WO2006060051A2 - Enzyme chimeras and methods of their use for the treatment of infection by bacillus anthracis - Google Patents

Abstract

The present invention provides chimeras of lytic enzymes selective for Bacillus anthracis that can be directed to lung tissue of a subject infected with the bacterium, or at risk for such an infection. Since the lytic enzyme is capable of selectively killing the bacterium, the chimeras are useful for delivering therapeutics to lung tissue of a subject to treat or prevent infection by Bacillus anthracis.

Description

ENZYME CHIMERAS AND METHODS OF THEIR USE FOR THE TREATMENT OF INFECTION BY BACILLUS ANTHRACIS

1. FIELD OF THE INVENTION

[0001] The present invention provides compositions and methods useful for the treatment of Bacillus anthracis infection. The compositions of the invention are capable of delivering a lytic molecule to the site of infection by a Bacillus anthracis cell under conditions wherein the lytic molecule can lyse and even kill the Bacillus anthracis cell. The compositions of the invention are also capable of delivering a lytic molecule to tissue or blood where the lytic molecule can lyse and even kill a Bacillus anthracis cell.

2. BACKGROUND OF THE INVENTION

[0002] In October, 2001, one or more bioterrorists attacked government and media targets in the United States with spores of the deadly bacterium Bacillus anthracis. Over a period of several weeks, spores were spread in envelopes sent via the U.S. Postal Service. Unfortunately, at least nineteen people were infected, and five of these people died as a result of the attacks. The attacks disrupted the infrastructure of the country including numerous postal and government facilities. One the nation's largest postal facilities was closed for more than one year to complete decontamination of its B. anthracis spores. [0003] B. anthracis, a spore forming gram positive bacterium commonly known as anthrax, has been developed into an agent of bioterrorism for widespread distribution (Alibek and Handelman, 1999, Biohazard: The Chilling True Story of the Largest Covert Biological Weapons Program in the World Told from the Inside by the Man Who Ran It, Random House Trade). In the United States, anthrax is a Category A Critical Biological Agent, an agent that is a high priority risk to United States national security. Such agents include those that can be easily disseminated or transmitted person to person; result in high mortality rates and have the potential for major public health impact; might cause public panic and social disruption; and requires special action for public health preparedness (Khan et al, 2000, Morbidity Mortality Weekly Report 49(RR04):l-14). Worldwide, nations are equally vigilant to the threat of anthrax.

[0004] Humans can become infected with anthrax following exposure to infected animals or tissue from infected animals or following direct exposure to the bacterium B. anthracis (Oncu et al, 2003, Med. Sci. Monit. 9:RA276-283). Depending on the route of infection, anthrax can occur in at least three forms: cutaneous, gastrointestinal, and inhalation (Oncu et al.). Resulting from the inspiration of a few thousand spores, inhalation anthrax is the most deadly form (Oncu et al.). The illness can be biphasic with initial i symptoms that are flu-like followed by a sudden onset of respiratory failure, acute dyspnea, circulatory collapse, cyanosis, pleural effusion and fever (Oncu et al). Following the onset of the second phase, death usually occurs within 24 hours (Oncu et al). Fatality rates from cases before the availability of antibiotics or vaccines ranged from 86% to 97% (Modlin et al, 2000, Morbidity Mortality Weekly Report 49(RR15):l-20).

[0005] B. anthracis produces toxins that are formed from three proteins, protective antigen (PA), lethal factor (LF) and edema factor (EF). LF is a protease that inhibits mitogen-activated protein kinase-kinase (Duesbury et al , 1998, Science 280:734-5). EF is an adenylate cyclase that generates cyclic adenosine monophosphate in the cytoplasm of eukaryotic cells (Pezard et al, 1991, Infection and Immunity 59:3472-7). PA is an 82 kD protein that binds to receptors on mammalian cells and is required for binding and translocating LF and EF into host cells (Modlin et al). Although each individual protein can be nontoxic by itself, they are toxic in combinations (Oncu et al). The combination of PA and EF forms the edema toxin that causes edema and decreases neutrophil function (Oncu et al). The combination of PA and LF forms the lethal toxin that can cause death when injected intravenously (Oncu et al).

[0006] A single vaccine is available in the United States for the prevention of anthrax infection. AVA, the only licensed human anthrax vaccine in the United States, is produced by BioPort Corporation in Lansing, Michigan, and is prepared from a cell-free filtrate of B. anthracis culture that contains no dead or live bacteria (Modlin et al). [0007] Following infection, a few antibiotics have been approved for treatment.

Although penicillin had been the first-line treatment, it is no longer recommended due to concerns about resistance (Oncu et al). Currently, ciprofloxacin and doxycycline are recommended for treatment, and that treatment can be last for up to 60 days in the context of a bioterrorist attack (Oncu et al). Unfortunately, B. anthracis can be engineered to resist one or more, or even all, of these antibiotics. Therefore, additional agents that are capable of inhibiting infection by B. anthracis are urgently needed.

[0008] Recently, a protein that is capable of lysing and killing B. anthracis cells has been isolated (Schuch et al, 2002, Nature 418:884-889). The protein is a bacteriophage lysin produced by the phage gamma, a bacteriophage highly specific for B. anthracis (Schuch et al). Lysins are used by bacteriophage to lyse their host cells and release phage particles. The isolated PIyG lysin from the gamma phage was shown to selectively lyse cells of fourteen B. anthracis strains and a closely related B. cereus strain (Schuch et al). It is believed that the PIyG lysin can be used to selectively kill B. anthracis cells in an infected subject if delivered to the site of infection. However, methods of efficiently delivering the lysin, especially to a large population of subjects that are infected or at risk for infection, have not been developed.

3. SUMMARY OF THE INVENTION

[0009] The present invention is based, in part, on the discovery of PIyG chimeras that can be used to efficiently deliver PIyG to, for example, to tissue of a subject that might be infected with B. anthracis. Such tissue includes epithelial and parenchymal tissue of the lung and other tissue such as blood. The chimeras, compositions comprising the chimeras and methods of their use are useful for the treatment or prevention of anthrax infection in subjects infected with the bacterium or at risk of infection with the bacterium. [0010] In one aspect, the present invention provides a chimera useful for the treatment or prevention of B. anthracis infection in a subject. The chimera comprises a lytic enzyme selective for B. anthracis and a targeting element capable of directing the chimera to tissue that is infected with B. anthracis or that is at risk for infection with B. anthracis. [0011] In certain embodiments, the lytic enzyme is a polypeptide that is capable of selectively binding and lysing a B. anthracis cell. One of skill in the art will recognize that such an enzyme should not bind or lyse cells of the subject, or at least not a sufficient number of cells to be unacceptably toxic to the host. Preferred lytic enzymes can be isolated or derived from phage that are capable of lysing a B. anthracis cell, as described in detail herein. Particularly preferred lytic enzymes include the PIyG enzyme isolated from phage gamma, and related enzymes, as describe in PCT publication no. WO 2004/027020, the contents of which are hereby incorporated by reference in their entirety. Also preferred are engineered PIyG enzymes and fragments thereof, as described in the sections below. Exemplary lytic enzymes are described in the examples below, including SEQ ID NO:1. [0012] The targeting element can be any element that is capable of directing the chimera to tissue of a subject that is infected with B. anthracis or that is at risk of infection with B. anthracis. For instance, the targeting element can be an element that is capable of directing the chimera to cutaneous tissue that is infected or at risk of infection, to gastrointestinal tissue that is infected or at risk of infection or to lung tissue that is infected or at risk of infection. For instance, the targeting element can be an element that is capable of binding a target that is characteristic of the surface of a lung cell, including an epithelial cell. The target can be on any surface of the cell, such as an epithelial cell, including the apical or basolateral surface of the cell. Advantageously, in certain embodiments the target can be one that is transported into or across the epithelial cell into the parenchyma of the lung. [0013] In preferred embodiments, the target is the polymeric immunoglobulin receptor ("plgR"), or a portion thereof, that is capable of binding and transporting immunoglobulins across lung epithelial cells. Polymeric immunoglobulin receptor ("plgR") is present on mucosal surfaces in organs that need to be protected from various biological threats and insults, such as from bacteria, viruses, foreign proteins, etc. plgR in all of these tissues and organs may act as a target. PIgR is a transporter for antibodies, such as IgA and IgM. Other transporters, either known now or yet to be discovered, may also be used as targets. In certain embodiments, the targeting element is capable of binding any portion of plgR. In particular embodiments, the targeting element is capable of binding the stalk of plgR, which can be found on the apical surface of a lung epithelial cell. Since the stalk of plgR is capable of transport from the apical surface of the cell to the basolateral surface of the cell, a chimera comprising a targeting element that is capable of binding the stalk of plgR is capable of delivering the lytic enzyme from the apical surface to the basolateral surface. Significantly, such delivery is capable of bringing the chimera to tissue infected with, or at risk for infection with, B. anthracis.

[0014] Preferred targeting elements are described in the sections below, in the examples herein and in U.S. patent application publication no. US 2003/0161809, the contents of which are hereby incorporated by reference in their entirety. In certain embodiments, the present invention provides targeting elements that comprise complementarity determining regions derived from antibodies. Preferred targeting elements include antibodies, including diabodies and single chain Fv fragments such as SEQ ID NOS :2 and 3 and variants thereof, that are capable of binding the stalk of plgR as described in the sections below. In certain embodiments, the targeting element comprises a peptide that may bind to transporters, such as plgR.

[0015] The targeting element can be linked to the lytic enzyme by any means known to those of skill in the art, so long as the linkage does not interfere with the function of the lytic enzyme or the targeting element. Indeed, the present invention is based, in part, on the discovery of the portions of structures of lytic enzymes that can be used for linking targeting elements to form the chimeras of the invention. In certain embodiments, the targeting element is linked to the amino terminus of the lytic enzyme, either directly or via a linker, linkage to the carboxy terminus of the lytic enzyme is not preferred according to the invention. Further portions of the lytic enzyme for linkage are discussed in other aspects of the invention in the paragraphs below.

[0016] In certain embodiments of the invention, the linker is labile under conditions useful for the treatment or prevention of infection by B. anthracis. While not intending to be bound by any particular theory of operation, in some of these embodiments, the chimera can be described as a prodrug of the lytic enzyme that is capable of delivering the enzyme to tissue infected or at risk of infection. In such chimeras, the targeting element, together with other elements if any that are also linked to the lytic enzyme, can reduce or mask the activity of the lytic enzyme until the linker is cleaved at the tissue. Preferred linkers include those that are labile to proteases that can be found at the site of infection. In certain embodiments, the protease is elastase that might be secreted by macrophages and neutrophils at a site of B. anthracis infection. In other embodiments, the protease is B. anthracis lethal factor that might be secreted by the bacterial cell itself. Remarkably, one of the discoveries of the invention are these chimeras that cause a B. anthracis cell to kill itself by secreting the protease that activates the lytic enzyme. In further embodiments, the protease can be another protease, such as a cathepsin, that may be present or induced to be present at the site of infection.

