding domain (e.g.

multimer forming, effector, or activating protein binding domains); mutations in cellular localization domain, etc. A mutant polypeptide may interact with wild-type polypeptides (made from the other allele) and form a non-functional multimer. In certain embodiments, the mutant polypeptide will be overproduced. Point mutations are made that have such an effect. In addition, fusion of different polypeptides of various lengths to the terminus of a protein, or deletion of specific domains can yield dominant negative mutants. General strategies are available for making dominant negative mutants (see for example, Herskowitz (1987) Nature 329:219, and the references cited above). Such techniques are used to create loss of function mutations, which are useful for determining protein function. Methods that are well known to those skilled in the art can be used to construct expression vectors containing coding sequences and appropriate transcriptional and translational control signals for increased expression of an exogenous gene introduced into a cell. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Alternatively, RNA capable of encoding gene product sequences may be chemically synthesized using, for example, synthesizers. See, for example, the techniques described in Oligonucleotide Synthesis", 1984, Gait, M. J. ed., IRL Press, Oxford.

Candidate agents are also found among small molecules. Naturally occurring or synthetic small molecule compounds of interest include numerous chemical classes, including, e.g., organic molecules, e.g. small organic compounds having a molecular weight of more than 50 and less than 2,500 daltons. Candidate agents comprise functional groups for structural interaction with proteins, particularly hydrogen bonding, and may include at least an amine, carbonyl, hydroxyl or carboxyl group, or in some instances, two or more of the functional chemical groups. The candidate agents may comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Examples of small molecule inhibitors that find particular use in methods of the invention include BI0121 1 (Abraham WM, et al. (2000) A small-molecule, tight-binding inhibitor of the integrin alpha(4)beta(1 ) blocks antigen-induced airway responses and inflammation in experimental asthma in sheep. Am J Respir Crit Care Med. 162(2 Pt 1 ):603-1 1 ); BI05192 (Ramirez P et al. (2009) BI05192, a small molecule inhibitor of VLA-4, mobilizes hematopoietic stem and progenitor cells. Blood. 2009 1 14(7): 1340-3); ELND002 (a PEGylated small molecule that inhibits α4β1 ); S18407 (Glasner J, et al. (2005) A small molecule alpha 4 beta 1 antagonist prevents development of murine Lyme arthritis without affecting protective immunity. Immunol.175(7):4724-34); S16197 (Glasner J, et al. (2005) supra); piperazinylphenylalanine derivatives (Saku et al. (2008) Synthetic study of VLA-4/VCAM-1 inhibitors: Synthesis and structure-activity relationship of piperazinylphenylalanine derivatives. Bioorganic & Medicinal Chemistry Letters 18(3): 1053-1057); JSM6427 (Heier, JS et al. (2008) JSM6427, a Small Molecule alpha(5)beta(1 ) Integrin Inhibitor. In: Proceedings of the 7^th International Symposium on Ocular Pharmacology and Therapeutics, p. 33 - 36, Medimond SRL); JSM8757 (2- aroylamino-3-{4-[(pyridin-2-ylaminomethyl)heterocyclyl]phenyl}propionic acid) (Tatsuma Okazaki et al. (2009) 5β1 Integrin Blockade Inhibits Lymphangiogenesis in Airway

Inflammation. Am. J. of Path. 2009;174:2378-2387); SJ749 (Maglott A, et al. (2006) The small alpha5beta1 integrin antagonist, SJ749, reduces proliferation and clonogenicity of human astrocytoma cells. Cancer Res. 66(12):6002-7); and SJ755 (David Cue et al. (2000) A nonpetide integrin antagonist can inhibit epithelial cell ingestion of Streptococcus pyogenes by blocking formation of integrin a531 -fibronectin-M1 protein complexes. PNAS vol. 97 no. 6:2858-2863).

Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. For example, peptides such as LLP2A (Li Peng et al. (2006)

Combinatorial chemistry identifies high-affinity peptidomimetics against 41 integrin for in vivo tumor imaging. Nature Chemical Biology 2, 381 - 389); ATN-161 (a peptide antagonist of α5β1 ); or cyclotetrapeptide mimetics comprising a PMRI Arg-Gly-Asp sequence, e.g. c[(R)-betaPhepsi(NHCO)Asppsi(NHCO)Gly-Arg; and c[(S)- betaPhepsi(NHCO)Asppsi(NHCO)Gly-Arg]. (Gentilucci L, et al. (2010) Antiangiogenic effect of dual/selective alpha(5)beta(1 )/alpha(v)beta(3) integrin antagonists designed on partially modified retro-inverso cyclotetrapeptide mimetics. J Med Chem. 53(1 ):106-18) may be employed. Other molecules may be identified, among other ways, by employing the screening protocols described below.

In some embodiments, the anti-anthrax agent that inhibits binding of PA to complexes comprising a β1 integrin subunit is an agent that inhibits the activation of a complex comprising a β1 integrin subunit to bind to PA. For example, CD44 (Genbank Accession Nos. NM_000610.3 (isoform 1 ); NM_001001389.1 (isoform 2); NM_001001390.1 (isoform 3); NM_001001391 .1 (isoform 4); NM_001001392.1 (isoform 5); NM_001202555.1 (isoform 6); NM_001202556.1 (isoform 7); NM_001202557.1 (isoform 8)) is a cell-surface glycoprotein involved in cell-cell interactions, cell adhesion, and migration, and is a known activator of integrin binding to ligand. As such, and as demonstrated in the examples section below, an agent that inhibits CD44 activity may be used as an agent that inhibits binding of PA to a complex comprising a β1 integrin subunit.

Binding of CD44 by certain CD44 ligands, e.g. osteopontin (OPN/SPP1 ) will activate CD44 to activate complexes comprising the β1 integrin subunit. As demonstrated in the examples below, inhibiting binding of such CD44 ligands to CD44 inhibits CD44 activation of complexes comprising the β1 integrin subunit. To inhibit binding between, e.g., OPN ligand and CD44 receptor, an agent that acts as member of a specific binding pair with CD44 receptor and inhibits binding of, e.g. OPN ligand to the CD44 receptor, may be employed. In certain embodiments, the specific binding member is an antibody or fragment thereof, e.g. an antibody or fragment thereof that is specific for OPN, e.g. 23C3 (Fan K, et al. (201 1 ) A humanized anti-osteopontin antibody protects from Concanavalin A induced- liver injury in mice. Eur J Pharmacol. 657(1 -3):144-51 ), or an antibody or fragment thereof that is specific for CD44, e.g. ARH460-16-2 (Young, D.S. et al. (2004) ARH460-16-2: a therapeutic monoclonal antibody targeting CD44 in Her2/neu negative breast cancer. J of Clin Oncol ASCO Annual Meeting Proceedings Vol 22, No 14S). In certain embodiments, the specific binding member is a ligand of CD44 that does not promote CD44 activation of complexes comprising a β1 integrin subunit, e.g. hyaluronic acid (HA). Other agents that inhibit binding of CD44 to, e.g. OPN, include dominant negative mutants of CD44, for example truncated versions of CD44 such as peptides comprising the extracellular domain of CD44. Such polypeptides can bind to OPN and prevent their binding to active CD44 complexes. Other agents include small molecules that bind to CD44 or OPN to inhibit their binding to one another or activation of CD44, and dominant negative mutants of CD44. Other such agents can be identified by standard experimentation, e.g. as described below.

In certain embodiments, the agent is an agent that inhibits expression of a functional β1 integrin subunit. Inhibition of β1 integrin subunit expression may be accomplished using any convenient means, including use of an agent that inhibits β1 integrin subunit expression, such as, but not limited to: antisense agents, RNAi agents, agents that interfere with transcription factor binding to a promoter sequence of the β1 integrin gene, etc., inactivation of the β1 integrin gene, e.g., through recombinant techniques, etc.

For example, antisense molecules can be used to down-regulate expression of β1 integrin subunit in the cell. The anti-sense reagent may be antisense oligodeoxynucleotides (ODN), particularly synthetic ODN having chemical modifications from native nucleic acids, or nucleic acid constructs that express such anti-sense molecules as RNA. The antisense sequence is complementary to the mRNA of the targeted repressor protein, and inhibits expression of the targeted repressor protein. Antisense molecules inhibit gene expression through various mechanisms, e.g., by reducing the amount of mRNA available for translation, through activation of RNAse H, or steric hindrance. One or a combination of antisense molecules may be administered, where a combination may comprise multiple different sequences.

Antisense molecules may be produced by expression of all or a part of the target gene sequence in an appropriate vector, where the transcriptional initiation is oriented such that an antisense strand is produced as an RNA molecule. Alternatively, the antisense molecule is a synthetic oligonucleotide. Antisense oligonucleotides may be 7 or more, such as 12 or more, including 20 or more nucleotides in length, and in certain embodiments are 500 or less, such as 50 or less, including 35 or less nucleotides in length, where the length is governed by efficiency of inhibition, specificity, including absence of cross-reactivity, and the like. It has been found that short oligonucleotides, of from 7 to 8 bases in length, can be strong and selective inhibitors of gene expression (see Wagner et al. (1996), Nature Biotechnol. 14:840-844).

A specific region or regions of the endogenous sense strand mRNA sequence is chosen to be complemented by the antisense sequence. Selection of a specific sequence for the oligonucleotide may use an empirical method, where several candidate sequences are assayed for inhibition of expression of the target gene in an in vitro or animal model. A combination of sequences may also be used, where several regions of the mRNA sequence are selected for antisense complementation.

Antisense oligonucleotides may be chemically synthesized by methods known in the art (see Wagner et al. (1993), supra, and Milligan et al., supra.) Oligonucleotides may be chemically modified from the native phosphodiester structure, in order to increase their intracellular stability and binding affinity. A number of such modifications have been described in the literature, which alter the chemistry of the backbone, sugars or heterocyclic bases.

Among useful changes in the backbone chemistry are phosphorothioates;

phosphorodithioates, where both of the non-bridging oxygens are substituted with sulfur; phosphoroamidites; alkyl phosphotriesters and boranophosphates. Achiral phosphate derivatives include 3'-0'-5'-S-phosphorothioate, 3'-S-5'-0-phosphorothioate, 3'-CH₂-5'-0- phosphonate and 3'-NH-5'-0-phosphoroamidate. Peptide nucleic acids replace the entire ribose phosphodiester backbone with a peptide linkage. Sugar modifications are also used to enhance stability and affinity. The oc-anomer of deoxyribose may be used, where the base is inverted with respect to the natural β-anomer. The 2'-OH of the ribose sugar may be altered to form 2'-0-methyl or 2'-0-allyl sugars, which provides resistance to degradation without comprising affinity. Modification of the heterocyclic bases must maintain proper base pairing. Some useful substitutions include deoxyuridine for deoxythymidine; 5-methyl- 2'-deoxycytidine and 5-bromo-2'-deoxycytidine for deoxycytidine. 5- propynyl-2'- deoxyuridine and 5-propynyl-2'-deoxycytidine have been shown to increase affinity and biological activity when substituted for deoxythymidine and deoxycytidine, respectively.

As an alternative to anti-sense inhibitors, catalytic nucleic acid compounds, e.g. ribozymes, anti-sense conjugates, etc. may be used to inhibit gene expression. Ribozymes may be synthesized in vitro and administered to the patient, or may be encoded on an expression vector, from which the ribozyme is synthesized in the targeted cell (for example, see International patent application WO 9523225, and Beigelman et al. (1995), Nucl. Acids Res. 23:4434-42). Examples of oligonucleotides with catalytic activity are described in WO 9506764. Conjugates of anti-sense ODN with a metal complex, e.g. terpyridylCu(ll), capable of mediating mRNA hydrolysis are described in Bashkin et al. (1995), Appl.

Biochem. Biotechnol. 54:43-56.