[0017] In another aspect, the present invention provides split chimeras of lytic enzymes that are based on the discovery of a second location in the lytic enzymes where a moiety can be inserted. Surprisingly, as described in detail below, insertions can be made between two domains of the lytic enzyme with little or no disruption of its activity. Accordingly, the split chimeras comprise a lytic enzyme having one or more inserted moieties between its domains. In particular, the split chimeras comprise an inserted moiety between the cell wall binding domain and the lysis domain of the lytic enzyme, as is described in detail herein. The activity of lytic enzymes, such as PIyG, can be improved by inserting longer peptides between the cell wall binding domain and the lysis domain. [0018] The inserted moiety can be any moiety known to those of skill in the art that can be inserted into a chimera. In certain embodiments, the inserted moiety can be any moiety that does not eliminate or interfere substantially with the activity of the lytic enzyme. Preferred inserted moieties are those that are useful for the delivery of the split chimera to tissue infected, or at risk of infection, with an infectious agent that the lytic enzyme is capable of lysing. For example, when the lytic enzyme is PIyG, preferred inserted moieties include those that target the split chimera to tissue infected, or at risk of infection, with B. anthracis. Preferred inserted moieties include peptides and polypeptides that are capable of binding plgR, or a portion thereof, such as the stalk. Other aspects of the invention include the insertion of moieties that confer other biological activity, such as an activity that modulates transport and/or binding of a chimera into and/or across a cell. For instance, the present invention provides insertion of peptides and polypeptides that may localize the chimera at a site of infection or within an organ or tissue. Such insertions might change or improve the specificity of the enzyme, including redirecting the enzyme to heretofore resistant bacteria and different bacteria.

[0019] As discussed in the sections below, the insertion is located at or near a site in the primary sequence of PIyG between its two domains. PIyG can be divided into an enzymatic domain and a cell wall binding domain. In certain embodiments, the insertion is at the boundary between the domains as defined in the sections below. In further embodiments, the insertion is one, two, three, four, five, ten or fifteen residues from the boundary. In certain embodiments, the present invention provides replacement of a cell wall binding domain with peptides that have unique biological functions, including binding to new bacterial targets.

[0020] In further aspects, the present invention provides compositions useful for making the chimeras of the invention. For example, the present invention provides nucleic acids encoding the chimeras and vectors comprising the nucleic acids. The present invention also provides host cells comprising the nucleic acids or vectors. [0021] The chimeras of the invention can be made by any technique apparent to one of skill in the art for making the chimeras. For instance, the chimeras can be made recombinantly, synthetically or semi-synthetically. In preferred embodiments, the chimeras are made recombinantly with a nucleic acid, vector or host cell of the invention. [0022] In another aspect, the present invention provides pharmaceutical compositions comprising a chimera of the invention. The pharmaceutical compositions are useful for the administration of the chimeras to subjects in need thereof. The pharmaceutical compositions comprise a chimera of the invention and a pharmaceutically acceptable carrier, diluent or excipient. In preferred embodiments, the pharmaceutical composition is formulated for pulmonary administration.

[0023] In a further aspect, the present invention provides methods of treating or preventing conditions modulated by a target of a lytic enzyme. For instance, the present invention provides methods of treating or preventing conditions mediated by B. anthracis. The methods comprise administering a chimera or a pharmaceutical composition of the invention to a subject in need thereof. The chimera or pharmaceutical composition can be administered by any technique apparent to those of skill in the art, as discussed in detail in the sections below.

4. BRIEF DESCRIPTION OF THE FIGURES

[0024] FIG. IA provides the amino acid sequence of bacteriophage gamma PIyG

(SEQ ID NO:!); [0025] FIG. IB provides the amino acid sequence of human polymeric immunoglobulin receptor (plgR; SEQ ID NO:2);

[0026] FIGS. 2 A and 2B provides the lytic activity of chimeras of the invention compared to the lysin PIyG;

[0027] FIG. 3 A illustrates the activation of a chimera of the invention; and

[0028] FIG. 3B provides the lytic activity of a chimera of the invention, activated with elastase, compared to the lysin PIyG.

5. DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention is based, in part, on the discovery of PIyG chimeras that can be administered to a subject infected or at risk of infection with B. anthracis. In particular, the invention is based, in part, on the discovery of the tolerance of lytic enzymes such as PIyG for fusions or insertions at certain locations in their primary structure as described in the sections below.

5.1 Definitions

[0030] "Chimera" is a convenient term that refers to a molecule that comprises two or more recognizable moieties that are not found linked together in nature. For instance, a chimera can comprise a moiety with a first function that is linked to a moiety with a second function. Many chimeras of the invention comprise a moiety with an enzymatic function and a moiety with a targeting function. The term chimera should not be construed to limit a molecule to any particular method of preparation. However, certain chimeras of the invention are capable of being prepared by linking a first moiety to a second moiety. In certain embodiments discussed below, a linker is used to link the moieties together in the chimera.

[0031] "Split chimera" is a convenient term that refers to a particular chimera wherein one moiety can be located within, or is surrounded by, the second moiety. Although term should not be construed to limit a split chimera to any particular method of preparation, a split chimera can conveniently be described as a chimera that can be prepared by inserting a first moiety within the two portions of the second moiety. [0032] "PIyG" refers to the lytic enzyme of B. anthracis bacteriophage gamma. The enzyme comprises a domain that is specific for the cell wall of B. anthracis and a domain that is capable of lysing a B. anthracis. Examples of PIyG include those described in the examples below, including SEQ ID NO:1 and those described in PCT publication WO 2004/027020. [0033] "Targeting element" refers to any moiety that is capable of binding a target molecule. For example, a targeting element for plgR is moiety that is capable of binding plgR. Such a moiety can be any moiety capable of such binding known to those of skill in the art. Examples include immunological molecules such as antibodies, polyclonal antibodies, monoclonal antibodies, humanized antibodies, single chain antibodies and other constructs related to single chain antibodies such as diabodies. As is known to those of skill in the art, such immunological molecules comprise one or more complementarity defining regions. Preferred immunological molecules are described in the examples herein and in U.S. Patent Nos. 6,072,041 and 6,287,817, the contents of which are hereby incorporated by reference in their entirety. Non-immunological examples include lipids, carbohydrates, small molecules, nucleic acids and peptides or polypeptides. Such molecules are described extensively in U.S. patent publication no. US 2003/0161809, the contents of which are hereby incorporated by reference in their entirety.

[0034] "Capable of binding" is intended to have its ordinary meaning known to those of skill in the art. It can refer to the function of a moiety that enables it to selectively interact with a target. Such interactions are familiar to those of skill in the art including antibody: antigen interactions, ligand:receptor interactions, enzyme :substrate or enzyme :inhibitor interactions, lectin:carbohydrate interactions and the like.

[0035] "Protease sensitive" refers to the property of a molecule, familiar to those of skill in the art, that enables it to be cleaved by a protease under conditions suitable for protease activity. Cleaved products may be detected by any one of several methods, including polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE), size exclusion chromatography, high performance liquid chromatography (HPLC), mass spectrometry, etc.

[0036J The term "subject" refers to an animal such as a mammal, including, but not limited to, primate {e.g., human, monkey), cow, sheep, goat, horse, dog, cat, rabbit, hamster, rat, mouse and the like. In preferred embodiments, the subject is a human. The terms "human," "subject" and "patient" are used interchangeably herein. [0037] "Treating" or "treatment" of any disease or disorder refers, in one embodiment, to ameliorating the disease or disorder (i.e., arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment "treating" or "treatment" refers to ameliorating at least one physical parameter, which may not be discernible by the subject. In yet another embodiment, "treating" or "treatment" refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In yet another embodiment, "treating" or "treatment" refers to delaying the onset of the disease or disorder.

[0038] "Preventing" or "prevention" refers to a reduction in risk of acquiring a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop in a subject that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease).

[0039] As used herein, the term "about" refers to a range of tolerance above or below a quantitative amount known to be acceptable to those of skill in the art. For instance, a dose of about 1000 mg indicates a dose typically administered under the guidance of a practitioner when a dose of 1000 mg is indicated. In certain embodiments, the term "about" refers to ± 10% or ± 5%.

5.2 Chimeras of the Invention

[0040] The present invention provides chimeras of lytic enzymes that are useful for the treatment or prevention of infections by Bacillus bacteria such as Bacillus anthracis. While such lytic enzymes have been discovered recently, methods of delivering them effectively and economically to tissue infected have yet to be developed. The chimeras of the invention are based, in part, on the discovery of locations on the lytic enzymes that can be modified to form the chimeras.

[0041] In certain aspects of the invention, the chimeras comprise a lytic enzyme linked to a targeting element. Lytic enzymes and targeting elements are described in detail below. In this aspect of the invention, the targeting element is linked to the amino terminus of the lytic enzyme.

[0042] They can be linked by any means for linking moieties known to those of skill in the art. For instance, they can be linked by a direct bond, by an amide linkage or by a linker. In preferred embodiments, they are linked by an amide linkage or by a peptide linker. Preferred linkers are discussed in detail below.

5.2.1 Lytic Enzymes

[0043] The lytic enzyme can be any enzyme capable of lysing an infectious organism known to those of skill in the art. Lytic enzymes include enzymes that are capable of cleaving bonds that are present in the peptidoglycan of bacterial cells. In preferred embodiments, the lytic enzyme is an enzyme capable of selectively binding and lysing a Bacillus bacterium, for example, a B. anthracis bacterium.

[0044] Preferred lytic enzymes include PIyG enzymes such as those described in

Schuch et al., 2002, Nature 418:884-889 and in PCT publication WO 2004/027020, the contents ol which are hereby incorporated by reference in their entireties. The preferred lytic enzyme, PIyG, is provided by FIG. 1 and SEQ ID NO:1.

[0045] In further embodiments, the present invention provides chimeras of other lytic enzymes, such as PIyV 12 isolated and shown to effectively kill both E.faecalis and E. faecium (Yoong et al, 2004, J Bact. 186:4808-4812) and CpI-I, isolated from pneumococcal bacteriophage (Loeffler et al, 2003, Infect. Immun. 71 :6199-6204). As will be recognized by those of skill in the art, the present invention also provides methods of killing cells of these organisms and methods of treating or preventing infection by these organisms with the appropriate chimera. The contents of the preceding lytic enzyme references are hereby incorporated by reference in their entirety. [0046] Further useful lytic enzymes include, for example, autolysins, cell wall hydrolases, bacteriocins and colicins. Autolysins degrade different bonds in peptidoglycans (Lopez et al, 1997, Microbial Drug Resistance 3: 199-211). Cell wall hydrolases include N-acetylmuramoyl-L-alanine amidases, DL-endopeptidases, muramidases (lysozymes), and glucosaminidases. Bacateriocins are protein toxins that are synthesized by bacteria and kill only bacteria that are closely related to the producing species (Pilsl et al, 1996, J. Bact. 178: 2431-2435). Colicins form pores in cytoplasmic membranes that disrupt transmembrane potential or by hydrolyzing DNA or 16S rRNA (Braun et al, 1994, Arch. Microbiol. 161 : 199-206). Some colicins produced by E. coli disrupt murein and O-antigen giosynthesis by inhibiting the regeneration of the common lipid carrier (colicin M). Some colicins do not inhibit the bacterial species that produce them, but they inhibit other related species (allelopathy) as described by Chao and Levin (Chao and Levin, 1981, Proc. Nat. Acad. Sci. 78, 6324—6328). Examples of colicins include colicins G, D., M., Ia, Ib, U, and V. Colicin U is produced by Shigella boydii (Smajs et al, 1981, J Bacteriol. 179: 4919- 4928) is active against E. coli and some species of Shigella. Pesticin inhibits Yersinia pestis (black plague). Pesticin, hydrolyses murein by N-acetyhlglucosaminidase activity (Ferber and Brubaker, 1979, J. Bacteriol. 139: 495-501). Streptococcus pneumoniae produces autolytic amidases (LytA) that acts as a murein hydrolyase. Lytic enzymes exist in gram positive microorganisms such as Clostridium acetobutylicum and Lactococcus lactis (Lopez et al, supra). Bacillus subtilis produces cell wall hydrolyases such as LytC (an amidase), LytD (an glucosaminidase), LytE (an endopeptidase), and LytF( an endopeptidase) (Yamamoto et al, 2003, J. Bacteriol. 185: 6666-6677).

[0047] In certain embodiments, the lytic enzyme can be an variant, mutant or fragment of a lytic enzyme described above. In the context of the invention, a mutant PIyG is a functionally active PIyG encoded by a bacteriophage specific for B. anthracis. A variant is aiso a runctionaiiy active HyG that is not necessarily encoded by such a bacteriophage. For instance, variants can be generated by directed or random mutagenesis by one of skill in the art. Useful mutant or variant PIyGs have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% amino acid sequence identity with SEQ ID NO:1. Sequence identity can be determined by any method familiar to those of skill in the art. Preferred techniques include BLAST sequence alignments (Genetics Computer Group, Madison, Wisconsin).

[0048] Such variants include, for instance, polypeptides wherein one or more amino acid residues are added, or deleted at the amino or carboxy terminus of SEQ ID NO: 1. In certain embodiments, one or more amino acids are substituted, deleted, and/or added to any position (s) in the sequence, or sequence portion. Although preferred substitutions are conservative substitutions, any substitution that does not eliminate PIyG activity is within the scope of the invention.

[0049] As used herein, a fragment is a variant polypeptide having an amino acid sequence that entirely, or substantially, is the same as a portion but not all of the amino acid sequence of the aforementioned polypeptides. Fragments may include, for example, truncation polypeptides having a portion of the amino acid sequence of SEQ ID NO: 1. A fragment can be a continuous series of residues that includes the amino terminus, or a continuous series of residues that includes the carboxyl terminus. Fragments that are substantially the same as a portion of a corresponding amino acid sequence have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% amino acid sequence identity to the corresponding portion of the aforementioned polypeptide. [0050] Mutants, variants and fragments of other lytic enzymes are similarly defined, as will be apparent to those of skill in the art.

5.2.2 Targeting Elements

[0051] In addition to the lytic enzyme, the chimeras of the invention comprise at least one targeting element. As discussed above, the targeting element, in this aspect of the invention, is linked to the amino terminus of the lytic enzyme via a direct bond, an amide linkage or a linker. Preferred linkers and methods of linking the targeting element to the chimera are discussed in the sections below.

[0052] In certain embodiments, the targeting element is directed to a ligand that is capable of conferring transcellular, transcytotic or paracellular transporting properties to the chimera. The ligand can be any such ligand known to those of skill in the art. However, preferred ligands include the polymeric immunoglobulin receptor (plgR) and portions of plgR. Particularly preferred portions of plgR include the stalk of plgR. Such ligands are described extensively in U.S. Patent Nos. 6,042,833, 6,072,041, 6,287,817 and U.S. patent application publication nos. US2002/0136732 and US2003/0161809. The contents of each reference is hereby incorporated by reference in its entirety.

[0053] A polyimmunoglobulin receptor (plgR) molecule has several structurally and functionally distinct regions that are defined as follows. In the art, a plgR molecule is generally described as consisting of two different, loosely defined regions called the "stalk" and the "secretory component" (SC). A plgR molecule binds polymeric immunoglobulins (IgA or IgM) on the basolateral side, and then transports the immunoglobulin to the apical side. Proteolyic cleavage of plgR takes place on the apical side of an epithelial cell between the SC and the stalk. The SC molecule is released from the cellular membrane and remains bound to and protects the immunoglobulins, whereas the stalk molecule remains bound to the cellular membrane (see "Mucosal Immunoglobulins" by Mestecky et al. in: Mucosoal Immunology, edited by P. L. Ogra, M. E. Lamm, J. Bienenstock, and J. R. McGhee, Academic Press, 1999).

[0054] Particularly preferred plgR molecules are those described in U.S. Pat. No.

6,042,833, and the simian plgR described in U.S. patent application Serial No. 60/266,182 (attorney docket No. 057220.0701) entitled "Compositions and Methods for Identifying, Characterizing, Optimizing and Using Ligands to Transcytotic Molecules" by Houston, L. L., and Sheridan, Philip L., which was filed on Feb. 2, 2001. However, it is understood that, in the context of this invention, plgR also refers to any of that receptor's family or superfamily members, any homolog of those receptors identified in other organisms, any iso forms of these receptors, any plgR-like molecule, as well as any fragments, derivatives, mutations, or other modifications expressed on or by cells such as those located in the respiratory tract, the gastrointestinal tract, the urinary and reproductive tracts, the nasal cavity, buccal cavity, ocular surfaces, dermal surfaces and any other mucosal epithelial cells. Preferred plgR and plgR-like proteins are those that direct the endocytosis or transcytosis of proteins into or across epithelial cells. An exemplary human plgR is provided in FIG. IA.

[0055] As used herein, the terms "secretory component" and "SC" refer to the extracellular domain of plgR, as known to those of skill in the art. In certain embodiments, it is the smallest (shortest amino acid sequence) portion of an apical proteolyzed plgR molecule that retains the ability to bind immunoglobulins (IgA and IgM). After proteolytic cleavage of plgR, some amino acid residues remain associated with SC:immunoglobulin complexes but are eventually degraded and/or removed from such complexes (Ahnen et al., 1986, J. Clin. Invest. 77:1841-1848). According to the definition of the secretory component used herein, such amino acids are not part of the SC. In certain embodiments of the invention, plgR-targeting elements that do not recognize or bind to the SC are preferred. [0056] As used herein, the term "stalk" refers to a molecule having an amino acid sequence derived from a plgR, wherein the stalk sequence does not comprise amino acid sequences derived from the SC. A stalk molecule comprises amino acid sequences that remain bound to the apical membrane following the apical proteolytic cleavage when such cleavage occurs and amino acid sequences required for such cleavage. Preferred stalk molecules confer one or more transcytotic properties to a ligand bound thereto. Most preferred are stalk molecules that confer the ability to undergo apical to basolateral transcytosis to a ligand bound thereto.

[0057| Another way in which different portions of a plgR molecule, and SC and stalk molecules derived therefrom, can be delineated is by reference to the domains thereof. A protein "domain" is a relatively small {i.e., <about 150 amino acids) globular unit that is part of a protein. A protein may comprise two or more domains that are linked by relatively flexible stretches of amino acids. In addition to having a semi-independent structure, a given domain may be largely or wholly responsible for carrying out functions that are normally carried out by the intact protein. In addition to domains that have been determined by in vitro manipulations of protein molecules, it is understood in the art that a "domain" may also have been identified in silico, i.e, by software designed to analyze the amino acid sequences encoded by a nucleic acid in order to predict the limits of domains. The latter type of domain is more accurately called a "predicted" or "putative" domain but, in the present disclosure, the term domain encompasses both known and predicted domains unless stated otherwise.

[0058] Domains of plgR molecules include a leader sequence, extracellular domains

1 through 6, a transmembrane domain and an intracellular domain {see FIG. 3 of Piskurich et al., 1995, J. Immunol. 154:1735-1747). The intracellular domain contains signals for transcytosis and endocytosis. Domains of a plgR molecule that are of particular interest in the present disclosure include but are not limited to domain 5, domain 6, the transmembrane domain and the intracellular domain. Preferred domains confer the ability to undergo apical to basolateral transcytosis to a ligand bound thereto.

[0059] Another way in which different portions of a plgR molecule can be defined is by reference to amino acid sequences that are conserved between plgR homologs {i.e., plgR molecules isolated from non-human species; see below). Non-limiting examples of conserved amino acid sequences include the following portions of SEQ ID NO: 1 , inclusively: 297-301, 325-331, 410-414, 476-480, 522-526, 624-629, 658-662 and 732-737. [0060] Preferred target elements confer the ability to undergo apical to basolateral transcytosis to a ligand bound to a plgR molecule or a stalk molecule, wherein the ligand does not bind specifically to an SC molecule. Other preferred target elements comprise sequences from a stalk molecule.

[0061] Homologs of plgR are also useful in the invention. Homologs of plgR are plgR proteins from species other than Homo sapiens. By way of non-limiting example, plgR proteins from various species include those from humans, monkeys, the rat, mouse, rabbit, cow and possum (see below). (See also Mostov and Kaetzel, Chapter 12, "Immunoglobulin Transport and the Polymeric Immunoglobulin Receptor" in Mucosal Immunity, Academic Press, 1999, pages 181-211; and Piskurich et al., J. Immunol. 154: 1735-1747, 1995). Relevant sequences include those described at the following accession numbers, which are hereby incorporated by reference in their entireties: Zebrafish {Brachydanio reriό) 9863256, 8713834, 8282255, & 7282118; Mouse (Mus musculus) 8099664, 2804245, 6997240, 4585867, 4585866, 2688814, 2688813, 2688812, 2688811, 2688810, 2688809, 2688808, 2688807, 3097245, 3046754, 3046752, 3046751, 3046756, 3046755, 3046750, 3046748, 3046747 and 2247711; Rat (Rattas norvegicus) 2222806, 475572, 475571, 473408, 603168 and 603167; Cow (Bos taunts) 388279; Possum (Trichosuras vulpeculά) 5305520, 5305518, 5305514 and 5305512.

[0062] Also useful in the invention are plgR-like proteins. A "plgR-like protein" is a protein that has an amino acid sequence having homology to a known plgR protein. In many instances, the amino acid sequences of such plgR-like molecules have been generated by the in silico translation of a nucleic acid, wherein the nucleotide sequence of the nucleic acid has been determined but is not known to encode a protein. By way of non-limiting example, plgR-like proteins include PIGRLl (U.S. Pat. No. 6,114,515); PIGR-I (U.S. Pat. No. 6,232,441); a mouse gene having an exon similar to one of plgR's (GenBank Accession No. 6826652); human proteins translated in silico that have homology to plgR proteins (GenBank Accession Nos. 1062747 and 1062741); and Digrl (Luo et al, 2001, Biochem Biophys Res Commun 287:35-41, 2001)

[0063] As used herein, a "homolog" of a plgR protein or a plgR-like protein is an isoform or mutant of human plgR, or a protein in a non-human species that either (i) is "identical" with or is "substantially identical" (determined as described below) to an amino acid sequence in human plgR, or (ii) is encoded by a gene that is identical or substantially identical to the gene encoding human plgR. Non-limiting examples of types of plgR isoforms include isoforms of differing molecular weight that result from, e.g., alternate RNA splicing or proteolytic cleavage; and isoforms having different post-translational modifications, such as glycosylation; and the like.