In addition, the transcription level of β1 integrin subunit can be regulated by gene silencing using RNAi agents, e.g., double-strand RNA (Sharp (1999) Genes and

Development 13: 139-141 ). RNAi, such as that which employs double-stranded RNA interference (dsRNAi) or small interfering RNA (siRNA), has been extensively documented in the nematode C. elegans (Fire, A., et al, Nature, 391 , 806-81 1 , 1998) and routinely used to "knock down" genes in various systems. RNAi agents may be dsRNA or a transcriptional template of the interfering ribonucleic acid which can be used to produce dsRNA in a cell. In these embodiments, the transcriptional template may be a DNA that encodes the interfering ribonucleic acid. Methods and procedures associated with RNAi are also described in WO 03/010180 and WO 01 /68836, all of which are incorporated herein by reference. dsRNA can be prepared according to any of a number of methods that are known in the art, including in vitro and in vivo methods, as well as by synthetic chemistry approaches. Examples of such methods include, but are not limited to, the methods described by Sadher et al. (Biochem. Int. 14:1015, 1987); by Bhattacharyya (Nature 343:484, 1990); and by Livache, et al. (U.S. Patent No. 5,795,715), each of which is incorporated herein by reference in its entirety. Single-stranded RNA can also be produced using a combination of enzymatic and organic synthesis or by total organic synthesis. The use of synthetic chemical methods enable one to introduce desired modified nucleotides or nucleotide analogs into the dsRNA. dsRNA can also be prepared in vivo according to a number of established methods (see, e.g., Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed.; Transcription and Translation (B.D. Hames, and S.J. Higgins, Eds., 1984); DNA Cloning, volumes I and II (D.N. Glover, Ed., 1985); and Oligonucleotide Synthesis (M.J. Gait, Ed., 1984, each of which is incorporated herein by reference in its entirety). A number of options can be utilized to deliver the dsRNA into a cell or population of cells such as in a cell culture, tissue, organ or embryo. For instance, RNA can be directly introduced intracellular^. Various physical methods are generally utilized in such instances, such as administration by microinjection (see, e.g., Zernicka-Goetz, et al. (1997)

Development 124:1 133-1 137; and Wianny, et al. (1998) Chromosoma 107: 430-439). Other options for cellular delivery include permeabilizing the cell membrane and electroporation in the presence of the dsRNA, liposome-mediated transfection, or transfection using chemicals such as calcium phosphate. A number of established gene therapy techniques can also be utilized to introduce the dsRNA into a cell. By introducing a viral construct within a viral particle, for instance, one can achieve efficient introduction of an expression construct into the cell and transcription of the RNA encoded by the construct.

In certain embodiments, the agent that inhibits expression of functional β1 integrin subunit is an agent that inactivates the β1 integrin gene so that it no longer expresses a functional protein. By inactivated is meant that the gene, e.g., coding sequence and/or regulatory elements thereof, is genetically modified so that it no longer expresses functional β1 integrin subunit polypeptides, e.g., at least with respect to cellular entry of an anthrax toxin that is mediated by complexes comprising a β1 integrin subunit. The alteration or mutation may take a number of different forms, e.g., through deletion of one or more nucleotide residues, through exchange of one or more nucleotide residues, and the like. One means of making such alterations in the coding sequence is by homologous recombination. Methods for generating targeted gene modifications through homologous recombination include those described in: U.S. Patent Nos. 6,074,853; 5,998,209;

5,998,144; 5,948,653; 5,925,544; 5,830,698; 5,780,296; 5,776,744; 5,721 ,367; 5,614,396; 5,612,205; the disclosures of which are herein incorporated by reference.

Inhibitors of P A/toxin endocytosis mediated by complexes comprising β 1 integrin subunits

In some embodiments, the anti-anthrax agent that inhibits cellular entry of an anthrax toxin is an agent that inhibits endocytosis of PA-bound toxin that is mediated by a complex comprising a β1 integrin subunit. To inhibit PA/toxin endocytosis, an agent that modulates the activity of a protein that promotes receptor endocytosis may be used. For example, calpains (e.g. calpain 1 (CAPN1 , Genbank Accession Nos. NM 001 198868 (isoform 1 ); NM_005186 (isoform 2) and NM_001 198869 (isoform 3)), calpain 2 (CAPN2; Genbank Accession Nos. NM_001748 (isoform 1 ) and NM_001 146068 (isoform 2)), calpain 3 (CAPN3, Genbank Accession Nos. NM_000070 (isoform a), NM_024344 (isoform b), NM_173087 (isoform c), NM_173088 (isoform d), NM_173089 (isoform e)), etc. are calcium-dependent proteases that promote endocytosis of ligand by β1 integrin by cleaving proteins in focal adhesions (e.g. Talin, Genbank Accession No. NM 006289.3) that link complexes comprising β1 integrins to the cytoskeleton. As such, and as demonstrated in the examples section below, an agent that inhibits the endocytosis mediated by complexes comprising a β1 integrin subunit may be an agent that inhibits calpain cleavage of proteins, e.g. in focal adhesions. For example, as demonstrated in the examples section below, inhibiting calpain cleavage of talin, e.g. by contacting cells either in vitro or in vivo with the small molecule MDL28170 or the polypeptide calpastatin, inhibits cellular entry of anthrax toxin that is mediated by complexes comprising a β1 integrin subunit. Another example includes nucleic acids encoding a dominant negative Talin mutant, e.g. Talin-L432G, a Talin mutant in which leucine at codon 432 is mutated to glycine. Other agents that inhibit endocytosis may be readily identified using the assays described in the examples below.

Inhibitors of mediators of signaling by complexes comprising a β 1 integrin subunit

In some embodiments, the agent that inhibits cellular entry of an anthrax toxin mediated by a complex comprising a β1 integrin subunit is an agent that inhibits a mediator of signaling by a complex comprising a β1 integrin subunit. Examples of mediators that may be inhibited include the endocytosed toxin and the proteins that are activated by that toxin once it has been internalized, for example, proteins that are activated by LF (a metalloproteinase) to cleave MAPKK proteins, or proteins that are activated by EF (an adenylyl cyclase) that cause accumulation of fluids within and between cells, Mediators that may be inhibited also include focal adhesion proteins, i.e. proteins such as Talin, paxillin, alpha-actinin, vinculin, FAK, etc., that are found in focal adhesions. As another example, mediators that may be inhibited include kinases. For example, the protein tyrosine kinase FAK (PTK2, GenBank Accession Nos. NM_153831 (isoform a), NM_005607 (isoform b), and NM 001 199649 (isoform c)) is a known mediator of β1 integrin signaling. Other nonlimiting examples of kinases that mediate signaling by complexes comprising a β1 integrin subunit include FYN, Src, and MAP kinases. An additional example of mediators of signaling by a complex comprising a β1 integrin is the receptors that are activated by β1 integrins, e.g. by clustering with β1 integrins. Nonlimiting examples of such receptors include other integrins, growth factor receptors (e.g. FGFR, VEGFR), and CMG2.

Any convenient modulator of a mediator of signaling by a complex comprising a β1 integrin subunit that achieves the desired effect of inhibiting cellular entry of anthrax toxin and/or inhibiting anthrax toxin toxicity may be employed in the subject methods, e.g.

antibodies, polypeptides, nucleic acids, small molecules, etc. Contact of the cell with the anti-anthrax agent that inhibits cellular entry of an anthrax toxin mediated by complexes comprising a β1 integrin subunit may occur using any convenient protocol. The protocol may provide for in vitro or in vivo contact of the modulatory agent with the target cell, i.e. the cell expressing the receptor that is mediating cellular entry, depending on the location of the target cell. Contact may or may not include entry of the agent into the cell. For example, where the target cell is an isolated cell and the modulatory agent is an agent that modulates expression of β1 integrin subunit, the modulatory agent may be introduced directly into the cell under cell culture conditions permissive of viability of the target cell. Such techniques include, but are not necessarily limited to: viral infection, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, viral vector delivery, and the like. The choice of method is generally dependent on the type of cell being contacted and the nature of the modulatory agent, and the circumstances under which the transformation is taking place (i.e. in vitro, ex vivo, or in vivo). A general discussion of these methods can be found in Ausubel, et al, Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995.

Alternatively, where the target cell or cells are part of a multicellular organism, the modulatory anti-anthrax agent may be administered to the organism or subject in a manner such that the agent is able to contact the target cell(s), e.g., via an in vivo or ex vivo protocol. By "in vivo," it is meant in the target construct is administered to a living body of an animal. By "ex vivo" it is meant that cells or organs are modified outside of the body. Such cells or organs may be returned to a living body.

In the subject methods, the active agent(s) may be administered to the targeted cells using any convenient means capable of resulting in the desired activity. Thus, the agent can be incorporated into a variety of formulations, e.g., pharmaceutically acceptable vehicles, for therapeutic administration. More particularly, the agents of the present invention can be formulated into pharmaceutical compositions by combination with appropriate,

pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments (e.g., skin creams), solutions, suppositories, injections, inhalants and aerosols. As such, administration of the agents can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intracheal, etc., administration.

In pharmaceutical dosage forms, the agents may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. The following methods and excipients are merely exemplary and are in no way limiting.

For oral preparations, the agents can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

The agents can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

The agents can be utilized in aerosol formulation to be administered via inhalation.

The compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.

Furthermore, the agents can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. The compounds of the present invention can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more inhibitors. Similarly, unit dosage forms for injection or intravenous administration may comprise the inhibitor(s) in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.

The term "unit dosage form," as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the novel unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host. The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.

Where the anti-anthrax agent is a polypeptide, polynucleotide, analog or mimetic thereof, it may be introduced into tissues or host cells by any number of routes, including viral infection, microinjection, or fusion of vesicles. Jet injection may also be used for intramuscular administration, as described by Furth et al. (1992), Anal Biochem 205:365- 368. The DNA may be coated onto gold microparticles, and delivered intradermal^ by a particle bombardment device, or "gene gun" as described in the literature (see, for example, Tang et al. (1992), Nature 356:152-154), where gold microprojectiles are coated with the DNA, then bombarded into skin cells. For nucleic acid therapeutic agents, a number of different delivery vehicles find use, including viral and non-viral vector systems, as are known in the art.

When contacting cells, an effective amount of the subject anti-anthrax agent, i.e. an agent that inhibits cellular entry of an anthrax toxin mediated by complexes comprising a β1 integrin, is used. Biochemically speaking, an effective amount or effective dose of a subject anti-anthrax agent is an amount of inhibitor to decrease or attenuate toxin entry into the cell by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or by 100%. In some embodiments, the effective amount will decrease or attenuate the effects of the toxin on the cell by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or by 100%. In other words, the responsiveness of a cell to an anthrax toxin that has been contacted with an effective amount or effective dose of an anti-anthrax agent that inhibits cellular entry mediated by complexes comprising a β1 integrin subunit will be 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or will be 0%, i.e. negligible, the strength of the responsiveness observed of a cell that has not been contacted with an effective amount/dose of the anti-anthrax agent. The amount of cellular responsive to anthrax toxin, that is, the responsiveness of a cell to the presence of anthrax toxin, can be determined by any convenient way. For example, the amount of endocytosis of the toxin may be measured; the amount of FAK phosphorylation can be measured; the viability of the cell can be measured, etc. In this way, the inhibitory, or antagonistic, effect of the agent may be confirmed.

In a clinical sense, an effective dose of a subject anti-anthrax agent is the dose that will prevent or treat, i.e. reduce, an anthrax toxin mediated condition in a host. In some instances, the sensitivity of the host will be 70% or less, 60% or less, or 50% or less the sensitivity of a host not treated with the agent, in some instances, 40% or less, 30% or less, 20% or less the sensitivity of a host not treated with the agent, for example 10% or less, 5% or less, or even 0%, i.e. negligible, the strength of the responsiveness observed of a host not administered an effective amount/dose of the agent. The sensitivity of the host to anthrax toxin can be determined by any convenient way. For example, the host's temperature may be measured, chest pain or the level of difficulty of breathing may be assessed, the presence of cutaneous infections observed and monitored, etc. In some embodiments, a single dose will be administered. In some embodiments, several doses will be administered for a suitable period of time, e.g. over 6 hours or more, 12 hours or more, 24 hours or more, 48 hours or more, 72 hours or more, e.g. a week or more, two weeks or more, such as four weeks or more, in some instances 3 months or more, 6 months or more, or a year or more, to prevent or treat an anthrax toxin mediated condition in a host. It will be understood by those of skill in the art that an initial dose may be administered for such periods of time, followed by maintenance doses, which, in some cases, will be at a reduced dosage.

In some instances, an effective dose of a subject anti-anthrax agent is the dose that, when provided prophylactically, will prevent an anthrax toxin mediated condition in a host. In some instances, the sensitivity of the host following such prophylactic treatment will be 50% or less of the sensitivity of a host not treated with the agent, for example, 40% or less, 30% or less, 20% or less the sensitivity of a host not treated with the agent, e.g. 10% or less, 5% or less, or 0%, i.e. negligible, the sensitivity observed of a host not administered a prophylactic amount/dose of the agent. In such instances, the dose may be administered prior to or at the time of exposure to the anthrax toxin. If administered prior to the exposure, it may be administered 5 minutes or more before exposure, e.g. 10 minutes, 20 minutes, 30 minutes, or 1 hour or more before exposure, sometimes 3 hours or more, 6 hours or more, 12 hours or more, or 24 hours or more before exposure, for example 48 hours or 72 hours or more before exposure. In some instances, a single dose is provided. In some instances, multiple doses are provided. In some instances, the prophylactic dose is combined with a treatment dose, i.e. one or more doses provided after exposure, as described above. It will be understood by those of skill in the art that an initial dose may be administered for such periods of time, followed by maintenance doses, which, in some cases, will be at a reduced dosage.