[0064] In certain embodiments, the ligand may be a polypeptide that corresponds to an amino acid sequence that is conserved in plgR proteins from a variety of species, e.g., the ligand is a polypeptide having an amino acid sequence selected from the group consisting of LRKED, QLFVNEE, LNQLT, YWCKW, GWYWC, STLVPL, SYRTD, and KRSSK. In a related aspect, the ligand may be a polypeptide that corresponds to an amino acid sequence present in a defined region of plgR, e.g. SEQ ID NO:1, selected from the group consisting of:

1 Rl From KRSSK to the carboxy terminus,

R2a From SYRTD to the carboxy terminus,

R2b From SYRTD to KRSSK,

R3a From STLVPL to the carboxy terminus,

R3b From STLVPL to KRSSK,

R3c From STLVPL to SYRTD,

R4a From GWYWC to the carboxy terminus,

R4b From GWYWC to KRSSK,

R4c From GWYWC to SYRTD,

R4d From GWYWC to STLVPL,

R5a From YWCKW to the carboxy terminus,

R5b From YWCKW to KRSSK,

R5c From YWCKW to SYRTD,

R5d From YWCKW to STLVPL,

R5e From YWCKW to GWYWC,

R6a From LNQLT to the carboxy terminus,

R6b From LNQLT to KRSSK,

R6c From LNQLT to SYRTD,

R6d From LNQLT to STLVPL,

R6e From LNQLT to GWYWC,

R6f From LNQLT to YWCKW,

R7a From QLFVNEE to the carboxy terminus,

R7b From QLFVNEE to KRSSK,

R7c From QLFVNEE to SYRTD,

R7d From LNQLT to STLVPL, R7e From QLFVNEE to GWYWC,

R7f From QLFVNEE to YWCKW,

R7g From QLFVNEE to LNQLT,

R8a From LRKED to the carboxy terminus,

R8b From LRKED to KRSSK,

R8c From LRKED to SYRTD,

R8d From LRKED to STLVPL,

R8e From LRKED to GWYWC,

R8f From LRKED to YWCKW,

R8g From LRKED to LNQLT, and

R8h From LRKED to QLFVNEE.

[0065] The targeting element can be any moiety capable of binding the ligand known to those of skill in the art. Preferred ligands include immunological molecules capable of binding the ligand. For instance, the targeting element can be an antibody or an antibody derivative. Antibodies per se include, but are not limited to, polyclonal, monospecific, and monoclonal antibodies. Antibody derivatives include those prepared by recombinant DNA technology, e.g., single-chain (sFv) antibodies and diabodies, and those prepared from whole antibodies by chemical manipulation, e.g., Fab, Fab¹ and (Fab)₂ fragments.

[0066] In further embodiments, the targeting element can be a moiety other than an immunological molecule, including those targeting elements described in U.S. patent application publication no. 2003/0161809. Such targeting elements include lipids, carbohydrates, small molecules and nucleic acids. In particular, a polypeptide that functions as a targeting element directed to the plgR stalk may be derived from a polypeptide derived from a calmodulin, an AP-I Golgi adaptor or a bacterial polypeptide. Non limiting examples of polypeptides from bacterial proteins that may be used as plgR-stalk-directed targeting elements are those amino acid sequences from CbpA that are described in U.S. patent application publication no. 2003/0161809.

[0067] In a related aspect, the ligand may be a polypeptide that corresponds to an amino acid sequence that is conserved in plgR proteins from a variety of species, e.g., a polypeptide having an amino acid sequence selected from the group consisting of LRKED, QLFVNEE, LNQLT, YWCKW, GWYWC, STLVPL, SYRTD, and KRSSK. [0068] In a related aspect, the ligand may be a polypeptide that corresponds to an amino acid sequence present in a defined region, e.g., a region of a plgR, wherein said plgR can be from any animal, and wherein said region is selected from the group consisting of: 2 Rl From KRSSK to the carboxy terminus of plgR,

R2a From SYRTD to the carboxy terminus of plgR,

R2b From SYRTD to KRSSK,

R3a From STLVPL to the carboxy terminus of plgR,

R3b From STLVPL to KRSSK,

R3c From STLVPL to SYRTD,

R5e From YWCKW to GWYWC,

R6e From LNQLT to GWYWC,

R6f From LNQLT to YWCKW,

R7e From QLFVNEE to GWYWC,

R7f From QLFVNEE to YWCKW,

R7g From QLFVNEE to LNQLT,

R8e From LRKED to GWYWC,

R8f From LRKED to YWCKW,

R8g From LRKED to LNQLT, and

R8h From LRKED to QLFVNEE.

5.2.3 Targeting Elements Comprising Immunological Molecules [0069] Wild type antibodies have four polypeptide chains, two identical heavy chains and two identical light chains. Both types of polypeptide chains have constant regions, which do not vary or vary minimally among antibodies of the same class (i.e., IgA, IgM, etc.), and variable regions. As is explained below, variable regions are unique to a particular antibody and comprise a recognition element for an epitope. [0070] Each light chain of an antibody is associated with one heavy chain, and the two chains are linked by a disulfide bridge formed between cysteine residues in the carboxy-terminal region of each chain, which is distal from the amino terminal region of each chain that constitutes its portion of the antigen binding domain. Antibody molecules are further stabilized by disulfide bridges between the two heavy chains in an area known as the hinge region, at locations nearer the carboxy terminus of the heavy chains than the locations where the disulfide bridges between the heavy and light chains are made. The hinge region also provides flexibility for the antigen-binding portions of an antibody. [0071] An antibody's specificity is determined by the variable regions located in the amino terminal regions of the light and heavy chains. The variable regions of a light chain and associated heavy chain form an "antigen binding domain" that recognizes a specific epitope; an antibody thus has two antigen binding domains. The antigen binding domains in a wildtype antibody are directed to the same epitope of an immunogenic protein, and a single wildtype antibody is thus capable of binding two molecules of the immunogenic protein at the same time.

[0072] Compositions of antibodies have, depending on the manner in which they are prepared, different types of antibodies. Types of antibodies of particular interest include polyclonal, monospecific and monoclonal antibodies.

[0073] Polyclonal antibodies are generated in an immunogenic response to a protein having many epitopes. A composition of polyclonal antibodies thus includes a variety of different antibodies directed to the same and to different epitopes within the protein. Methods for producing polyclonal antibodies are known in the art (see, e.g., Cooper et al., Section III of Chapter 11 in: Short Protocols in Molecular Biology, 2nd Ed., Ausubel et al., eds., John Wiley and Sons, New York, 1992, pages 11-37 to 11-41). [0074] Monospecific antibodies (a.k.a. antipeptide antibodies) are generated in a humoral response to a short (typically, 5 to 20 amino acids) immunogenic polypeptide that corresponds to a few (preferably one) isolated epitopes of the protein from which it is derived. A plurality of monospecific antibodies includes a variety of different antibodies directed to a specific portion of the protein, i.e, to an amino acid sequence that contains at least one, preferably only one, epitope. Methods for producing monospecific antibodies are known in the art (see, e.g., Cooper et al., Section III of Chapter 11 in: Short Protocols in Molecular Biology, 2nd Ed., Ausubel et al., eds., John Wiley and Sons, New York, 1992, pages 11-42 to 11-46).

[0075] A monoclonal antibody is a specific antibody that recognizes a single specific epitope of an immunogenic protein. In a plurality of a monoclonal antibody, each antibody molecule is identical to the others in the plurality. In order to isolate a monoclonal antibody, a clonal cell line that expresses, displays and/or secretes a particular monoclonal antibody is first identified; this clonal cell line can be used in one method of producing the antibodies of the invention. Methods for the preparation of clonal cell lines and of monoclonal antibodies expressed thereby are known in the art (see, for example, Fuller et al., Section II of Chapter 11 in: Short Protocols in Molecular Biology, 2nd Ed., Ausubel et al., eds., John Wiley and Sons, New York, 1992, pages 11-22 to 11-11-36). [0076] Variants and derivatives of antibodies include antibody and T-cell receptor fragments that retain the ability to specifically bind to antigenic determinants. Preferred fragments include Fab fragments (i.e, an antibody fragment that contains the antigen- binding domain and comprises a light chain and part of a heavy chain bridged by a disulfide bond); Fab' (an antibody fragment containing a single anti-binding domain comprising an Fab and an additional portion of the heavy chain through the hinge region); F(ab')₂ (two Fab¹ molecules joined by interchain disulfide bonds in the hinge regions of the heavy chains; the Fab' molecules may be directed toward the same or different epitopes); a bispecific Fab (an Fab molecule having two antigen binding domains, each of which may be directed to a different epitope); a single chain Fab chain comprising a variable region, a.k.a., a sFv (the variable, antigen-binding determinative region of a single light and heavy chain of an antibody linked together by a chain of 10-25 amino acids); a disulfide-linked Fv, or dsFv (the variable, antigen-binding determinative region of a single light and heavy chain of an antibody linked together by a disulfide bond); a camelized VH (the variable, antigen- binding determinative region of a single heavy chain of an antibody in which some amino acids at the VH interface are those found in the heavy chain of naturally occurring camel antibodies); a bispecific sFv (a sFv or a dsFv molecule having two antigen-binding domains, each of which may be directed to a different epitope); a diabody (a dimerized sFv formed when the VH domain of a first sFv assembles with the VL domain of a second sFv and the VL domain of the first sFv assembles with the VH domain of the second sFv; the two antigen-binding regions of the diabody may be directed towards the same or different epitopes); and a triabody (a trimerized sFv, formed in a manner similar to a diabody, but in which three antigen-binding domains are created in a single complex; the three antigen binding domains may be directed towards the same or different epitopes). Derivatives of antibodies also include one or more CDR sequences of an antibody combining site. The CDR sequences may be linked together on a scaffold when two or more CDR sequences are present.

[0077] The term "antibody" also includes genetically engineered antibodies and/or antibodies produced by recombinant DNA techniques and "humanized" antibodies. Humanized antibodies have been modified, by genetic manipulation and/or in vitro treatment to be more human, in terms of amino acid sequence, glycosylation pattern, etc., in order to reduce the antigenicity of the antibody or antibody fragment in an animal to which the antibody is intended to be administered (Gussow et al., Methods Enz. 203:99-121, 1991).

[0078] Diabodies are dimeric antibody fragments (Hollinger et al., "Diabodies": small bivalent and bispecific antibody fragments, Proc Natl Acad Sci USA JuI. 15, 1993;90(14):6444-8). In each polypeptide, a heavy-chain variable domain V(H) is linked to a light-chain variable domain V(L) but unlike single-chain Fv fragments, each antigen- binding site is formed by pairing of one V(H) and one V(L) domain from the two different polypeptides. Diabodies thus have two antigen-binding sites, and can be bispecific or bivalent. (Perisic et al., Crystal structure of a diabody, a bivalent antibody fragment, Structure Dec. 15, 1994;2(12):1217-26).

[0079] The length of the linker(s) between V-domains influences the size, flexibility and valency of single chain Fv antibody fragments (sFv's). sFv molecules are predominantly monomeric when the V(H) and V(L) domains are joined by polypeptide linkers of at least 12 amino acid residues. An sFv molecule with a linker of 3 to 12 amino acid residues is less likely to fold into a monomer, i.e., a single chain Fv in which the V(H) and V(L) domains are paired intramolecularly. However, sFv's that do not easily form monomers may interact with a second sFv molecule to form a "diabody". Diabodies may be bispecific (Muller et al., "A dimeric bispecific miniantibody combines two specificities with avidity", Federation of European Biochemical Societies, 432 (1998), pp. 45-49) or bivalent. A bivalent diabody is formed from two sFv's that are identical to, or substantially the same as, each other; it has two binding [V(H):: V(L)] regions directed to the same target molecule. A bispecific diabody is formed from two sFv's that are different from each other, and has two binding [V(H):: V(L)] regions, each of which is directed to a different target molecule . [0080] Reducing the linker length below three amino acid residues can force sFv molecules to associate to form multimers (e.g., trimers a.k.a. triabodies, tetramers a.k.a., tetrabodies, etc.) depending on linker length, composition and V-domain orientation (see, e.g., U.S. Pat. No. 5,837,242). The increased valency in sFv multimers may result in higher avidity (low off-rates) (Hudson et al., High avidity scFv multimers; diabodies and triabodies, J Immunol Methods Dec. 10, 1999;231(l-2):177-89; Todorovska et al., Design and application of diabodies, triabodies and tetrabodies for cancer targeting, J. Immunol Methods Feb. 1, 2001;248(l-2):47-66; Hudson et al., High avidity scFv multimers; diabodies and triabodies, J Immunol Methods Dec. 10, 1999;231(1 -2): 177-89). [0081] Single-chain antibodies having V(H) and V(L) domains with 10-residue

(Gly4Ser)₂ or five-residue (Gly4Ser) linkers, or no linkers, have been examined. In one report (Kortt et al., Single-chain Fv fragments of anti-neuramimidase antibody NClO containing five- and ten-residue linkers form dimers and with zero-residue linker a trimer, Protein Engineering, 10:423-433, 1997), the zero-residue linker sFv formed a trimer with three active antigen combining sites. BIAcore biosensor experiments showed that the affinity of each individual antigen combining site in both the 10- and five-residue linker sFv dimers and zero-residue liner sFv trimer was essentially the same when the sFvs were immobilized onto the sensor surface. However, when the sFv was used as the analyte, the dimeric and trimeric sFv's showed an apparent increase in binding affinity due to the avidity of binding the multivalent sFv's. [0082] In general, sFv molecules in which the number of amino acid residues between the V(H) and V(L) domains is 0 to 15 are less likely to form monomers and are more likely to form some type of multimer. When the linker length is 1 or 2 amino acids, trimers and/or other multimers are more likely to form. Linker lengths of 3 to 12 amino acids favor the formation of dimers, where sFv's having linkers of 12 or more more amino acids are more likely to form monomers. These rules are not absolute, however, those skilled in the art can prepare and analyze sFv's with differing linker lengths and test them for the presence of monomers and various multimers.

[0083] Higher multimers of sFv molecules may be polyvalent, polyspecific, or both

(see, e.g., Muller et al., "A dimeric bispecifϊc miniantibody combines two specificities with avidity", Federation of European Biochemical Societies, 432 (1998), pp. 45-49). Using triabodies as a non-limiting example of higher multimers of sFv's, it can be seen that there are three possible combinations of sFv molecules. First, a triabody may comprise three identical or substantially identical sFv molecules, each of which is directed to the same target molecule, and is thus a trivalent triabody. Second, a triabody may comprise three different sFv molecules, each of which is directed to a different target molecule, and is thus a trispecific triabody. Third, a triabody may comprise two types of sFv molecules, a pair of which (sFvla and sFvlb) is directed to a target molecule #1, whereas the third sFv in the triabody is directed to target molecule #2. The latter triabody is both bispecific, as it specifically binds both target molecule #1 and target molecule #2, and bivalent, as it has two binding regions directed to target molecule #1.

[0084] Disulfide-stabilized sFv's (dsFv's) are recombinant Fv fragments of antibodies in which the unstable variable heavy V(H) and variable light V(L) heterodimers are stabilized by disulfide bonds engineered at specific sites that do not appreciably alter the binding activity of the sFv. Such sites lie between structurally conserved framework positions of V(H) and V(L). It should be possible to use positions in conserved framework regions to disulfide-stabilize many different sFv's (Reiter et al., Stabilization of the Fv fragments in recombinant immunotoxins by disulfide bonds engineered into conserved framework regions, Biochemistry May 10, 1994;33(18):5451-9). In addition to influencing the tendency of a sFv molecule to form monomers or multimers, sFv molecules into which Cys residues have been introduced into may in some instances have altered production and stability characteristics.

[0085] To improve the stability of Fv molecules, a cysteine residue is introduced into conserved framework regions of both the heavy and light variable domains at positions compatible with the formation of an interdomain disulfide linkage. A disulfide-stabilized Fv (dsFv) may be more resistant to denaturation by heat or urea treatment than the corresponding single-chain Fv (sFv). Moreover, the yield of dsFv may be higher than that of the sFv (Webber et al., Preparation and characterization of a disulfide-stabilized Fv fragment of the anti-Tac antibody: comparison with its single-chain analog, MoI Immunol 1995 March;32(4):249-58; Reiter et al., Antitumor activity and pharmacokinetics in mice of a recombinant immunotoxin containing a disulfide-stabilized sFv fragment, Cancer Res May 15, 1994;54(10):2714-8).

[0086] Molecular graphic modeling may be used to identify sites for the introduction of interchain disulfide bonds in the framework region of sFv molecules. Mutations that result in the Cys-modification of the sites are introduced in the reading frame that encodes the sFv molecule using any appropriate method, e.g., PCR-mediated mutagensis. The disulfide-stabilized Fv (dsFv) is expressed and tested for its binding activity (Luo et al., Vl -linker- Vh orientation-dependent expression of single chain Fv- containing an engineered disulfide-stabilized bond in the framework regions, J Biochem (Tokyo) 1995 October; 118(4):825-31).

5.3 Linkers

[0087] The targeting element and the lytic enzyme can be linked by any linker known to those of skill in the art. In certain embodiments, they are linked directly, for instance, via a direct bond, via an amide linker. In other embodiments, they are linked via a linker.

[0088] Any means of linking two moieties known to those of skill in the art can be used in the invention. For instance, amino acid residues present in the natural sequence of a first protein member can be directly covalently linked to amino acid residues in the natural amino acid sequence of a second protein member as in, e.g., a disulfide bridge. In addition, mutant amino acids useful for covalent linkages can be introduced into one or more protein members by using molecular genetics to alter the reading frame encoding such protein members or, in the case of synthetic oliogopeptides, directly during the in vitro synthesis thereof. Furthermore, natural or mutant amino acid sequences present in isolated proteins can be "derivatized" (i.e., chemically modified in vitro) so as to include chemical groups not present in natural amino acids but useful for the chemical conjugation of oligopeptides, polypeptides, and proteins in a related methodology, unnatural amino acids having moieties useful for chemical conjugation are introduced into oligopeptides or peptidomimetics during their synthesis in vitro. In addition, a cross-linking reagent (a.k.a. "cross-linker"), typically a bifunctional (two-armed) chemical linker that forms covalent linkages between two or more conjugate members, can be used to covalently link conjugate members to each other. Such bifunctional linkers can be homobifunctional (wherein both "arms" of the linker are the same chemical moiety) or heterobifunctional (wherein each of the two "arms" is a different chemical moiety than the other).

[0089] Hermanson (Bioconjugate Techniques, Academic Press, 1996), herein incorporated by reference, summarizes many of the chemical methods used to link proteins and other molecules together using various reactive functional groups present on various cross-linking or derivatizing reagents. Polypeptide cross-linking agents are based on reactive functional groups that modify and couple to amino acid side chains of proteins and peptides, as well as to other side groups and other macromolecules. Bifunctional cross- linking reagents incorporate two or more functional reactive groups. The functional reactive groups in a bifunctional cross-linking reagent may be the same (homobifunctional) or different (heterobifunctional). Many different cross-linkers are available to cross-link various proteins, peptides, and macromolecules.

[0090] Preferred linkers include are amino acid sequences that can be included in a chimera in between other portions of a chimera. Linkers can be included for a variety of reasons. For example, a linker can provide some physical separation between two parts of a protein that might otherwise interfere with each other via, e.g., steric hinderance. One example of a linker of this type is the repeating amino acid sequence (Gly4-Ser)_x, wherein x is 1 to 10, and preferably 1 to 4.

[0091] In certain embodiments of the invention, the chimeras can be designed so as to contain a site (a "protease sensitive site" or simply "protease site") that is amenable to being cleaved by agents or under conditions that cause or promote such cleavage. In some preferred embodiments of the invention, the cleavage site is contained within a linker, so that cleavage separates, e.g., the targeting element of a chimera from the lytic enzyme, which is useful for in vivo therapeutic methods.

[0092] The nature and arrangement of a cleavage site or of a spacer containing a cleavage site will depend on the nature of the in vivo or in vitro method(s) of interest. It is understood by those skilled in the art that the amino acids sequences of chimeras that one wishes to have cleaved by a protease must be designed so as to retain the protease cleavage site of choice. Non-limiting examples of in vitro and in vivo cleavage sites and systems are as follows.

[0093] In certain preferred embodiments of the invention, a linker comprises a protease sensitive site that can be cleaved by an elastase enzyme of a subject to which the chimera might be administered. Elastase is an enzyme that can be secreted by macrophages at sites of infection in a subject. While not intending to be bound by any particular theory of operation, it is believed that elastase cleavage of a chimera of the invention can release the lytic enzyme at the site of infection by a B. anthracis cell. Preferred elastase sensitive sites include, but are not limited to, the amino acid sequence GAAPVG (SEQ ID NO:3). [0094] In further preferred embodiments of the invention, a linker comprises a protease sensitive site that can be cleaved by lethal factor (LF) secreted by a B. anthracis cell. LF is an enzyme that can be secreted by B. anthracis and can form a portion of the B. anthracis lethal toxin. While not intending to be bound by any particular theory of operation, it is believed that lethal factor cleavage of a chimera of the invention can release the lytic enzyme at the site of infection by a B. anthracis cell. Remarkably, chimeras comprising a lethal factor sensitive site use the biology of the B. anthracis cell itself to activate the chimera and kill the cell or neighboring cells. Preferred lethal factor sensitive sites include, but are not limited to, amino acid sequences from the amino terminus of mitogen-activated protein kinase-kinase (see Vitale et al, 1998, Biochem. Biophys. Res. Commun. 248:706-711). Preferred peptide sequences that can be cleaved by lethal factor are described in the examples below and also include those described in Cummings et al, 2003, Proc. Natl. AcadSci. USA 99:6603-6606; Tonello et al, 2002, Nature 418:386; Rosetto et al, 2000, Clin. Chim. Acta 291 : 189-199; Gupta et al, 1998, Infect. Immun. 66:862-865; Vitale et al, 2000, Biochem J. 352:739-745; Pellizzari et al, 1999, FEBS Lett. 462:199-204; and Hammond & Hanna, 1998, Infect. Immun. 66:687-691. The contents of each reference are hereby incorporated by reference in their entireties. Preferred peptide sequences also include those that are cleaved by cathepsins, such as cathepsin G. A preferred peptide cleaved by cathepsin G is Thr-Pro-Phe-Ser-Ala-Leu-Gln (Rehault et al, 1999, J. Biol. Chem. 274:13810-13817).