The calculation of the effective amount or effective dose of the subject anti-anthrax agent to be administered is within the skill of one of ordinary skill in the art, and will be routine to those persons skilled in the art. Needless to say, the final amount to be administered will be dependent upon the route of administration and upon the nature of the disorder or condition that is to be treated. The effective amount of a therapeutic

composition to be given to a particular patient will depend on a variety of factors, several of which will differ from patient to patient. A competent clinician will be able to determine an effective amount of a therapeutic agent to administer to a patient to halt or reverse the progression the disease condition as required. Utilizing LD50 animal data, and other information available for the agent, a clinician can determine the maximum safe dose for an individual, depending on the route of administration. For instance, an intravenously administered dose may be more than an intrathecal^ administered dose, given the greater body of fluid into which the therapeutic composition is being administered. Similarly, compositions which are rapidly cleared from the body may be administered at higher doses, or in repeated doses, in order to maintain a therapeutic concentration. Utilizing ordinary skill, the competent clinician will be able to optimize the dosage of a particular therapeutic in the course of routine clinical trials.

In some embodiments, the method further comprises the step of contacting the cell or administering to the individual an effective amount of a second anti-anthrax agent. For example, an anti-anthrax agent may be an agent that inhibits entry of B. anthracis spores into a cell. As another example, the anti-anthrax agent is an agent that inhibits entry of anthrax toxin into a cell. Anti-anthrax agents are described in greater detail in the

Compositions section below. Any convenient anti-anthrax agent may be employed.

COMPOSITIONS

Aspects of the invention include compositions for preventing or treating anthrax toxicity in a subject ,i.e."anti-anthrax compositions". By treating anthrax toxicity it is meant that the subject, or host, is less sensitive to the toxicity of B. anthracis than if the subject had not been administered the composition. By preventing anthrax toxicity in a subject, it is meant that the subject, or host, exhibits no symptoms of the toxicity of B. anthracis.

Sensitivity to toxicity, as measured using any convenient protocol for measuring B.

anthracis toxicity, e.g. as described in the examples section below, is decreased by an amount of 2-fold or more, usually by 5-fold or more and including by 10-, 25-, 50-, 100-fold or more, as compared to a control, i.e., a subject that is not subjected to the methods of the present invention. In certain embodiments, the compositions inhibit entry of anthrax toxin into the subject's cells, such that cells do not die upon exposure to anthrax toxin. In some embodiments, the compositions inhibit entry of B. anthracis spores into the subject's cells, such that the cells cannot produce anthrax toxin. In some embodiments, the compositions inhibit B. anthracis multiplication. Compositions of the invention may comprise an anti-anthrax agent that inhibits cellular entry of anthrax mediated by complexes comprising a β1 integrin subunit. In some embodiments, the anti-anthrax agent is an agent that inhibits the binding of PA and PA- comprising polypeptides to a complex comprising the β1 integrin subunit. In some embodiments, the composition comprises an agent that inhibits activation of the complex comprising a β1 integrin subunit to bind to PA. In some embodiments, the composition comprises an agent that inhibits PA/toxin endocytosis mediated by a complex comprising a β1 integrin subunit. In some embodiments, the composition comprises an agent that inhibits a mediator of signaling by a complex comprising a β1 integrin subunit. In some embodiments, the composition comprises an agent that inhibits the expression of the β1 integrin subunit. Agents that inhibit cellular entry of toxin are described in greater detail above with regard to methods of the invention.

In some embodiments, the anti-anthrax composition comprises a second anti- anthrax agent. In other words, a second anti-anthrax agent is agent provided in

combination with an anti-anthrax agent that inhibits cellular entry of anthrax toxin mediated by complexes comprising a β1 integrin subunit. Any convenient second anti-anthrax agent may be employed.

For example, a second anti-anthrax agent may be an agent that inhibits entry of B. anthracis spores into a cell. Examples of receptors that permit B. anthracis spores to enter cells include complexes comprising β1 integrin, e.g. α2β1 (Xue et al. (2001 ) Entry of

Bacillus anthracis spores into epithelial cells is mediated by the spore surface protein BcIA, integrin a2b1 and complement component C1 q. Cell Microbiol 13(4):620-34), and complexes comprising β2 integrin, e.g. αηιβ2 (Mac-1 ) (Oliva CR et al. The integrin Mac-1 (CR3) mediates internalization and directs Bacillus anthracis spores into professional phagocytes. Proc Natl. Acad Sci 105(4):1261 -6). As such, agents that inhibit entry of B. anthracis spores into a cell will inhibit binding of spore proteins, e.g. BcIA, to β1 or β2 integrin complexes and endocytosis/phagocytosis of spores mediated by β1 or β2 integrin complexes (see, e.g., US 2009/0221094 and WO 2008/063147).

As another example, a second anti-anthrax agent may be an agent that prevents B. anthracis from multiplying, e.g. an antibiotic. Any convenient antibiotic may be employed, e.g. penicillin, doxycycline, ciprofloxacin, etc. Regimens may vary depending on the type of exposure to B. anthracis. For example, for inhalation anthrax, antibiotics are typically given for about 60 days, as it may take that long for spores to germinate. For cutaneous anthrax, treatment is usually for 7-10 days. Such regimens will be known to the ordinarily skilled artisan or can be readily determined. As another example, a second anti-anthrax agent may be an agent that inhibits entry of anthrax toxin into a cell. Anti-anthrax agents that inhibit entry of anthrax toxin into a cell include agents that inhibit binding of anthrax toxin to cell surface receptors or that inhibit the endocytosis of the anthrax toxin by those cell surface receptors. Examples of receptors other than β1 integrin that also mediate anthrax toxin into cells include CMG2 (Liu S, et al. (2009) Capillary morphogenesis protein-2 is the major receptor mediating lethality of anthrax toxin in vivo. Proc Natl Acad Sci USA. 106:12424-12429), LRP6 (Wei W, et al. (2006) The LDL receptor-related protein LRP6 mediates internalization and lethality of anthrax toxin. Cell 124(6):1 141 -54), and TEM8 (Liu S, Leppla SH. (2003) Cell surface tumor endothelium marker 8 cytoplasmic tail-independent anthrax toxin binding, proteolytic processing, oligomer formation, and internalization. J Biol Chem. 278(7) :5227-34.) As such, agents that inhibit anthrax toxin from entering cells include agents that inhibit CMG2-, LRP6- or TEM8-mediated entry of anthrax toxin. These include, for example, antibodies or fragments thereof that are specific for CMG2, LRP6 or TEM8 (see, e.g., Li G, et al. (2009) The inhibition of the interaction between the anthrax toxin and its cellular receptor by an anti-receptor monoclonal antibody. Biochem Biophys Res Commun 385(4):591 -5; and Duan, HF et al. (2007) Antitumoral Activity of TEM8-Fc: An engineered Antibody-like Molecule Targeting Tumore Endothelial Marker 8. J. Natl Cancer Inst 99:1551 -6), peptides that bind to and block ligand binding to CMG2, LRP6 or TEM8 (see, e.g. Basha S, et al. (2006) Polyvalent inhibitors of anthrax toxin that target host receptors. Proc Natl Acad Sci U S A. 2006 Sep 5;103(36):13509-13) and small molecule inhibitors that inhibit receptor activity (see, e.g., Zhu PJ et al. (2009) Quantitative high throughput screening identifies inhibitors of anthrax-induced cell death. Bioorg Med Chem. 17(14): 5139-5145.)

Other agents that inhibit binding of anthrax toxin to cell surface receptors that may be used in combination with the agents of the present invention include agents that bind to PA, LF, or EF of anthrax toxin. Examples of such agents include antibodies and fragments thereof that are specific for PA, LF, or EF (see, e.g., Mabry, R. et al. (2005) Passive

Protection against anthrax by Using a High-Affinity Antitoxin Antibody Fragment Lacking an Fc Region. Infect and Immun 73(12):8362-8368; Leysath, CE et al. (2009) Crystal Structure of the Engineered Neutralizing Antibody M18 Complexed to Domain 4 of the Anthrax Protective Antigen. J Mol Biol. 387(3): 680-693; US 2010/01 19520; and US

2010/01 1 1868); dominant negative polypeptides ,e .g., dominant negative polypeptides of CMG2, LRP6 or TEM8 (e.g., Cai C, et al. (201 1 ) Tumor endothelium marker-8 based decoys exhibit superiority over capillary morphogenesis protein-2 based decoys as anthrax toxin inhibitors. PLoS One. 201 1 ;6(6):e20646; Sharma S, et al. (2009) Efficient

neutralization of antibody-resistant forms of anthrax toxin by a soluble receptor decoy inhibitor. Antimicrob Agents Chemother 53(3):1210-2; and Wei W, et al. (2006) supra), and the like. Additional agents may be readily identified based upon the understanding of the interaction domain between anthrax and its cellular receptors; see, e.g. Liu S. et al. (2007) Characterization of the interaction between anthrax toxin and its cellular receptors. Cell Microbiol. 9(4): 977-987). Anti-anthrax agents that inhibit entry of anthrax toxin into a cell also include agents that inhibit the cleavage of protective antigen, e.g. polypeptide agents (e.g. US 201 1/0014236).

As yet another example, the second anti-anthrax agent may be an agent that inhibits the activity of the anthrax toxin once inside the cell. Examples of agents that would have such an effect include, for example, small molecule agents that inhibit toxin protease activity (e.g. Johnson, SL et al. (2009) Structure-activity relationship studies of a novel series of anthrax lethal factor inhibitors. Bioorg Med Chem.17(9):3352-68; US 2010/0298390), and the like.

In some instances, the effect of the composition upon the sensitivity of the host to anthrax toxicity is additive. In other words the sensitivity of the individual to toxicity by administering the combination of a first anti-anthrax agent and a second anti-anthrax agent is reduced by the sum of the reduction in sensitivity of the individual to toxicity that would be achieved if only the first anti-anthrax agent was administered plus the reduction in sensitivity of the individual to toxicity that would be achieved if only the second anti-anthrax agent was administered. In some instances, the effect of the composition upon the sensitivity of the host to anthrax toxicity is synergistic. In other words, the sensitivity of the individual to toxicity by administering the combination of a first anti-anthrax agent and a second anti- anthrax agent is reduced by more than the sum of the reduction in sensitivity of the individual to toxicity that would be achieved if only the first anti-anthrax agent was administered plus the reduction in sensitivity of the individual to toxicity that would be achieved if only the second anti-anthrax agent was administered. In some instance, the sensitivity is at least 1 .5 -fold less 2 than the sum, for example 2-fold less, 3-fold less, 4-fold less, 5-fold less, 10-fold less, or more, including 20-fold less, 50-fold less, or 100-fold less.

Also provided are pharmaceutical preparations of the subject compositions. The subject compounds can be incorporated into a variety of formulations for administration to a subject. More particularly, the compounds of the present invention can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols. The formulations may be designed for administration via a number of different routes, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intracheal, etc., administration.

In pharmaceutical dosage forms, the compositions may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. The following methods and excipients are merely exemplary and are in no way limiting.

The pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily

suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such

compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example, magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated by the technique described in the U.S. Pat. Nos. 4,256,108; 4,166,452; and 4,265,874 to form osmotic therapeutic tablets for control release.

Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredients is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active material in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethyl-cellulose, methylcellulose, hydroxy- propylmethycellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethylene-oxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as

polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more

preservatives, for example ethyl, or n-propyl, p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, saccharin or aspartame.

Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.

The pharmaceutical compositions of the invention may also be in the form of oil-in- water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the the partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavouring agents.

Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents. The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally- acceptable diluent or solvent, for example as a solution in 1 ,3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The compositions can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

The compositions can be utilized in aerosol formulation to be administered via inhalation. The compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.

Furthermore, the compositions can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. The compositions of the present invention can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.

The compositions of this invention and their pharmaceutically acceptable salts which are active on topical administration can be formulated as transdermal compositions or transdermal delivery devices ("patches"). Such compositions include, for example, a backing, active compound reservoir, a control membrane, liner and contact adhesive. Such transdermal patches may be used to provide continuous or discontinuous infusion of the compositions of the present invention in controlled amounts. The construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art. See, e.g., U.S. Pat. No. 5,023,252, issued Jun. 1 1 , 1991 , herein incorporated by reference in its entirety. Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents.

Optionally, the pharmaceutical composition may contain other pharmaceutically acceptable components, such a buffers, surfactants, antioxidants, viscosity modifying agents, preservatives and the like. Each of these components is well-known in the art. See, for example, U.S. Pat. No. 5,985,310, the disclosure of which is herein incorporated by reference.

Other components suitable for use in the formulations of the present invention can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985). In an embodiment, the aqueous cyclodextrin solution further comprise dextrose, e.g., about 5% dextrose.

Dosage levels of the order of from 0.01 mg to 140 mg/kg of body weight per day are useful in representative embodiments, or alternatively 0.5 mg to 7 g per patient per day. For example, inflammation may be effectively treated by the administration of from 0.01 to 50 mg of the compound per kilogram of body weight per day, or alternatively 0.5 mg to 3.5 g per patient per day. Those of skill will readily appreciate that dose levels can vary as a function of the specific compound, the severity of the symptoms and the susceptibility of the subject to side effects.