5.4 Split Chimeras

[0095] In another aspect, the present invention provides split chimeras of a lytic enzyme. The split chimeras comprise a lytic enzyme and an insertion between two domains of the lytic enzymes.

[0096] This aspect of the invention is based, in part, on the discovery that the lytic enzyme PIyG can be fully functional with insertions when they are located in a region between the two functional domains of the enzyme.

[0097] For the purposes of this invention, the domains of PIyG are the amino terminal, enzymatic domain and the carboxy terminal, cell wall binding domain. The amino terminal domain is amino acids 1 through approximately 165 of SEQ ID NO:1. The carboxy terminal domain is approximately amino acid 166 through amino acid 233 of SEQ ID NO:1. Since corresponding domains of other lytic enzymes will be apparent to those of skill in the art, for instance, upon examining sequence alignments with PIyG, this aspect of the invention encompasses split chimeras of such lytic enzymes.

[0098] In certain embodiments, the insertion can be located at any site within 20, 19,

18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 residues of amino acid 165 or 166 of PIyG. Preferred locations for the insertion are described in the examples below. [0099] The insertion can be any moiety that can be inserted between the domains of the lytic enzyme without limitation. Preferred insertions are those that do not interfere with the function of the lytic enzyme. Particularly preferred insertions are described in the sections above. Also within the scope of the invention are chimeras comprising elements that confer novel biological properties to the chimera. Examples include elements that stabilize the chimera to degradation or loss of activity, enhance enzymatic activity or reactivity for example towards inhibiting and/or killing a target cell, extend the reaction half life of the chimera, enable or enhance the transport of the chimera into and/or across cellular barriers and the like.

[00100] In further embodiments, peptides that are recognized by antibodies and other agents can be inserted between the two domains. These peptides can be used, for example, for purification or for specific binding. For example, a bispecific sFv can be constructed that incorporates one sFv specific for plgR and a second sFv specific for a PIyG chimera of the invention. The PIyG chimera comprises an inserted element that is recognized by the second sFv. The complex of bispecific sFv : PIyG chimera can be transported from the apical to the basolateral surface of plgR-containing epithelial cells. Human sFv directed toward the epitope contained in the PIyG chimera can be made following the procedures and protocols outlined by Marks and Bradbury, 2004, Met. MoI. Biol. 248: 161-176 and Sheets et al, 1998, Proc. Natl. Acad ScI USA 95: 6157-6162.

[00101] The insertion can be linked to each domain of the lytic enzyme directly or with a linker. Preferred linkers are discussed in the sections above. In certain embodiments, this aspect of the invention provides a split chimera wherein two linkers are used to link the insertion to the lytic enzyme, one to each domain of the chimeras. [00102] In preferred embodiments, the split lysin chimers are stable under the conditions in which they are to be used. However, in certain embodiments, one might wish to use a split chimera with one or more protease sensitive sites in order to disrupt the split lysin under suitable conditions. Hence, a split chimera can comprise one or more linkers with a protease sensitive site, as discussed in the sections above. 5.5 Methods of Making the Chimeras

[00103] The chimeras of the invention can be made according to any method for making the molecules that is apparent to one of skill in the art without limitation. The chimeras can be made recombinantly, synthetically or semi-synthetically. The entire chimera can be prepared as if it were a single molecule, or portions of the chimera can be prepared separately and linked together. Standard techniques for the recombinant, synthetic or sem-synthetic preparation of lytic enzymes, targeting elements and linkers are well known to those of skill in the art and need not be reproduced extensively here. [00104] The antibodies and antibody fragments of the invention may be produced by any suitable method, for example, in vivo (in the case of polyclonal and monospecific antibodies), in cell culture (as is typically the case for monoclonal antibodies, wherein hybridoma cells expressing the desired antibody are cultured under appropriate conditions), in in vitro translation reactions, in recombinant DNA expression systems (the latter method of producing proteins is disclosed in more detail herein) and even synthetically. Antibodies and antibody variants can be produced from a variety of animal cells, preferably from mammalian cells, with murine and human cells being particularly preferred. Antibodies that include non-naturally occurring antibody and T-cell receptor variants that retain only the desired antigen targeting capability conferred by an antigen binding site(s) of an antibody can be produced by known cell culture techniques and recombinant DNA expression systems (see, e.g., Johnson et al., Methods in Enzymol. 203:88-98, 1991 ; Molloy et al., MoI. Immunol. 32:73-81, 1998; Schodin et al., J. Immunol. Methods 200:69-77, 1997). Recombinant DNA expression systems are typically used in the production of antibody variants such as, e.g., bispecifϊc antibodies and sFv molecules. Preferred recombinant DNA expression systems include those that utilize host cells and expression constructs that have been engineered to produce high levels of a particular protein. Preferred host cells and expression constructs include Escherichia coli; harboring expression constructs derived from plasmids or viruses (bacteriophage); yeast such as Saccharomyces cerevisiae or Pichia pastoris harboring episomal or chromosomally integrated expression constructs; insect cells and viruses such as Sf9 cells and baculovirus; and mammalian cells harboring episomal or chromosomally integrated (e.g., retroviral) expression constructs (for a review, see Verma et al., J. Immunol. Methods 216:165-181, 1998). Antibodies can also be produced in plants (U.S. Pat. No. 6,046,037; Ma et al., Science 268:716-719, 1995) or by phage display technology (Winter et al., Annu. Rev. Immunol. 12:433-455, 1994). [00105] XenoMouse strains are genetically engineered mice in which the murine IgH and Igk loci have been functionally replaced by their Ig counterparts on yeast artificial YAC transgenes. These human Ig transgenes can carry the majority of the human variable repertoire and can undergo class switching from IgM to IgG isotypes. The immune system of the xenomouse recognizes administered human antigens as foreign and produces a strong humoral response. The use of XenoMouse in conjunction with well-established hybridomas techniques, results in fully human IgG mAbs with sub-nanomolar affinities for human antigens (see U.S. Pat. Nos. 5,770,429, entitled "Transgenic non-human animals capable of producing heterologous antibodies"; 6,162,963, entitled "Generation of Xenogeneic antibodies"; 6,150,584, entitled "Human antibodies derived from immunized xenomice"; 6,114,598, entitled Generation of xenogeneic antibodies; and 6,075,181, entitled "Human antibodies derived from immunized xenomice"; for reviews, see Green, Antibody engineering via genetic engineering of the mouse: XenoMouse strains are a vehicle for the facile generation of therapeutic human monoclonal antibodies, J. Immunol. Methods 231 :11-23, 1999; Wells, Eek, a XenoMouse: Abgenix, Inc., Chem Biol 2000 August;7(8):R185-6; and Davis et al., Transgenic mice as a source of fully human antibodies for the treatment of cancer, Cancer Metastasis Rev 1999; 18(4):421-5). [00106] After synthesis, it is preferred that a composition or compound isolated or purified, preferably substantially purified. By "isolated" it is meant that the composition or compound has been separated from any molecule that interferes with the biological activity or plgR-targeting capacity thereof. As used herein the term "substantially purified" means at least about 95%, preferably at least about 99%, free of other components used to produce and/or modify the protein conjugate. The term "purified" refers to a composition or compound that has been separated from at least about 50% of undesirable elements. Techniques and methods for the separation and isolation of functional conjugates comprising sFv5A are used herein as non-limiting examples, but the techniques any be applied to any stalk-binding protein conjugate of the invention.

[00107] The purification of the sFv's and the conjugated material is achieved using any of the methods that are known by those skilled in the art to purify proteins, peptides, and macromolecules. Such methods include gel filtration, HPLC using ion exchange chromatography, immobilized metal affinity chromatography, hydrophobic interaction chromatography, selective precipitation, and crystallization.

[00108] Chromatography methods are selected for their ability to remove unreacted reagents, including unreacted derivatized proteins, peptides, and macromolecules and unreacted plgR binding ligands. Chromatography methods are also selected for their ability to separate conjugates having different molar rations or protein, peptide, or macromolecule to plgR binding ligands. Such conjugates are often referred to as l-'mers (1 :1 conjugates), 2-'mers (2:1 conjugates), 3-'mers (3:1 conjugates), etc. The production of different 'mers is a function of the number of reactive groups present on each molecule incubated in the conjugation mixture.

[00109] Optional protein elements can be incorporated into a chimera, which may be a compound of the invention or a member of a protein conjugate of the invention, or which may be comprised in a composition of the invention, and used during its purification and/or preparation. For example, as is discussed in more detail above, a protein member may include a protein purification element such as, for example, a "His tag" (His6). A His- tagged protein member or conjugate thereof can be isolated, or at least partially purified, from a composition that further comprises undesirable compounds by contacting the composition with a column of nickel immobilized on a metal -binding matrix. The His- tagged protein member or conjugate will bind to the nickel column and will thus be retained in the column; undesirable compounds pass through the column. As is explained above in more detail, various methods may be used to remove the protein purification element from the protein member or conjugate after such steps. Further optional elements include those discussed in the sections on components of the chimeras, above.

[00110] Post-translational modifications to a polypeptide may be created in vitro or in vivo. Various chemical treatments can be used for in vitro modifications of pure or semi- pure proteins; whereas in vivo modifications result from the choice of expression system and host cells. Post-translational modifications include, by way of non-limiting example, glycosylation, cleavage, phophorylation, cross-linking, formation or reduction of disulfide bridges, and the like.

[00111] Polypeptides that contain plgR-derived amino acid sequences that are identical or similar to the epitopes to which sFv molecules that bind plgR are prepared according to known methods. The epitope-containing polypeptides are covalently coupled to thiol Sepharose (activated thio Sepharose 4B contains a thiol group to which peptides may be attached covalently). A thiol containing peptide is reacted with Ellman's reagent (DTNB) to form a mixed disulfide. The TNB-peptide is separated from 2-nitro-5- thiobenzoic acid by gel sizing column chromatography. The TNB-peptide is reacted with thiol Sepharose to form a mixed disulfide of the peptide covalently bound to the resin. [00112] As another example, a maleimido group is placed at the amino or carboxyl terminal of the peptide. The maleimido group on the peptide is reacted with thiol Sepharose to form a thioether bond. Alternatively, the epitope-containing polypeptides are covalently coupled to activated supports that react with primary amines present on the polypeptide. Such supports include cross-linked agarose or acrylic matrices that have functional groups such as N-hydroxysuccinimide. These activated supports includeAffi-Gel 10 (Bio-Rad), Affi-Gel 15 (Bio-Rad), Affi-Prep 10 (Bio-Rad) and NHS-activated Sepharose 4 Fast Flow (Pharmacia). Immobilization of the polypeptide may also be performed with epoxy- activated matrices such as Epoxy-activated Sepharose 6B (Pharmacia) or cyanogen bromide-activated matrices such as CnBr-activated Sepharose 4 Fast Flow (Pharmacia). [00113] The peptide-Sepharose resin is used to bind an sFv, or other antibody derivative that binds the epitope in plgR that is recognized by the antibody, or a conjugate comprising such an antibody. Depending on the epitope to which the sFv binds in plgR, the amino acid sequence may be modified to provide the epitope in an amino acid sequence that inlcudes a residue that may be covalently linked to thiol Sepharose.