The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a formulation intended for the oral administration of humans may contain from 0.5 mg to 5 g of active agent compounded with an appropriate and convenient amount of carrier material which may vary from 5 to 95 percent of the total composition. Dosage unit forms will generally contain between from 1 mg to 500 mg of an active ingredient, e.g. 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 800 mg, or 1000 mg.

It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

As such, unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more inhibitors. Similarly, unit dosage forms for injection or intravenous administration may comprise the inhibitor(s) in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier. The term "unit dosage form," as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compositions of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the novel unit dosage forms of the present invention depend on the particular peptidomimetic compound employed and the effect to be achieved, and the

pharmacodynamics associated with each compound in the host. The above methods and compositions find use in a variety of different applications. Certain applications are now reviewed in the following Utility section.

UTILITY

The methods find use in a variety of therapeutic applications in which it is desired to decrease anthrax toxin entry into a target cell or collection of cells, where the collection of cells may be a whole animal or portion thereof, e.g., tissue, organ, etc. In such methods, an effective amount of an anti-anthrax agent that inhibits, or antagonizes, cellular entry of anthrax toxin mediated by complexes comprising a β1 integrin subunit is administered to the target cell or cells, e.g., by contacting the cells with the agent, by administering the agent to the animal, etc. By effective amount is meant a dosage sufficient to modulate cellular entry of the anthrax toxin, as desired; e.g. as discussed above. In some embodiments, an effective amount of a second anti-anthrax agent is also provided, this agent being any convenient anti-anthrax agent.

The subject methods find use in the treatment of a variety of different conditions in which the modulation, e.g., enhancement or decrease, of cellular entry of anthrax toxin mediated by complexes comprising a β1 integrin subunit is desired. In representative embodiments, the methods are employed to modulate an anthrax toxin mediated condition in a subject. In certain of these embodiments, the methods are methods of prophylactically conferring an anthrax toxin resistant phenotype on the subject, such that the subject can later be exposed to B.anthracis and not suffer from subsequent anthrax toxin mediated disease conditions, as reviewed above. In certain embodiments, the methods are employed to treat a subject suffering from an anthrax mediated disease condition resulting from exposure to B.anthracis. In certain of these embodiments, the methods include first diagnosing the presence of such a condition in the subject. By treatment is meant that at least an amelioration of the symptoms associated with the condition afflicting the host is achieved, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g. symptom, associated with the condition being treated. As such, treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g. prevented from happening, or stopped, e.g. terminated, such that the host no longer suffers from the condition, or at least the symptoms that characterize the condition. In certain of the embodiments, the subject methods find use in the treatment of host having a "late-stage" disease condition, where a substantial amount of anthrax toxin is present in the host and the condition is no longer treatable by targeting the pathogen itself. A "subject" or "patient" for the purposes of the present invention includes both humans and other animals, particularly mammals. Thus the methods are applicable to both human therapy and veterinary applications. In the preferred embodiment the patient is a mammal, e.g. a primate, e.g. a human.

In certain embodiments, the subject anti-anthrax agent can be used in combination with other therapeutic agents in the methods of the invention. For example, agents that inhibit cellular entry of anthrax toxin mediated by complexes comprising a β1 integrin subunit may be used with other anti-anthrax agents to further reduce sensitivity of cells to B. anthracis, e.g. by further reducing cellular entry of toxins, reducing cellular entry of spores, reducing the ability of the bacteria to multiply, etc..

A variety of hosts are treatable according to the subject methods. Generally such hosts are "mammals" or "mammalian," where these terms are used broadly to describe organisms which are within the class mammalia, including the orders carnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In many embodiments, the hosts will be humans.

SCREENING ASSAYS

Also provided by the subject invention are screening assays designed to find anti- anthrax agents, where such agents may find use in a variety of applications, including as therapeutic agents as described above and to treat other diseases in which β1 integrin signaling has been implicated, e.g. cancer. The screening methods may be assays which provide for qualitative/quantitative measurements of β1 integrin subunit-mediated anthrax toxin cell entry activity in the presence of a particular candidate therapeutic agent. The screening method may be an in vitro or in vivo format. Depending on the particular method, one or more of, usually one of, the components of the screening assay may be labeled, where by labeled is meant that the components comprise a detectable moiety, e.g. a fluorescent or radioactive tag, or a member of a signal producing system, e.g. biotin for binding to an enzyme-streptavidin conjugate in which the enzyme is capable of converting a substrate to a chromogenic product.

For example, in screening assays for biologically active agents, cells expressing the cell receptor of interest, e.g. a β1 integrin, are contacted with a toxin and a candidate agent of interest and the effect of the candidate agent on the toxicity of the toxin is assessed by monitoring one or more output parameters. Parameters are quantifiable components of cells, particularly components that can be accurately measured, desirably in a high throughput system. A parameter can be any cell component or cell product including cell surface determinant, receptor, protein or conformational or posttranslational modification thereof, lipid, carbohydrate, organic or inorganic molecule, nucleic acid, e.g. mRNA, DNA, etc. or a portion derived from such a cell component or combinations thereof. While most parameters will provide a quantitative readout, in some instances a semi-quantitative or qualitative result will be acceptable. Readouts may include a single determined value, or may include mean, median value or the variance, etc. Characteristically a range of parameter readout values will be obtained for each parameter from a multiplicity of the same assays. Variability is expected and a range of values for each of the set of test parameters will be obtained using standard statistical methods with a common statistical method used to provide single values. Thus, for example, one such method may comprise contacting a cell that expresses a receptor of interest with the toxin and a candidate agent; and comparing the parameter to the parameter in a control, e.g. a cell that expresses the receptor and that was contacted with the toxin but was not contacted with the candidate agent, wherein a difference in the parameter in the cell contacted with the candidate agent as compared to the control indicates that the candidate agent has an effect on cell sensitivity to the toxin.

One example of an output parameter that may be quantified when screening for, e.g., agents that modulate cellular sensitivity to a toxin, would be the amount of toxin internalized by, i.e. endocytosed by, the cell. Another example would be an output parameter that is reflective of an apoptotic state, such as the amount of DNA fragmentation, the amount of cell blebbing, the amount of phosphatidylserine on the cell surface as visualized by Annexin V staining, and the like; and/or an output parameter that is reflective of the viability of the culture, e.g. the number of cells in the culture, the rate of proliferation of the culture. Other output parameters could include those that are reflective of the function of the cells in the culture, e.g. the cytokines and chemokines produced by the cells, the rate of chemotaxis of the cells, the phagocytic activity of the cells, etc. In some instances, one parameter is measured. In some instances, multiple parameters are measured.

Cells useful for screening include any cell that expresses the receptor of interest, e.g. an integrin complex comprising, a31 integrin subunit. For example, the cell may be a macrophage, NK cell, bronchial epithelial cell, cardiomyocyte, smooth muscle, adipocyte, etc. As another example, the cell may be a cell that does not endogenously express the receptor of interest but ectopically expresses the receptor of interest, e.g. by supplying the gene encoding the receptor to the cell as a nucleic acid such that the cell will express the gene of interest.

Candidate agents of interest for screening include known and unknown compounds that encompass numerous chemical classes, primarily organic molecules, which may include organometallic molecules, inorganic molecules, genetic sequences, etc. Candidate agents of interest for screening also include nucleic acids, for example, nucleic acids that encode siRNA, shRNA, antisense molecules, or miRNA, or nucleic acids that encode polypeptides, e.g. antibodies or peptides. Candidate agents of interest for screening also include polypeptides. An important aspect of the invention is to evaluate candidate drugs, including toxicity testing; and the like.

A variety of other reagents may be included in the screening assay. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc that are used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc. may be used. A variety of different candidate agents may be screened by the above methods, including any of the representative agents described above. Using the above screening methods, a variety of different therapeutic agents may be identified. Such agents may be find use in a variety of therapeutic applications, as reviewed above.

KITS

Kits with unit doses of the compositions, such as in oral or injectable doses, are provided. For example, kits and systems for practicing the subject methods may include an anti-anthrax agent that inhibits cellular entry of an anthrax toxin mediated by complexes comprising a β1 integrin subunit. In some embodiments, the kit will comprise at least a second anti-anthrax agent. In some embodiments, the anti-anthrax agent(s) are provided in pharmaceutical formulations.

In addition to the above components, the subject kits may further include instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, etc. Yet another means would be a computer readable medium, e.g., diskette, CD, etc., on which the information has been recorded. Yet another means that may be present is a website address which may be used via the internet to access the information at a removed site. Any convenient means may be present in the kits.

While the description above is focused on cellular entry of an anthrax toxin mediated by β1 integrin, this description is not meant to be limiting. As such, embodiments of the subject invention include those in which the above described modulatory agents target different cell surface receptors and/or different toxins or the pathogen that produce them. For example, certain embodiments of the invention are drawn to agents that inhibit β1 integrin-mediated entry of a toxin including toxins other than an anthrax toxin into a cell. As such, aspects of the invention include embodiments of modulating β1 -mediated entry of a toxin into a cell. Certain other embodiments of the invention are drawn to agents that inhibit receptor-mediated entry of an anthrax toxin into a cell, wherein the receptor is a receptor other than β1 integrins, e.g. CMG2, TEM8, and the like. In these embodiments, these modulatory agents function to modulate the entry of an anthrax toxin into a cell. Certain other embodiments of the invention are drawn to agents that inhibit receptor-mediated entry of a toxin or pathogen including a toxin or pathogen other than an anthrax toxin into a cell, e.g. S. pyogenes adenylate cyclase toxin, B. pertussis adenylate cyclase toxin, Clostridium septicum a-toxin, Shiga toxin, H. pylori VacA and the like. As such, aspects of the invention include embodiments of modulating receptor mediated entry of a toxin or pathogen into a cell.

The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL

Example 1

I. Materials & Methods

Bioinformatics Analysis of Gene Expression. GABRIEL software (Pan KH, Lih CJ, Cohen SN. Analysis of DNA microarrays using algorithms that employ rule-based expert knowledge. Proc Natl Acad Sci USA. 2002;99:21 18-2123) separated cancer cell lines (SK- MEL-5, SK-MEL28, M-14, MALME-3M, MDA-MB-435, MDA-N, SK-MEL-2, HL-60, MCF7, SW-620, EKVX, and A549) into two groups, LF-PA-sensitive and LF-PA-resistant, based on previous reports (Abrami L, et al. Functional interactions between anthrax toxin receptors and the WNT signalling protein LRP6. Cell Microbiol. 2008;10:2509-2519; Abi-Habib RJ, et al. A urokinase-activated recombinant anthrax toxin is selectively cytotoxic to many human tumor cell types. Mol Cancer Ther. 2006;5:2556-2562; Abi-Habib RJ, et al. BRAF status and mitogen-activated protein/extracellular signal-regulated kinase kinase 1/2 activity indicate sensitivity of melanoma cells to anthrax lethal toxin. Mol Cancer Ther.

2005;4:1303-1310; Chen KH, et al. Selection of anthrax toxin protective antigen variants that discriminate between the cellular receptors TEM8 and CMG2 and achieve targeting of tumor cells. J Biol Chem. 2007;282:9834-9845; Kassam A, et al. Differentiation of human monocytic cell lines confers susceptibility to Bacillus anthracis lethal toxin. Cell Microbiol. 2005;7:281 -292; Koo HM, et al. Apoptosis and melanogenesis in human melanoma cells induced by anthrax lethal factor inactivation of mitogen-activated protein kinase kinase. Proc Natl Acad Sci USA. 2002;99:3052-3057; Liu S, et al. Matrix metalloproteinase- activated anthrax lethal toxin demonstrates high potency in targeting tumor vasculature. J Biol Chem. 2008;283:529-540). GABRIEL (Ross DT, et al. Systematic variation in gene expression patterns in human cancer cell lines. Nat Genet. 2000;24:227-235.) was used for correlation of steady-state levels of expression with toxin lethality.

Chemicals and Reagents. PA and LF were purchased from List Biological

Laboratories. The fluorescently labeled antibodies purchased from BioLegend were anti— CD44-APC, anti-31 -integrin-APC, anti-a4-integrin/Alexa Fluor 647, and anti-a5

integrin/Alexa Fluor 647. The preservative-free monoclonal antibodies purchased from

BioLegend were anti-CD44 (IM7), anti— β1 -integrin (ΗΜβ1 -1 ), anti-a4-integrin (R1 -2), anti— a5 integrin (MFR5), anti— β3 integrin (ΗΜβ3-1 ), and anti-av integrin (RMV-7). High molecular weight HA from human umbilical cord was purchased from Sigma-Aldrich and HA from S. pyogenes of high, medium, low, and ultra-low molecular weight were purchased from R&D Systems.