[00114] In the case of sFv5 and its derivatives (sFv5AF and sFv5AF-Cys), the amino acid sequence of the epitope in plgR is known, see U.S. patent application publication no. 2002/0102657, entitled "Ligands Directed To The Non-Secretory Component, Non-Stalk Region of plgR and Methods of Use Thereof filed Mar. 26, 2001 by Mostov et al. The amino acid sequence is, at a minimum, DPRLF. The maximum epitope amino acid sequence is QDPRLF in human and LDPRLF, which suggests that the most amino-terminal residue in the epitope sequence is not required for binding to sFv5. [00115] After the sFv or conjugate has been applied to the column, unreactive material is washed through the column. The sFv's, or conjugates comprising sFv's, remain attached to the column through specific interaction with the peptide. The specifically bound sFv or conjugate is separated from the column by low pH (pH 3 to 4) treatment for a brief time (preferably less than 15 minutes and preferably less than 5 minutes), by passing free peptide over the column, by reducing the covalently bound peptide with DTT or mercaptoethanol or by high concentrations OfMgCl₂, for example 3.0 M MgCl₂. When using a free peptide to obtain elution of the sFv or conjugate, the peptide need only contain the epitope to which the sFv binds or it may contain the same peptide sequence (without the cysteine) used to conjugate to the resin.

[00116] For maleimide conjugated peptide to the thiol Sepharose resin, reduction will not release the peptide:sFv or conjugate complex. Therefore, elution with free peptide or low pH may be used.

[00117] The sequence within the epitope may be varied such that the interaction is weakened compared to the native epitope. By substituting different amino acids within the sequence, a weaker binding peptide sequence may be identified. Weak binding to the immobilized peptide on thiol Sepharose is used to obtain some retention of sFv and conjugates on the column and to allow nonbinding components to pass straight through the column without binding. Therefore, no strenuous conditions may be required for elution and free peptide may not be required for elution. Tribbick et al. (J. Immunol. Methods 139: 155- 166, 1991) have described a similar approach. A weak binding peptide epitope is identified by performing alanine scans on the epitope to identify the amino acid side chains that provide most of the binding specificity and strength.

[00118] A peptide epitope is identified using a set of peptides designed to explore all of the binding regions of a protein, a general net. All overlapping peptides of a defined length, homologous with the protein, are synthesised. The offset is set from 1 to 5 residues, and preferably 3 to 4 residues in the first trials. The peptides should be sufficiently long so as not to miss an epitope by "dividing if between two peptides in the nested set. The peptides should be preferably 8 to 12 amino acids in length and preferably 10 to 15 amino acids in length. The boundaries of the epitope may be more precisely identified using a process that examines the linear sequence of the protein through a series of moving windows of a different size—a window net. The contributions of each amino acid side chain in the epitope are estimated by substituting each amino acid position in the epitope with all of the other 19 amino acids and determining the effect on the binding characteristics of the sFv to the peptide— a replacement net. Such strategies are described by Geysen et al. (MoI. hnmunol. 23: 7090715, 1986), Geysen et al. (J. Immunol. Methods 102: 259-274, 1987), Tribbick et al. (J. Immunol. Methods 139: 155-166, 1991), and Geysen et al. (J. MoI. Recog. 1 : 32-41, 1988).

[00119] In ion exchange chromatography, charged substances are separated via column materials that carry a charge. In cation exchange, the solid phase carries a negative charge whereas, in anion exchange, the stationary phase carries a positive charge. The solid phase of the columns is composed of ionic groups that are covalently bound to a gel matrix. Before a sample is passed through the column, the ionic charges in the solid phase are compensated by small concentrations of counter-ions present in the column buffer. When a sample is added to the column, an exchange occurs between the weakly bound counter-ions in the column buffer and more strongly bound ions present in the sample. Bound molecules do not elute from the column until a solution of varying pH or ionic strength is passed through the column. If desired, the degree of separation may be improved by a change in the gradient slope. If a compound of interest does not bind to the column under the selected conditions, the concentration and/or the pH value of the starting buffer can be changed. [00120] Ion chromatography of polypeptides occurs because polypeptides are multivalant anions or cations. Under strongly basic conditions, polypeptides are anions because the amino group is a free base and the carboxy group is dissociated. Under strongly acidic conditions polypeptides are cations as a result of suppression of the dissociation of the carboxy group and protonation of the amino group. Due to the net charge of the polypeptides it is possible to bind them to a corresponding charged stationary phase as long as the salt concentration is kept low.

[00121] Various ion-exchange resins, cellulose derivatives and large-pore gels are available for chromatographic use. Ion-exchange materials are generally water insoluble polymers containing cationic or anionic groups. Non-limiting examples of cation exchange matrices have anionic functional groups such as ~SO₃.sup.-, -OPO₃ ^" and —COO^", and anion exchange matrices may contain the cationic tertiary and quaternary ammonium groups having the general formulae -NHR^{+4^} and -NR⁺^. Proteins become bound by exchange with the associated counter-ions.

[00122] For reviews of ion-exchange chromatography, see Bollag, Ion-exchange chromatography, Methods MoI Biol 36:11-22, 1994; Holthuis et al., Chromatographic techniques for the characterization of proteins, Pharm Biotechnol 7:243-99, 1995; and Kent, Purification of antibodies using ion-exchange chromatography, Methods MoI Biol 115:19- 22, 1999.

[00123] Separation of polypeptides and other compounds by hydrophobic interaction chromatography (HIC) is based on the hydrophobicity of the compounds presented to the solvents. HIC separates compounds by mechanisms similar to reversed-phase chromatography (RPC) but under gentle reverse salt gradient elution conditions in aqueous buffers. Because no organic solvent is used in HIC, the biological activity of polypeptides and other compounds is more likely to be retained.

[00124] HIC involves sequential adsorption and desorption of protein from solid matrices mediated through non-covalent hydrophobic bonding. Generally, sample molecules in a high salt buffer are loaded on the HIC column. The salt in the buffer interacts with water molecules to reduce the solvation of the molecules in solution, thereby exposing hydrophobic regions in the sample molecules which are consequently adsorbed by the HIC column. The more hydrophobic the compound, the less salt needed to promote binding. A decreasing salt gradient may be used to elute samples from the column. As the ionic strength decreases, the exposure of the hydrophilic regions of the molecules increases, and compounds elute from the column in order of increasing hydrophobicity. Sample elution may also be achieved by the addition of mild organic modifiers or detergents to the elution buffer. Non-limiting examples of HIC-immobilized functional groups that can function to separate compounds include octyl groups, such as those on Octyl Sepharose CL4B media from Pharmacia, and propyl groups, such as those on High Propyl media from Baker. Alkoxy, butyl, and isoamyl functional group resins may also be used. [00125] Hydrophilic interaction chromatography (HILIC) separates compounds by passing a hydrophobic or mostly organic mobile phase across a neutral hydrophilic stationary phase, causing solutes to elute in order of increasing hydrophilicity. For example, with neutral peptides one may use 15 mM ammonium formate and reverse organic conditions. Highly charged molecules require low amounts (e.g., 10 mM) of salt for ion suppression, and a slight perchlorate or sulfate gradient (in a high organic solvent concentration) to effect desorption. HILIC has been used successfully with phosphopeptides, crude extracts, peptide digests, membrane proteins, carbohydrates, histones, oligonucleotides and their antisense analogs, and polar lipids. [00126] In hydrophobic-interaction chromatography, compounds of relatively greater hydrophobicity are retained longer on the column relative to those compounds that are more hydrophilic. Conversely, using hydrophilic-interaction chromatography, hydrophilic compounds are retained longer on the column relative to those compounds that are more hydrophobic.

[00127] For reviews and exemplary uses of hydrophobic interaction chromatography

(HIC), see Ghosh, Separation of proteins using hydrophobic interaction membrane chromatography, J Chromatogr A 923(l-2):59-64, 2001; Queiroz et al., Hydrophobic interaction chromatography of proteins, J Biotechnol 87(2): 143-59, 2001 ; Arakawa et al., Solvent modulation in hydrophobic interaction chromatography, Biotechnol Appl Biochem 13(2):151-72, 1991; el Rassi et al., Reversed-phase and hydrophobic interaction chromatography of peptides and proteins, Bioprocess Technol 9:447-94, 1990; Kato, High- performance hydrophobic interaction chromatography of proteins, Adv Chromatogr 26:97- 115, 1987; Hjerten, Hydrophobic interaction chromatography of proteins, nucleic acids, viruses, and cells on noncharged amphiphilic gels, Methods Biochem Anal 27:89-108, 1981; Ochoa, Hydrophobic (interaction) chromatography, Biochimie 60(1): 1-15, 1978; and in Protein Purification, 2d Ed., Springer- Verlag, New York, pgs 176-179 (1988). [00128] For reviews and exemplary uses of hydrophilic interaction chromatography .

(HILIC), see Zhu et al., Hydrophilic-interaction chromatography of peptides on hydrophilic and strong cation-exchange columns, J Chromatogr 548(1-2): 13-24, 1991; Olsen, Hydrophilic interaction chromatography using amino and silica columns for the determination of polar pharmaceuticals and impurities, J Chromatogr A. 913(1 -2): 113-22, 2001 ; Olsen, Hydrophilic interaction chromatography using amino and silica columns for the determination of polar pharmaceuticals and impurities, J Chromatogr A. 913(1 -2): 113- 22, 2001; and Alpert et al., Hydrophilic-interaction chromatography of complex carbohydrates, J Chromatogr A. 676(1): 191 -22, 1994; and Alpert, Hydrophilic-interaction chromatography for the separation of peptides, nucleic acids and other polar compounds, J Chromatogr. 499:177-96, 1990.

[00129] During or after the purification process, it is often desirable to monitor both the amount and biological activity of the composition, complex or compound being purified. The amount of the composition or compound can be detected by using antibodies directed to an epitope thereof. Additionally or alternatively, a composition or compound of the invention may comprise a detectable polypeptide by which the protein conjugate may be monitored.

[00130] Some of the biological activities of a composition or compound of the invention will vary depending on the nature of the biologically active polypeptide(s) included therein, and assays specific for the biological activities of the parent proteins are used. The compositions or compounds are also assayed for their ability to bind plgR and undergo various forms of cellular trafficking. Assays for these and plgR-related attributes are described herein and are applicable to any of the compositions or compounds of the invention.