Cell Culture. RAW264.7 mouse macrophage cells were maintained in DMEM (Invitrogen) supplemented with 10% FBS (HyClone) and 100 μg/mL penicillin and 100 μg/mL streptomycin. In this study we isolated single clones from a pool of ARAP3, LRP6 knockdown, and their respective WT RAW264.7 cell lines, which were described previously (Lu Q, et al. EST-based genome-wide gene inactivation identifies ARAP3 as a host protein affecting cellular susceptibility to anthrax toxin. Proc Natl Acad Sci USA. 2004;101 :17246- 17251 ; Wei W, et al. The LDL receptor-related protein LRP6 mediates internalization and lethality of anthrax toxin. Cell. 2006;124:1 141 -1 154). For CMG2 knockdown experiment, lentiviral-based shRNAmir vector pGIPZ with sequence targeted for CMG2 was purchased from Open Biosystems. Viral infection of CMG2 shRNA was performed using lentiviral- based methods (Lu Q, et al., supra; Wei W, et al. supra). Single RAW264.7 colonies were selected after puromycin treatment (4 g/mL).

Toxin Treatment and Cell Viability Assays. Cells were treated with toxin for 4 or 24 h, and determination of cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed as described (Lu Q, et al., supra; Wei W, et al. supra). Each data point shown in figures for MTT assays represents the average of results from four wells in each of at least three separate experiments. Cell viability is shown as the percentage of survivors obtained relative to treatment by PA alone (100%).

Antibody/lntegrin Protection Assay. RAW264.7 cells (3 ^χ 10³ per well) were seeded in 96-well plates (100 μΙ_Λ/νβΙΙ) 24 h before the assay. The cells were then incubated at 37 ^<C for 24 h with 3 ng/mL of PA, 500 ng/mL of LF, and serial dilutions of antibodies or purified α5β1 -integrin (R&D Systems), followed by 24-h incubation in fresh, toxin-free media. The preservative-free monoclonal antibodies were suspended in the media at various concentrations. Toxin sensitivity was determined by MTT assay.

Immunofluorescence Microscopy. PA protein was labeled with Alexa Fluor 488 using the A10235 protein labeling kit (Molecular Probes). We determined by MTT assay that such labeling did not affect the ability of PA-LF to kill macrophages. For experiments determining the effects of β1 and β3 integrin inhibition or HA on toxin cell entry, cells were preincubated with 100 μg/mL HA or with 10 μg/mL of anti-mouse β1 or β3 integrin inhibitory monoclonal antibodies for 1 h in serum-free Iscove modified Dulbecco medium (IMDM; Invitrogen). Fresh serum-free IMDM media was added that contained 1 μg/mL PA/Alexa Fluor 488 alone or 1 μg/mL PA/Alexa Fluor 488 mixed with fresh 100 μg/mL HA or with 10 μg/mL of anti-mouse β1/β3 integrin inhibitory monoclonal antibodies. Cells were incubated for 60 min at 4 ^<C for PA-binding analysis and for 20 min at 37 °C for determination of PA internalization.

For localization of integrins and CD44, cells were incubated with or without 1 μg/mL

PA/Alexa Fluor 488, and with 0.5 μg/mL fluorescently labeled antibodies for 60 min at 4 °C for PA-binding analysis and for 20 min at 37 °C for PA-internalization analysis in serum-free IMDM media (Invitrogen). Cells were washed, fixed, and examined by using a fluorescence microscope (Leica).

Binding Kinetics. Experiments were performed using the Bio-Rad ProteOn XPR36 system. The extracellular portions of a5 and β1 -integrins, devoid of their transmembrane and cytoplasmic domains, were cloned upstream of acidic and basic tails, and the heterodimer was expressed in and purified from CHO cell line by R&D Systems. The α5β1 heterodimer was functional based on its ability to bind fibronectin with Kd of nanomolar range (R&D Systems). Protein concentration of α5β1 -integrin was 0.2 μηιοΙ/Ι_ and the concentrations of PA ranged from 148 nmol/L to 12 μηιοΙ/Ι_ in PBS buffer with 1 mmol/L MnCI₂. Equilibrium dissociation constants were calculated on the basis of the kinetic measurements of the association and dissociation rate constants according to the formula Kd = kd / ka.

Biochemical Assay of PA Binding and Internalization. Cells were grown to confluence and preincubated in serum-free IMDM media (Invitrogen) with 10 μg/mL of inhibitory monoclonal antibodies in β1 or β3 integrins or with 100 μg/mL of HA for 1 h before LF-PA exposure. Cells were exposed to 1 μg/mL of PA at 4 °C for 1 h for binding assay or 100 ng/mL of PA at 37 ^<C for 20 min for internalization assay in the presence or in the absence of HA or inhibitory antibody in serum-free IMDM media. Cells were then washed with cold PBS solution five times and lysed in RIPA buffer containing a protease inhibitor mixture (Roche). Western blot analysis was performed using rabbit polyclonal anti-PA antibody 7349.3 (kindly provided by G. Bokoch, Scripps Research Institute, La Jolla, CA) to detect monomeric PA, rabbit polyclonal anti-PA antibody (Abeam) to detect heptameric PA, or mouse antiactin monoclonal antibody (Sigma-Aldrich). Chemiluminescence of bands and their relative intensities were revealed using a VersaDoc 1000 instrument (Bio-Rad).

II. Results

Identification of Genes that Are Differentially Expressed in LF-PA-Sensitive Versus LF-PA-Resistant Cell Lines. Using pattern-search algorithms of GABRIEL (Genetic

Analysis By Rules Incorporating Expert Logic), a rule-based system of computer programs designed for genetic analyses (Pan KH, et al., supra), to detect such correlations in a previously published dataset of gene expression profiles for NCI 60 tumor cell lines (Ross DT, et al., supra), we identified nine genes whose reduced expression in LF-PA-resistant versus LF-PA-sensitive cell lines exceeded the variation in global gene expression in those cell lines by at least twofold (Fig. 6). The false-positive rate for this threshold choice was 0.09. Using the same search parameters, no genes were found by GABRIEL to be up- regulated in LF-PA-resistant and down-regulated in LF-PA-sensitive NCI 60 cell lines.

Quantitative RT-PCR analysis of mRNA from two additional cell lines found previously to be highly sensitive [RAW264.7 mouse macrophages (Hanna PC, Acosta D, Collier RJ. On the role of macrophages in anthrax. Proc Natl Acad Sci USA.

1993;90:10198-10201 )] or highly resistant [M2182 human prostate cancer cells (Lu Q, et al., supra; Wei W et al., supra)] to LF-PA showed that expression of three of the nine GABRIEL-detected genes, osteopontin (OPN), DAB2, and cystatin B, in RAW264.7 cells exceeded expression in M2182 cells by 350,000-, 240-, and 80-fold, respectively, consistent with possible association of gene expression with anthrax toxicity; statistically significant differential expression of the other six genes was not observed. OPN is a secreted phosphoprotein known to activate three distinct integrin complexes (α4β1 , α5β1 , and ανβ3) through its interaction with the cell surface protein CD44 (Bayless KJ, et al., Osteopontin is a ligand for the α4β1 integrin. J Cell Sci. 1998;1 1 1 :1 165-1 174; Katagiri YU, et al. CD44 variants but not CD44s cooperate with β1 -containing integrins to permit cells to bind to osteopontin independently of arginine-glycine-aspartic acid, thereby stimulating cell motility and chemotaxis. Cancer Res. 1999;59:219-226; Ponta H, et al. CD44: From adhesion molecules to signalling regulators. Nat Rev Mol Cell Biol. 2003;4:33^15; Redondo-Munoz J, et al. α4β1 integrin and 190-kDa CD44v constitute a cell surface docking complex for gelatinase B/MMP-9 in chronic leukemic but not in normal B cells. Blood. 2008;1 12:169- 178; Scatena M, et al. NF-kappaB mediates alphavbeta3 integrin-induced endothelial cell survival. J Cell Biol. 1998;141 :1083-1093; Yokosaki Y, et al. Distinct structural

requirements for binding of the integrins alphavbeta6, alphavbeta3, alphavbeta5, α5β1 and α9β1 to osteopontin. Matrix Biol. 2005;24:418^127). Importantly, the β components of these integrin complexes contain the vWA domain, which has been shown to be a site for PA binding in the two previously identified anthrax toxin receptors. Together, these

considerations raised the possibility that integrins β1 and/or β3, like TEM8 and CMG2, may function as receptors for PA. The following results were obtained in experiments designed to test this conclusion.

α4β 1 and α5β 1 -integrin Complexes Colocalize with PA at the Macrophage Cell Surface and During Endocytosis. Fluorescence microscopy of RAW264.7 macrophages exposed to PA and anti— β1 -integrin antibodies that have been labeled differentially with fluorescent dyes showed colocalization of PA with integrin at the surface of cells maintained at 4 °C but no detectable integrin or PA in the cytoplasm (Fig. 1 A). Shift of the cells to 37 ^<C, which leads to PA uptake (Lu Q, et al., supra; Wei W et al., supra) resulted in the conjoint internalization of β1 -integrin and PA and their continued colocalization in the cytoplasm. However, in the absence of PA, β1 -integrin remained at the surface after shift of macrophages to 37 °C, indicating that its uptake was dependent on PA. This finding, together with earlier evidence that integrins routinely undergo endocytosis upon binding to specific ligands (Caswell PT, et al. Integrins: Masters and slaves of endocytic transport. Nat Rev Mol Cell Biol. 2009;10:843-853), argues that PA and β1 -integrin are constituents of a PA internalization complex. Supporting this conclusion, fluorescence microscopy using differentially labeled PA and antibodies to integrin a4 or a5, which are binding partners of β1 -integrin, showed that these a integrins also colocalized with PA at the cell surface, and upon uptake of PA at 37 °C, also in the cytoplasm (Fig. 1 B and C). Macrophages treated with antibodies to integrin β3 showed no corresponding fluorescence, consistent with earlier investigations showing that this integrin is not expressed in RAW264.7 macrophages (Cuetara BL, et al. Cloning and characterization of osteoclast precursors from the

RAW264.7 cell line. In Vitro Cell Dev Biol Anim. 2006;42:182-188).

Unexpectedly, given the concurrent presence on the surface of RAW264.7 cells of CMG2 (Dal Molin F, et al. Cell entry and cAMP imaging of anthrax edema toxin. EMBO J. 2006;25:5405-5413; Rainey GJ, et al. Receptor-specific requirements for anthrax toxin delivery into cells. Proc Natl Acad Sci USA. 2005;102:13278-13283), which has been described as the major PA receptor (Liu S, et al. Capillary morphogenesis protein-2 is the major receptor mediating lethality of anthrax toxin in vivo. Proc Natl Acad Sci USA.

2009 ;106:12424-12429), prior treatment of cells with a monoclonal anti-βΐ antibody known to highly specifically block the interaction of this integrin with its ligands (Noto K, et al. Identification and functional characterization of mouse CD29 with a mAb. Int Immunol. 1995;7:835-842) decreased the binding of PA to RAW264.7 cells by at least 60% as indicated by Western blotting, and also reduced the uptake of PA into the cytoplasm. In contrast, antibody against β3 integrin, which is not produced by these macrophages, did not detectably affect either the attachment (Fig. 2 A and B) or internalization (Fig. 2 A and C) of PA. These results indicate that integrin β1 affects the actions of CMG2 as a PA receptor.

Biochemical Evidence of Interaction of α5β 1 Complex with PA. Biochemical evidence of the ability of the purified βΐ -integrin-containing complex α5β1 to bind to PA in vitro, and the dissociation constant for the interaction, were obtained using a surface plasmon resonance (SPR)-based optical biosensor (ProteOn XPR36; Bio-Rad). The equilibrium dissociation constant (Kd) we determined for the PA/integrin complex, 1 μΜ, approximates that observed for the PA/TEM8 interaction using SPR methods (Scobie HM, et al. A soluble receptor decoy protects rats against anthrax lethal toxin challenge. J Infect Dis. 2005;192:1047-1 051 ), but both values are much lower than the Kd calculated from data obtained using a cell-based assay of the effects of PA inhibitors on toxin lethality (Liu S, et al. Capillary morphogenesis protein-2 is the major receptor mediating lethality of anthrax toxin in vivo. Proc Natl Acad Sci USA. 2009;106:1 2424-1 2429). We found no interaction between α5β1 -integrin and PA in the absence of divalent cation (Fig. 7), indicating that the interaction is mediated by the vW A/MIDAS domain of β1 -integrin.

Functional Role of ct-lntegrin Components οίβ 1 -integrin Complexes in RA W264.7

Macrophage Sensitivity to LF-PA. The ability β1 -integrin to bind to ligands depends on its prior interaction with a suitable a integrin partner (Hynes RO. Integrins: Bidirectional, allosteric signaling machines. Cell. 2002;1 10:673-687). Monoclonal antibodies directed against specific epitopes in a integrins can prevent this interaction (Kinashi T, Springer TA. Adhesion molecules in hematopoietic cells. Blood Cells. 1 994;20:25^14; Lobb RR, Hemler ME. The pathophysiologic role of a 4 integrins in vivo. J Clin Invest. 1 994;94:1722-1 728). We found that treatment of RAW264.7 macrophages with such anti-a4 (Lobb and Hemler (1 994), supra) or anti-a5 (Kinashi and Springer (1994), supra) integrin antibodies increased survival of macrophages exposed to LF-PA (Fig. 3A)— providing further confirmation of the role of integrins in PA-dependent anthrax toxin lethality and establishing the role of αβ integrin complex formation in this process. Similarly, the purified α5β1 -integrin complex also increased the ability of RAW264.7 macrophages to survive the lethality of LF-PA (Fig. 3B) indicating that free αβ integrin complexes competitively inhibits the action of such complexes at the cell surface. Monoclonal antibodies that inhibit the formation of αβ complexes containing integrin av (1 7) or β3 (19) had no detectable effect on the ability of these macrophages to survive exposure to LF-PA (Fig. 3A), consistent with the absence of detectable β3 integrin on the surface of RAW264.7 macrophages (Fig. 2) (36, 37) and the absence of av in integrin complexes containing β1 . The anti-a5 integrin monoclonal antibody we used has a relatively low affinity for the ligand-binding site on its targeted integrin (http://www.biolegend.com) and showed a correspondingly small effect on anthrax toxicity. Control experiments indicated that none of the antibodies we tested detectably affected macrophage survival in the absence of the toxin (Fig. 8)

Effects of CD44 and Hyaluronic Acid on LF-PA Lethality. The cell surface protein CD44 participates with OPN in the activation of integrins and increases their binding to ligands (Katagiri YU, et al. CD44 variants but not CD44s cooperate with β1 -containing integrins to permit cells to bind to osteopontin independently of arginine-glycine-aspartic acid, thereby stimulating cell motility and chemotaxis. Cancer Res. 1999;59:219-226; Ponta H, Sherman L, Herrlich PA. CD44: From adhesion molecules to signalling regulators. Nat Rev Mol Cell Biol. 2003;4:33-45; Redondo-Munoz J, et al. α4β1 integrin and 190-kDa CD44v constitute a cell surface docking complex for gelatinase B/MMP-9 in chronic leukemic but not in normal B cells. Blood. 2008;1 12:169-178). Fluorescence microscopy indicated that CD44 colocalizes with PA upon binding to the cell surface (Fig. 9), as do integrins; but unlike integrins, CD44 was not detectably endocytosed along with PA.

Additionally, as shown in Fig. 3A, a monoclonal antibody that blocks the interaction between CD44 and OPN (Mikecz K, et al. Anti-CD44 treatment abrogates tissue oedema and leukocyte infiltration in murine arthritis. Nat Med. 1995;1 :558-563) protected macrophages against late killing by LF-PA without affecting cell survival in the absence of the toxin (Fig. 8). These results provide still further evidence to support the role of integrins in PA internalization and toxicity, and additionally argue that integrin activation is required for its PA-internalizing function.

The integrin-activating function of the CD44/OPN complex is antagonized by hyaluronic acid (HA), an anionic glycosaminoglycan (40) that interacts with CD44 and largely negates the integrin-activating effects of OPN and CD44. As we observed that integrin activation (Knudson CB, Knudson W. Hyaluronan and CD44: Modulators of chondrocyte metabolism. Clin Orthop Relat Res. 2004;427:S152-S162) is necessary for anthrax toxicity, we tested the effects of high molecular weight HA from human umbilical cord [estimated molecular weight of 3,420,000 Da (Saari H, et al. Differential effects of reactive oxygen species on native synovial fluid and purified human umbilical cord hyaluronate. Inflammation. 1993;17:403-415)] on internalization of PA and on the lethality of the PA-LF complex. Addition of 100 μg/mL HA to cultures of RAW264.7 macrophages reduced PA binding and PA entry into the cytoplasm by 50% to 70% as shown by fluorescence microscopy and Western blotting (Fig. 4 A-C), and addition of HA also reduced βΙ -integrin-PA cytoplasmic colocalization during toxin internalization (Fig. 4D and Fig.10)— consistent with the previously reported ability of HA to interfere with CD44- mediated integrin activation. However, addition of HA had no detectable effect on survival of macrophages treated with more than 50 ng/mL LF (Fig. 4E), indicating that notwithstanding the modest decrease in binding and internalization of toxin resulting from anti-integrin measures, this toxin dose achieved sufficient internalization to achieve cell death. However, as was observed for anti-integrin antibodies, HA treatment at 100 μg/mL dramatically enhanced the ability of macrophages to survive exposure to toxin, increasing the IC50 by 32-fold (from 0.12 ng/mL to 3.85 ng/mL) and higher concentrations of HA did not further affect LF-PA lethality (Fig.1 1 ). Low molecular weight HA degradation products have been reported to not bind to CD44 (Gariboldi S, et al. Low molecular weight hyaluronic acid increases the self-defense of skin epithelium by induction of β-defensin 2 via TLR2 and TLR4. J Immunol. 2008;181 :2103-21 10), and we found that HA products of Streptococcus pyogenes fermentation (900,000 Da, 132,000 Da, 16,100 Da, and 6,400 Da; R&D Systems) did not detectably change the LF-PA sensitivity of RAW264.7 cells.

Combined use of anti— β1 -integrin monoclonal antibody with 100 μg/mL of HA did not result in any further reduction in cellular sensitivity to the toxin (Fig. 4E), supporting the conclusion that these agents target the same pathway; similarly, the effects of HA and the knockdown of expression of ARAP3, which previously has been implicated in PA entry by an unknown mechanism (Lu Q,e t al. (2004), supra), were nonadditive (Fig. 5A). In contrast, however, siRNA-induced deficiency of LRP6, which complexes with CMG2 at the cell surface and colocalizes with that receptor, reduced macrophage sensitivity to LF-PA beyond what was observed at the maximally effective concentration of HA (IC50 of 17.4 in LRP6-deficient cells vs. IC50 of 3.08 in parental cells; Fig. 5B).

Integrin Effects on Early and Late LF-Mediated Killing of Macrophages. RAW264.7 macrophages have been reported to undergo LF-mediated killing by two distinct

mechanisms: a rapid lytic death occurs in the presence of high LF concentrations, whereas a slow caspase-dependent apoptotic death is observed at low LF concentrations (Popov SG, et al. Lethal toxin of Bacillus anthracis causes apoptosis of macrophages. Biochem Biophys Res Commun. 2002;293:349-355; Wu W, et al. Resistance of human alveolar macrophages to Bacillus anthracis lethal toxin. J Immunol. 2009;183:5799-5806). A recent publication indicates that CMG2 is necessary for the lethality of high doses of anthrax toxin in mice and MEFs derived from them (Liu S, et al. Capillary morphogenesis protein-2 is the major receptor mediating lethality of anthrax toxin in vivo. Proc Natl Acad Sci USA.

2009 ;106:12424-12429). Using shRNA that reduced the steady-state level of CMG2 expression in RAW264.7 macrophages by 70%, we confirmed that CMG2 knockdown reduces the early death occurring after 4 h of exposure to more than 50 ng/mL LF (Fig. 5C). However, the same knockdown of CMG2 did not affect late death, as assayed by survival after 24-h exposure to the same LF concentration (Fig. 5D), implying that either the CMG2 expression remaining in these cells enabled entry of sufficient toxin to achieve late death, or alternatively, that late death can occur by a CMG2-independent mechanism. Only late death was observed in macrophages treated with a low concentration of LF (i.e., <1 ng/mL), as reported previously by Popov et al. (Popov SG, et al., supra), and CMG2 deficiency had no detectable effect on this lethality (Fig. 5D), arguing that late killing by low-dose toxin in macrophages is independent of CMG2. Interestingly, whereas the HA-mediated

interference with integrin had no detectable effect on early macrophage death (Fig. 5C), it further reduced the lethality to CMG2-deficent cells. The finding is consistent with our earlier evidence (Fig. 2) that antibody to integrin β1 reduces the ability of CMG2 to act as a PA receptor in RAW264.7 macrophages. III. Discussion

The results reported here indicate a dual role for integrins in the entry of anthrax toxin into mouse macrophages. The ability of integrin complexes to respond to PA binding by conjointly entering the cytoplasm with PA indicatesthat integrins function as a PA receptor. Like both of the previously identified anthrax receptors, TEM8 and CMG2, β1 - integrin includes a PA-binding vWA domain containing the MIDAS motif, which is absolutely conserved in cation-dependent ligand binding of integrins (Hynes RO, supra). Structural analyses indicate that the vWA domains of CMG2 and integrin fold similarly (Lacy DB, et al. Crystal structure of the von Willebrand factor A domain of human capillary morphogenesis protein 2: an anthrax toxin receptor. Proc Natl Acad Sci USA. 2004;101 :6367-6372) and PA-CMG2 complexes show mimicry of integrin-ligand interactions (Bradley KA, et al.

Binding of anthrax toxin to its receptor is similar to alpha integrin-ligand interactions. J Biol Chem. 2003;278:49342^19347; Santelli E, et al. Crystal structure of a complex between anthrax toxin and its host cell receptor. Nature. 2004;430:905-908). However, the finding that integrin inactivation by antibodies or HA reduces the ability of concurrently present CMG2 to internalize PA and mediate lethality in RAW264.7 macrophages argues that integrins can also act as modulators of CMG2 receptor function.

Two distinct mechanism of killing of macrophages exposed to LF have been reported (Hanna PC, et al. On the role of macrophages in anthrax. Proc Natl Acad Sci USA. 1993;90:10198-10201 ; Popov SG, et al. Lethal toxin of Bacillus anthracis causes apoptosis of macrophages. Biochem Biophys Res Commun. 2002;293:349-355), and our

investigations provide additional evidence of this. Macrophages treated with high doses of LF undergo rapid cell lysis and death (Hanna PC, et al., supra; Popov SG, et al., supra), and— not surprisingly, given similar results obtained for mouse embryonic fibroblasts (Liu S, et al. (2009), supra)— we show here that macrophage death occurring soon after exposure to the toxin is dependent on CMG2 function (Fig. 5C). HA-mediated interference with integrin activity had no detectable effect on CMG2-dependent early macrophage death (Fig. 5C). However, RAW264.7 macrophages defective in CMG2 expression nevertheless underwent eventual killing by toxin (Fig. 5D), implying that a second mechanism exists for toxin entry in these cells. This mechanism, which necessarily cannot involve TEM8 as TEM8 is not present on the surface of macrophages (Dal Molin F, et al. Cell entry and cAMP imaging of anthrax edema toxin. EMBO J. 2006;25:5405-5413; Rainey GJ, et al.

Receptor-specific requirements for anthrax toxin delivery into cells. Proc Natl Acad Sci USA. 2005;102:13278-13283) was inhibited by HA (Fig. 5D). Late apoptotic killing also occurs after exposure of macrophages to sublytic doses of LF (Hanna PC, et al., supra; Popov SG, et al., supra; and our data; see Fig. 5D), and such killing, although not affected by an shRNA-mediated decrease in CMG2 expression, was decreased by HA as well as by antibodies specific to β1 -integrin and its a integrin partners. It is worth noting that integrins also mediate the lethality of the a toxin of Staphylococcus aureus (Liang X, Ji Y.

Involvement of α5β1 -integrin and TNF-a in Staphylococcus aureus a-toxin-induced death of epithelial cells. Cell Microbiol. 2007;9:1809-1821 ; Liang X, Ji Y. a-toxin interferes with integrin-mediated adhesion and internalization of Staphylococcus aureus by epithelial cells. Cell Microbiol. 2006;8:1656-1668), and that such killing by lytic or apoptotic mechanisms also appears to be concentration-dependent (Jonas D, et al. Novel path to apoptosis: Small transmembrane pores created by staphylococcal a-toxin in T lymphocytes evoke

internucleosomal DNA degradation. Infect Immun. 1994;62:1304-1312).

The effects of maximal interference with integrin by HA are additive to those of knockdown of LRP6, a previously identified member of an anthrax toxin internalization complex (Wei W, et al., supra; Abrami L, et al. (2008), supra), whereas HA is non-additive to knockdown of ARAP3, another modulator of PA entry (Fig. 5 A and B). These findings indicate that ARAP3 and LRP6 affect different pathways of toxin internalization and that the ARAP3 pathway is at least partially congruent with the pathway of integrin-mediated internalization. During these experiments, we observed that interference with different entry- modulating proteins has differential effects on the death of RAW264.7 macrophages induced by exposure to PA plus LF versus PA plus FP59, a hybrid toxin containing the PA binding site of LF plus a toxin domain derived from Pseudomonas aeruginosa exotoxin A.

The synergistic— rather than simply additive— effects of HA and shRNA directed against CMG2 on late death by sublytic LF concentrations indicate that integrin enhances action of residual CMG2 remaining after shRNA knockdown, further supporting the conclusion that integrins facilitate the receptor functions of CMG2. Similarly, TEM8 may also potentiate CMG2 knockout in addition to serving as a low-affinity receptor (Liu S, et al. 2009, supra). TEM8 has been reported to cooperate with integrin in regulating the VEGFR2 receptor (Jinnin M, et al. Suppressed NFAT-dependent VEGFR1 expression and constitutive VEGFR2 signaling in infantile hemangioma. Nat Med. 2008;14:1236-1246), and analogous cooperation between TEM8 and integrins may occur in regulation of CMG2- mediated toxin entry. Example 2

Microbial toxins exploit a variety of host cell functions to enter and kill their targets. The protective antigen (PA) component of Bacillus anthracis toxins can interact with at least three distinct proteins on the host cell surface, and with the assistance of other cell surface proteins, is internalized by these receptors—carrying along PA-bound toxin moieties. Here we report the previously unsuspected role of calpain, a ubiquitous Ca2+- dependent cysteine protease, in enabling anthrax toxin internalization. We show that the lethality of PA-LF in macrophages is decreased by interference with calpain actins by calpain inhibiting protein calpastatin and also by a small molecule inhibitor. We further show that PA binding to the surface of RAW267.4 mouse macrophages is normal during calpain inhibition but that inhibition of calpain function results in sharply diminished internalization of the toxin. We also demonstrate that calpain-mediated cleavage of talin, an important linker protein between integrins and actin cytoskeleton, is required for anthrax toxin lethality by showing that expression of a talin mutant that is resistant to calpain cleavage rendered macrophages less susceptible to anthrax lethal toxin. Taken together, these data indicate an important role for calpain in anthrax toxin internalization, through talin cleavage.

I. Materials and Methods

Cell culture, transfection and infection. RAW264.7 mouse macrophage cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (HyClone) and 100 units/ml penicillin and 100 μg/ml streptomycin. Plasmid DNA transfection was performed using the FuGENE6 transfection reagent (Roche, Inc.). Viral Infection was performed using lentiviral-based methods (Lu et al., 2004). GFP- expression vector for talinl (wild type and L432G) were purchased from Addgene Inc (Addgene plasmid 26724 and 26725, respectively) (Franco, S.J., et al. (2004a). Calpain- mediated proteolysis of talin regulates adhesion dynamics. Nat Cell Biol 6, 977-983).

Transfection RAW264.7 cells expressing enhanced green fluorescent protein (EGFP) tagged human CMG2 (CMG2-EGFP) were generated by transfection with pLEGFP.N1 - CMG2 (Scobie, H.M., et al. (2003). Human capillary morphogenesis protein 2 functions as an anthrax toxin receptor. Proceedings of the National Academy of Sciences of the United States of America 100, 5170-5174). The transfected cells were then selected in the medium containing 1 mg/ml G418. Single clones were isolated from a pool of transfected cells, and EGFP-expressing cells were subsequently isolated by using a fluorescence microscopy.

Chemicals and reagents. PA and LF were purchased from List Biological

Laboratories (Campbell, CA). Calpain inhibitors III (MDL28170) was purchased from Calbiochem. The fluorescently labeled monoclonal antibody anti-βι integrin-APC (ΗΜβ1 -1 ) was purchased from BioLegend (San Diego, CA). Anti-N-term MEK-2 and anti-PA antibodies were purchased from Santa Cruz Biotechnology, and anti-calpastatin antibody was purchased from Cell Signaling Technology.

EST library screening. Screening of EST library was performed as described by Lu et al. (Lu et al., 2004) with a slight modification. Briefly, RAW264.7tTA cells established by introducing a gene encoding tetracycline- repressed transactivator (tTA) were infected with pLEST-based human EST library reported in Lu et al. (Lu et al., 2004). A pool of cells expressing EST was treated with 500 ng/ml PA-LF for 2 days, grown in toxin-free media for 10 days, and then surviving clones were picked and expanded. Genomic DNA was extracted from each clone using DNeasy kit (Qiagen) and the EST in each clone was identified by PCR amplification and sequencing.

Calpain activity assay. Calpain activity in total cell lysates was determined using calpain activity assay kit (Biovision) according to the manufacturer's instruction. Briefly, RAW264.7 cells were collected and lysed in Extraction buffer (Biovision). After quantitation of protein concentrations, equal amounts of lysates were added to calpain substrates, Ac- LLY-AFC (Biovision). Fluorescence was measured at 400 nm excitation and 505 nm emission wavelengths using a microplate reader Infinite 200 (TECAN).

Quantitative Real Time PCR. Total RNA was isolated using the RNeasy kit (Qiagen), and then reverse-transcribed using M-MLV reverse transcriptase (Invitrogen) and random primers. Real-time PCR with SYBR green detection was performed using the Bio- Rad iCycler iQ system (Bio-Rad) and the following primers:

CAST1 , 5^'-TCGCAAGTTGGTGGTACAAG-3^' (SEQ ID NO:1 );

CAST2, 5^'-CTCCCCAAACTTGCTGCTT-3^' (SEQ ID NO:2);

actinl , 5^'-CTAAGGCCAACCGTGAAAAG-3^' (SEQ ID NO:3);

actin2, 5^'-ACCAGAGGCATACAGGGACA-3^' (SEQ ID NO:4);

GFP1 F, 5^'-GCAGAAGAACGGCATCAAGGT-3^' (SEQ ID NO:5);

GFP1 R, 5^'-ACGAACTCCAGCAGGACCATG-3^' (SEQ ID NO:6). The fluorescence threshold value was calculated using iCycler iQ system software.

Toxin Treatment and Cell Viability Assay. Cells were seeded in a 96-well plate at a density of 2 x 10⁵ cells/ml 1 day prior to toxin treatment. Various concentrations of LF combined with a fixed concentration of PA (200 ng/ml) were added to the wells, and cells were incubated for 4 h at 37^QC. Cell viability was measured by adding 3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to a final concentration of 1 mg/ml for 2 h at 37^QC. The supernatant was carefully removed before the addition of 50 ul of lysis solution (10% SDS in 0.01 N HCI). After incubation at 37^QC for 30 min, we added 100 ul of PBS to cell lysates. Spectrophotometer readings at 570 nm were determined using a microplate reader Infinite 200 (TECAN). Cell viability was normalized to wells lacking toxin. Each data point shown in figures for MTT assays represents the averaging of results from three wells.

Immunofluorescence Microscopy. PA protein was labeled with Alexa Fluor 488 using the protein labeling kit (A10235, Molecular Probes, Inc.). The potency of PA after labeling was fully retained as indicated by the MTT assay. Immunostaining was performed as follows. RAW264.7 cells were preincubated with/without 80 μΜ MDL28170 for 1 h in serum-free Iscove modified Dulbecco medium (IMDM; Invitrogen), and then treated with 1 μ9/ι ιΙ PA-Alexa Fluor 488 in the presence of 0.5 μg/ml anti-βι integrin-APC (ΗΜβ1 -1 ) antibody for 20 min at 37°C, Cells were washed with PBS for three times, fixed in 4% paraformaldehyde, and examined under a fluorescence microscope (Leica).

Biochemical assay of PA binding and internalization. RAW264.7 was grown to confluence and preincubated with 80 μΜ MDL28170 for 1 h before exposed to PA. Cells were exposed to 0.5 μg ml of PA at 4^QC for 1 h for binding assay, or at 37^QC for the indicated time for internalization assay. After washed with cold PBS three times and lysed in RIPA buffer containing a Protease inhibitor cocktail (Roche). Cell lysates were quantitated using BCA protein assay kit (Pierce) and loaded onto 4-12% denaturing gels (Criterion XT Precast Gel, Bio-Rad). After transfer overnight, the membrane was probed with anti-PA or anti-actin antibodies. Quantitative Western blot analysis of the bands was done by using the Versadoc 1000 instrument (Bio-Rad) or Odyssey infrared imaging system (Li-Cor).

Animal studies. CAST/EiJ mice were treated with MDL28170 (10 ug/g of body weight of mouse) or vehicle (50% DMSO) alone by intraperitoneal injection 6 h prior to toxin challenge. Anthrax lethal toxin (PA+LF) was administrated once by intraperitoneal injection at a dose of 5 ug/g of body weight of mouse. 4 mice were tested for each group. II. Results

Identification and characterization of macrophage clones resistant to PA- LF. A previously reported approach that uses regulated transcription of a lentivirus-based human EST library to perturb host gene expression globally and randomly (Chang, A.C., et al. (2006) Phenotype-based identification of host genes required for replication of African swine fever virus. Journal of virology 80, 8705-8717; Lu, Q., et al. (2004) EST-based genome- wide gene inactivation identifies ARAP3 as a host protein affecting cellular susceptibility to anthrax toxin. Proc. Natl Acad. Sci U.S.A. 101 , 17246-17251 ; Piccone, et al. (2009).

Identification of cellular genes affecting the infectivity of foot-and-mouth disease virus. Journal of virology 83, 6681 -6688; Wei, W., et al. (2006) The LDL receptor-related protein LRP6 mediates internalization and lethality of anthrax toxin. Cell 124, 1 141 -1 154) yielded a group of 96 RAW267.4 macrophage cell clones that survived exposure for 2 days to 500 ng/ml PA-LF. Addition of doxycycline (Dox), which represses the modified CMV promoter controlling transcription of ESTs in this library, partially reversed insensitivity to PA-LF in three of these clones, implying a role for the cognitive ESTs in the observed phenotype. One of these clones, which showed a more than 8 fold increase in the toxin concentration required for 50% lethality in the absence of Dox (clone 3-12, Fig 12A) was selected for further study. PCR amplification of the EST inserted into the chromosome (Lu et al. 2004, supra) of clone 3-12 followed by sequencing of the amplicon indicated that this EST corresponds to an intron segment of transcripts encoding calpastatin (CAST; IMAGE clone no. 71286), oriented in sense direction relative to the promoter. The CAST protein acts broadly to inhibit the actions of calpains.

Reintroduction of the CAST EST into parental RAW264.7tTA cells in the sense direction behind the pLEST lentivirus promoter (Lu et al. 2004, supra) reproduced the resistance phenotype (Fig 12B), confirming the role of the EST in this phenotype. The ability of the introduced CAST EST to increase abundance of mRNA and protein for CAST was directly demonstrated by quantitative RT-PCR and Western blot analysis, respectively (Fig 12C and D). While the mechanism by which transcription of an intronic segment of CAST increases CAST expression at both the mRNA and protein has not been elucidated, the biological consequences of this increase are unambiguous, as described below: The only known cellular action of calpastatin is to inhibit the activity of calpains— an evolutionarily conserved family of Ca²⁺-dependent cysteine proteases, and as anticipated from the observed effects of the CAST EST on CAST abundance (Fig 12C and D), lysates of cells expressing the CAST EST showed a prominent decrease in calpain activity (Fig 12E).

Reduced activity of calpain diminishes the lethal effects of anthrax toxin. The above results indicate effect of cellular calpain deficiency on anthrax toxicity in RAW267.4 macrophages. To directly test this conclusion, we examined the effect of a cell-permeable calpain inhibitor, MDL28170, on PA-LF-mediated cytotoxicity. The ability of MDL28170 to broadly inhibit calpain activity in these cells was first confirmed by monitoring cleavage of the fluorogenic calpain substrate Ac-LLY-AFC; addition of 40 μΜ MDL28170 to RAW264.7 cells decreased overall calpain activity by 60% (Fig 18). MDL28170-treated RAW 264.7 cells were exposed to an ordinarily highly lethal dose of PA- LF (500 ng/ml), and cell viability was monitored by MTT assay. As shown in Fig 13A, treatment of macrophages with MDL28170 largely reversed LF lethality, enabling 80% survival of macrophages exposed to high dose PA-LF. Protection against LF killing by MDL28170 was observed when the calpain inhibitor was added either prior to exposure to the toxin, or concurrently with toxin administration, but not when the inhibitor was added only 75 minutes after toxin treatment (Fig 13B), indicating that the calpain activity that is inhibited by MDL28170 occurs promptly after macrophages encounter the toxin. Consistent with this conclusion, cleavage of MEK-2, a target of LF, was prevented in cells treated with MDL28170 (Fig 14A). This data also indicates a severe defect in delivery of toxin into cytoplasm.

Inhibition of calpain specifically reduces PA-LF internalization. The above results indicate that the functions of calpains are required for either binding of PA to its receptors(s) or for normal entry of receptor-bound PA into macrophages. PA binds to receptors and forms an SDS-sensitive heptameric oligomer on the cell surface, and subsequent endocytosis of the heptamer into acidic endosomes leads to a conformational change, yielding an SDS-resistant form (Liu, S., and Leppla, S.H. (2003) Cell surface tumor endothelium marker 8 cytoplasmic tail-independent anthrax toxin binding, proteolytic processing, oligomer formation, and internalization. J Biol Chem 278, 5227-5234; Miller, C.J., et al. (1999) Anthrax protective antigen: prepore-to-pore conversion. Biochemistry 38, 10432-10441 ). Thus, the surface-bound and internalized forms of PA are easily

distinguishable by their different motilities during electrophoresis on SDS-PAGE gels. To determine the effects of calpain inhibition specifically on PA binding, RAW264.7 cells were incubated with PA at 4 ^QC to prevent endocytosis of PA. As shown in Figure 14B,

MDL28170 did not significantly affect the amount of PA bound to the cell surface. In contrast, internalization of heptameric PA was dramatically reduced by the presence of MDL28170, as shown by a 73% reduction in SDS-resistant heptameric PA 30 min after incubation at 37 ^QC (Fig 14C). Fluorescence microscopy studies, which confirmed that inhibition of calpains by MDL28170 reduces the internalization of PA (Fig 15B and E), also showed that calpain inhibition prevents the PA-mediated internalization of integrin βι and CMG2 (Fig 15 and 16), which is shown in example 1 above to be internalized upon uptake of anthrax toxin from the cell surface. Calpain-mediated cleavage of talin is required for anthrax toxin lethality. Calpain has been shown to be important for efficient disassembly of integrin-mediated adhesion complex by cleavage of adhesion components (Franco, S.J., et al. (2004b). Calpain- mediated proteolysis of talin regulates adhesion dynamics. Nat Cell Biol 6, 977).

Cytoskeletal protein talin, an established substrate for calpain, plays a critical role in linkage integrins to actin cytoskeleton (Seino, S. (1999) ATP-sensitive potassium channels: a model of heteromultimeric potassium channel/receptor assemblies. Ann Rev Physiol 61 , 337-362). Therefore, we tested whether calpain-mediated talin cleavage is required for cytotoxicity of anthrax lethal toxin. Mutation of leucine to glycine at codon 432 of talin (L432G) renders it resistant to calpain cleavage, and expression of this talin mutant L432G perturbs focal adhesion turnover (Franco et al. 2004b, supra). RAW264.7 cells were transfected with pEGFP-C1 plasmid expressing wild type or calpain-resistant mutant talin, and the similar level of expression of EGFP-tagged talin was achieved in the transfected cells (Fig 17A). The expression of talin-L432G in macrophages resulted in decreased PA internalization when compared to wild type talin-expressing cells, but did not cause any significant change in PA binding to cell surface (Fig 17B). Consistent with deficiency in PA uptake, expression of talin-L432G rendered cells less susceptible to PA-LF, whereas the susceptibility was not significantly changed by expression of wild type talin. Our data demonstrate that calpain is required for toxin internalization, which is mediated by cleavage of talin.

Inhibition of calpain is protective against anthrax toxicity. The above results indicate that calpain inhibition will protect against anthrax toxicity in vivo. To test this directly, mice were administered either a prophylactic dose of the calpain inhibitor MDL28170 or vehicle. Six hours later, anthrax lethal toxin (PA+LF) was administered intraperitoneal^. 24 hours after administration of the toxin, 3 of the 4 control mice, i.e. those that received vehicle, were moribund, and the fourth mouse displayed symptoms of illness. In contrast, all four mice that received MDL28170 were still active. At 48 hours, all 4 control mice had died, whereas 2 of the mice that received MDL28170 were still alive and active. At 72 hours, these 2 mice were still alive and active. These data demonstrate that calpain inhibitors may be used to prevent anthrax toxicity in mammals.

III. Discussion

Calpain, a Ca²⁺-dependent cysteine protease, has been implicated in a variety of cellular processes, including cell migration, cell differentiation, cytoskeletal remodeling, and apoptosis (Franco, S.J., and Huttenlocher, A. (2005) Regulating cell migration: calpains make the cut. J Cell Sci 1 18, 3829-3838; Goll, D.E.,et al. (2003) The calpain system.

Physiol Rev 83, 731 -801 ; Lebart, M.C., and Benyamin, Y. (2006) Calpain involvement in the remodeling of cytoskeletal anchorage complexes. FEBS 273, 3415-3426; Moyen, C. et al. (2004) Involvement of micro-calpain (CAPN 1 ) in muscle cell differentiation. Int. J. of Biochem and Cell Biol. 36, 728-743). Calpains are involved in translocation of

Streptococcus pyogenes or Bordetella adenylate cyclase toxin via destabilization of intercellular junctions associated with the epithelial barrier or liberation of the toxin-receptor complex from binding to actin cytoskeleton, respectively (Bumba, L, (2010) Bordetella adenylate cyclase toxin mobilizes its beta2 integrin receptor into lipid rafts to accomplish translocation across target cell membrane in two steps. PLoS Pathog 6, e1000901 ;

Sumitomo, T. et al. (2010) Streptolysin S contributes to group A streptococcal translocation across an epithelial barrier. J Biol Chem 286, 2750-2761 ). In addition, calpains contribute to induction of apoptosis or necrosis by pathogens or toxins such as Streptococcus pneumonia, Shiga toxin, Neisserial porin, and Clostridium septicum oc-toxin (Kennedy, C.L. et al. (2009) Programmed cellular necrosis mediated by the pore-forming alpha-toxin from Clostridium septicum. PLoS Pathog 5, e1000516; Lee, S.Y., et al. (2008) Shiga toxin 1 induces apoptosis through the endoplasmic reticulum stress response in human monocytic cells. Cellular microbiology 10, 770-780; Muller, A., et al. (1999) Neisserial porin (PorB) causes rapid calcium influx in target cells and induces apoptosis by the activation of cysteine proteases. Embo J 18, 339-352; Schmeck, B., et al. (2004) Streptococcus pneumoniae-induced caspase 6-dependent apoptosis in lung epithelium. Infection and immunity 72, 4940-4947). However, the nature of the substrates cleaved by calpains in exerting these effects remains largely unknown. Here we propose a role of calpain on endocytosis of anthrax and other toxins through cleavage of talin.

Some of the processes involved in endocytosis of anthrax toxin have been well established. For example, it is known that to enter into cells, PA triggers activation of src- like kinases, and in turn the receptors are phosphorylated, which allows receptor ubiquitination and internalization of the receptor-toxin complex via a clathrin-mediated pathway (Abrami et al., 2010a, supra; Abrami, L. et al. (2010b) Anthrax toxin triggers the activation of src-like kinases to mediate its own uptake. Proc. Natl. Acad. Sci U.S.A. 107, 1420-1424; Abrami, L. et al. (2006) Receptor palmitoylation and ubiquitination regulate anthrax toxin endocytosis. J Cell Biol 172, 309-320). Actin has also been reported to be required for the endocytosis of PA, although its precise role is still unclear (Abrami et al., 2010a, supra). Previous studies indicate that the PA receptor TEM8, like integrins, interacts with the actin cytoskeleton as well as cytoskeletal proteins including talin, vinculin, and myosin II, and that the binding of PA to TEM8 releases TEM8 from the cytoskeleton in Hela cells. While it is tempting to speculate that calpains may have a role in PA endocytosis in, for example, RAW264.7 cells, RAW264.7 cells do not express TEM8, but rather use CMG2 as their principal anthrax toxin receptor (Dal Molin et al., 2006; example 1 above), and despite the similarities between the cytoplasmic tails of TEM8 and CMG2, CMG2 has not been found to bind to the actin cytoskeleton (Abrami et al., 2010a, supra). However, Example 1 above indicates that integrins can potentiate CMG2-mediated PA endocytosis, and it has been well established that integrins are linked to actin cytoskeleton via cytoskeletal proteins such as talin, and thus integrin-interacting proteins may be implicated in the effects of calpain on PA endocytosis. We observed that the expression of calpain- resistant talin renders cells more resistant to anthrax lethal toxin, indicating that calpain functions through talin cleavage. Collectively, our data indicate that cleavage of talin by calpain could weaken the linkage between integrin and actin cytoskeleton, which could play a role in PA endocytosis.

During these experiments, we observed that inhibition of calpain, which dramatically inhibited macrophage sensitivity to PA-LF, increased cellular susceptibility to the

Pseudomonas aeruginosa exotoxin (i.e. PE toxin). In contrast, calpain inhibition had little effect on the lethality of the hybrid toxin PA-FP59, despite the ability of calpain inhibition to impair the internalization of PA-mediated FP59 (Fig 19). These findings indicate that calpain affects PE catalytic function, as well as internalization of PA.

Here we have shown that MDL28170, a specific calpain inhibitor, provides significant protection against PA-LF mediated cytotoxicity in vitro and in vivo. This inhibitor previously has been tested in pre-clinical animal studies, where it has been shown to prevent motor disturbances in rats in a model of spinal cord injury (Arataki, S., et al (2005) Calpain inhibitors prevent neuronal cell death and ameliorate motor disturbances after compression-induced spinal cord injury in rats. Journal of neurotrauma 22, 398-406) and also to prevent amyloid β-induced neuronal death in mice (Lopes, J. P., et al.

Neurodegeneration in an Abeta-induced model of Alzheimer's disease: the role of Cdk5. Aging cell 9, 64-77). We show that calpain inhibition will also be of value in the reduction of anthrax toxicity in humans.

The above results and discussion demonstrate that the invention provides important new approaches to preventing and/or treating disease conditions resulting from exposure to B.anthracis. A significant advantage of the subject invention is that it is not based on targeting the pathogen itself, but instead to a host receptor. As such, the subject methods find use in the treatment of subjects in the late stage anthrax toxin mediated disease conditions, in which agents targeting the pathogen itself are no longer effective.

Accordingly, the subject invention represents a significant contribution to the art. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims.

Claims

WHAT IS CLAIMED IS:

1 . A method of inhibiting entry of an anthrax toxin into a cell, the method comprising:

contacting the cell with an effective amount of an agent that inhibits cellular entry of an anthrax toxin mediated by a complex comprising a β1 integrin subunit.

2. The method according to claim 1 , wherein the anthrax toxin is selected from lethal factor (LF) and edema factor (EF).

3. The method according to claim 1 , wherein the agent inhibits binding of protective antigen (PA) to the complex comprising the β1 integrin subunit.

4. The method according to claim 3, wherein the agent inhibits activation of the complex comprising a β1 integrin subunit.

5 The method according to claim 4, wherein the agent that inhibits activation of the complex comprising a β1 integrin subunit inhibits CD44 activation.

6. The method according to claim 1 , wherein the agent inhibits anthrax toxin endocytosis mediated by the complex comprising a β1 integrin subunit.

7. The method according to claim 6, wherein the anti-anthrax agent that inhibits anthrax toxin endocytosis inhibits calpain.

8. The method according to claim 7, wherein the anti-anthrax agent is

MDL28170.

9. The method according to claim 1 , wherein the anti-anthrax agent inhibits a mediator of signaling by the complex comprising a β1 integrin subunit.

10. The method according to claim 1 , wherein the anti-anthrax agent inhibits β1 integrin subunit expression.

1 1 . The method according to claim 1 , wherein the method is ex vivo

12. The method according to claim 1 , wherein the method is in vivo.

13. The method according to claim 1 , further comprising contacting the cell with an effective amount of a second anti-anthrax agent.

14. The method according to claim 13, wherein the second anti-anthrax agent is an agent that inhibits entry of B. anthracis spores into a cell.

15. The method according to claim 13, wherein the second anti-anthrax agent is an agent that inhibits B. anthracis multiplication.

16. The method according to claim 13, wherein the second anti-anthrax agent is an agent that inhibits entry of an anthrax toxin into a cell.

17. A method of inhibiting an anthrax toxin-mediated condition in a host, the method comprising:

administering to the host an effective amount of an agent that inhibits cellular entry of an anthrax toxin mediated by a complex comprising a β1 integrin subunit.

18. The method according to claim 17, wherein the method is a method of prophylactically conferring an anthrax toxin-resistant phenotype on the host.

19. The method according to claim 17, wherein the method is a method of treating a subject suffering from an anthrax-toxin mediated disease condition.

20. The method according to claim 17, further comprising administering to the host an effective amount of a second anti-anthrax agent.

21 . An anti-anthrax composition, comprising:

a first anti-anthrax agent that inhibits cellular entry of an anthrax toxin mediated by exes comprising the β1 integrin subunit ; and

a second anti-anthrax agent.

22. The method according to claim 21 , wherein the second anti-anthrax agent agent that inhibits entry of B. anthracis spores into a cell.

23. The method according to claim 21 , wherein the second anti-anthrax agent is an agent that inhibits B. anthracis multiplication.

24. The method according to claim 21 , wherein the second anti-anthrax agent is an agent that inhibits entry of an anthrax toxin into a cell.

25. The method according to claim 21 , wherein the second anti-anthrax agent is an agent that inhibits anthrax toxicity in a cell.

26. The anti-anthrax composition according to claim 21 , further comprising a pharmaceutically acceptable vehicle.

27. A method of a screening candidate agent for anti-toxin activity, the method comprising:

contacting a cell expressing a β1 integrin with a toxin and a candidate agent; and comparing the viability of the cell to the viability of a cell expressing a β1 integrin that was contacted with the toxin not contacted with the candidate agent;

wherein enhanced viability of the cell contacted with the candidate agent indicates that the candidate agent has anti-toxin activity.

28. The method according to claim 27, wherein the toxin is an anthrax toxin.

29. An anti-anthrax kit comprising:

an anti-anthrax agent that inhibits cellular entry of an anthrax toxin mediated by a complex comprising a β1 integrin subunit.

30. The anti-anthrax kit according to claim 29, wherein the kit further comprises a second anti-anthrax agent.