[00131] Purity can be assessed by any suitable method, including HPLC analysis and staining of gels through which an aliquot of the preparation containing the protein conjugate has been electrophoresed. Those practiced in the art will know what degree of isolation or purification is appropriate for a given application. For example, (in the U.S. at least) biologicals do not have to meet the same standard of purity for, e.g., a compound. [00132] Hermanson (Bioconjugate Techniques, Academic Press, 1996), herein incorporated by reference, summarizes many of the chemical methods used to link proteins and other molecules together using various reactive functional groups present on various cross-linking or derivatizing reagents. Polypeptide cross-linking agents are based on reactive functional groups that modify and couple to amino acid side chains of proteins and peptides, as well as to other side groups and other macromolecules. Bifunctional cross- linking reagents incorporate two or more functional reactive groups. The functional reactive groups in a bifunctional cross-linking reagent may be the same (homobifunctional) or different (heterobifunctional). Many different cross-linkers are available to cross-link various proteins, peptides, and macromolecules. Table 7 lists some of the cross-linkers that are available through commercial sources according to their class of chemical reactivity. Table 8 lists some of the properties of chemical cross-linkers and the types of functional groups with which they react. [00133] Protein purification elements (a.k.a. protein isolation elements) are amino acid sequences that can be incorporated into a chimera in order to facilitate the purification or isolation of a chimera from a mixture containing other molecules. [00134] In order to achieve recombinant expression of a chimera, an expression cassette or construct capable of expressing a chimeric reading frame is introduced into an appropriate host cell to generate an expression system. The expression cassettes and constructs of the invention may be introduced into a recipient prokaryotic or eukaryotic cell either as a nonreplicating DNA or RNA molecule, which may either be a linear molecule or, more preferably, a closed covalent circular molecule. Since such molecules are incapable of autonomous replication, the expression of the gene may occur through the transient expression of the introduced sequence. Alternatively, permanent expression may occur through the integration of the introduced DNA sequence into the host chromosome. [00135] Host cells which may be used in the expression systems of the present invention are not strictly limited, provided that they are suitable for use in the expression of the chimeric plgR-targeting peptide of interest. Suitable hosts may often include eukaryotic cells. Preferred eukaryotic hosts include, for example, yeast, fungi, insect cells, mammalian cells either in vivo, or in tissue culture.

[00136] Expression cassettes and constructs may be introduced into an appropriate host cell by any of a variety of suitable means, i.e, transformation, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate-precipitation- , direct microinjection, and the like. After the introduction of the vector, recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells. Expression of the cloned gene(s) results in the production of a chimeric plgR-targeting peptide of the invention, or fragments thereof.

[00137] The introduced nucleic acid molecule can be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors may be employed for this purpose. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species. [00138] A variety of recombinant DNA expression systems may be used to produce the chimeras of the invention. Expression systems of particular interest include prokaryotic systems, yeast expression systems, insect expression systems mammalian expression systems. [00139] Prokaryotic Expression Systems utilize plasmid and viral (bacteriophage) expression vectors that contain replication sites and control sequences derived from a species compatible with the host may be used. Suitable phage or bacteriophage vectors may include λgtlO, λgtl 1 and the like; and suitable virus vectors may include pMAM-neo, pKRC and the like. Appropriate prokaryotic plasmid vectors include those capable of replication in E. coli (such as, by way of non-limiting example, pBR322, pUCl 18, pUCl 19, CoIEl, pSClOl, pACYC 184, πVX; "Molecular Cloning: A Laboratory Manual", 1989, supra). Bacillus plasmids include pC194, pC221, pT127, and the like (Gryczan, In: The Molecular Biology of the Bacilli, Academic Press, NY, pp. 307-329, 1982). Suitable Streptomyces plasmids include plJlOl (Kendall et al., J. Bacteriol. 169:4177-4183, 1987), and streptomyces bacteriophages such as ΦC31 (Chater et al., In: Sixth International Symposium on Actinomycetales Biology, Akademiai Kaido, Budapest, Hungary, pp. 45-54, 1986). Pseudomonas plasmids are reviewed by John et al. (Rev. Infect. Dis. 8:693-704, 1986), and Izaki (Jpn. J. Bacteriol. 33:729-742, 1978). See also Brent et al., "Vectors Derived From Plasmids," Section II, and Lech et al. "Vectors derived from Lambda and Related Bacteriophages" Section III, in Chapter 1 of Short Protocols in Molecular Biology, 2nd Ed., Ausubel et al., eds., John Wiley and Sons, New York, 1992, pages 1-13 to 1-27; Lech et al. "Vectors derived from Lambda and Related Bacteriophages" Section III and Id. pages 1-28 to page 1-52.

[00140] Recognized prokaryotic hosts include bacteria such as E. coli, Bacillus,

Streptomyces, Pseudomonas, Salmonella, Serratia, and the like. However, in such hosts, the chimera will not be glycosylated. In any event, the host cell must be compatible with the replicon and control sequences in the expression cassette.

[00141] To express a chimeric plgR-targeting peptide of the invention (or a functional derivative thereof) in a prokaryotic cell, it is necessary to operably link the sequence encoding the chimeric plgR-targeting peptide of the invention to a functional prokaryotic promoter. Such promoters may be either constitutive or, more preferably, regulatable (i.e, inducible or derepressible). Examples of constitutive promoters include the int promoter of bacteriophage λ, the bla promoter of the P-lactamase gene sequence of pBR322, and the cat promoter of the chloramphenicol acetyl transferase gene sequence of pPR325, and the like. Examples of inducible prokaryotic promoters include the major right and left promoters of bacteriophage λ (PL and PR), the trp, recA, lacZ, lac, and gal promoters of E. coli, the a-amylase (Ulmanen et al., J. Bacteriol. 162:176-182, 1985) and promoters of B. subtilis (Gilman et al., Gene Sequence 32:1 1-20, 1984), the promoters of the bacteriophages of Bacillus (Gryczan, in: The Molecular Biology of the Bacilli, Academic Press, Inc., NY, 1982), and Streptomyces promoters (Ward et al., MoI. Gen. Genet. 203:468-478, 1986). Prokaryotic promoters are reviewed by Glick (Ind. Microbiot. 1 :277-282, 1987), Cenatiempo (Biochimie 68:505-516, 1986), and Gottesman (Ann. Rev. Genet. 18:415-442, 1984).

[00142] Proper expression in a prokaryotic cell also requires the presence of a ribosome-binding site upstream of the gene sequence-encoding sequence. Such ribosome- binding sites are disclosed, for example, by Gold et al. (Ann. Rev. Microbiol. 35:365-404, 1981). The selection of control sequences, expression vectors, transformation methods, and the like, are dependent on the type of host cell used to express the gene. As used herein, "cell", "cell line", and "cell culture" may be used interchangeably and all such designations include progeny. Thus, the words "transformants" or "transformed cells" include the primary subject cell and cultures derived therefrom, without regard to the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. However, as defined, mutant progeny have the same functionality as that of the originally transformed cell. [00143] Bacterial systems may also be used to create and produce large amounts of shuttle vectors. Shuttle vectors are constructs designed to replicate in a prokaryotic host such as E. coli but which contain sequences that allow the shuttle vector and a chimeric reading frame incorporated therein to be transferred to a eukaryotic viral vector or other vector such as baculovirus or adenovirus.

[00144] Yeast Expression Systems can be utilized which incorporate promoter and termination elements from the actively expressed sequences coding for glycolytic enzymes that are produced in large quantities when yeast are grown in mediums rich in glucose. Known glycolytic gene sequences can also provide very efficient transcriptional control signals. Yeast cells provide a substantial advantage over prokarytoic expression systems in that they can carry out post-translational modifications of chimeras. A number of recombinant DNA strategies exist utilizing strong promoter sequences and high copy number plasmids which can be utilized for production of the desired proteins in yeast. Yeast recognizes leader sequences on cloned mammalian genes and secretes peptides bearing leader sequences (i.e, pre-peptides).

[00145] Preferred yeast expression vectors include those derived from the episomal element known as the 2-micron circle as well as derivatives of yeast integrating (YIp), yeast replicating (YRp), yeast centromeric (YCp), yeast episomal (YEp), and yeast linear (YLp) plasmids (Broach, in: The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y., p. 445-470, 1981; Lundblad et al., Section II and, Becker et al., Section III, of Chapter 13 in: Short Protocols in Molecular Biology, 2nd Ed., Ausubel et al., eds., John Wiley and Sons, New York, 1992, pages 13-19 to 13-41).

[00146] Insect Expression Systems utilize insect host cells, e.g., Sf9 and Sf21 cells, both of which are derived from the iplbsf-21 cell line derive from the pupal ovarian tissue of the fall army worm spodoptera frugiperda (O'Reilly et al., Baculovirus expression vectors: A Laboratory Manual New York, N. Y., W. H. Freeman and Company. See also baculovirus expression protocols in Methods in Molecular Biology Vol. 39; Richardson ed. Humana Totowa N.J., 1992; and Vaughn et al., In vitro 13:213-217, 1977. The cell line bti-tn-5bl-4 (high 5 tm cell line), which originated from the ovarian cells of the cabbage luper, Trichoplusa ni (Davis et al., Biotechnology 10:1148-1150, 1992; Granados et al., J.Invertebr. Pathol. 64:260-266, 1994; Wickham et al., Biotechnology Prog. 8:391-396, 1992; Wickham et al., Biotechnol. Prog. 9:25-30, 1993). Other insect cell lines that can be used to express baculovirus vectors have been described (Hink et al., Biotechnol. Prog. 7:9- 14, 1991). See, also Piwnica- Worms "Expression of Proteins in Insect Cells Using Baculo Viral Vectors" section II in chapter 16 of Short Protocols in Molecular Biology, second edition, Ausubel et al, eds., John Wiley and Sons, New York, N. Y. 1992. Using insect cells as hosts, the Drosophila alcohol dehydrogenase promoter can be used (Rubin, Science 240:1453-1459, 1988). Alternatively, baculovirus vectors can be engineered to express large amounts of chimeric plgR-targeting peptides of the invention in insect cells (Jasny, Science 238:1653, 1987; Miller et al., in: Genetic Engineering, Vol. 8, Plenum, Setlow et al., eds., pp. 277-297, 1986).

[00147] Mammalian Expression Systems utilize host cells such as HeLa cells, cells of fibroblast origin such as VERO, CV-I monkey kidney cells and COS-I (CV-I cells transformed with large T antigen) or CHO-KI, or cells of lymphoid origin and their derivatives. Preferred mammalian host cells include SP2/0 and J558L, as well as neuroblastoma cell lines such as INR 332, which may provide better capacities for correct post-translational processing.

[00148] Several expression vectors are available for the expression of chimeric plgR- targeting peptides of the invention in a mammalian host. A wide variety of transcriptional and translational regulatory sequences may be employed, depending upon the nature of the host. The transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, cytomegalovirus, simian virus, or the like, where the regulatory signals are associated with a particular gene sequence which has a high level of expression. Alternatively, promoters from mammalian expression products, such as actin, collagen, myosin, and the like, may be employed. Transcriptional initiation regulatory signals may be selected which allow for repression or activation, so that expression of the gene sequences can be modulated. Of interest are regulatory signals which are temperature-sensitive so that by varying the temperature, expression can be repressed or initiated, or are subject to chemical (such as metabolite) regulation.

[00149] Preferred eukaryotic plasmids include, for example, BPV, vaccinia, SV40, 2- micron circle, and the like, or their derivatives. Such plasmids are well known in the art (Botstein et al., Miami Wntr. Symp. 19:265-274, 1982; Broach, in: The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., p. 445-470, 1981; Broach, Cell 28:203-204, 1982; Bollon et al., J. Clin. Hematol. Oncol. 10:39-48, 1980; Maniatis, In: Cell Biology: A Comprehensive Treatise, Vol. 3, Gene Sequence Expression, Academic Press, NY, pp. 563-608, 1980). [00150] Expression of chimeric plgR-targeting peptides of the invention in eukaryotic hosts requires the use of eukaryotic regulatory regions. Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis. Preferred eukaryotic promoters include, for example, the promoter of the mouse metallothionein I gene sequence (Hamer et al., J. MoI. Appl. Gen. 1:273-288, 1982); the TK promoter of Herpes virus (McKnight, Cell 31 :355-365, 1982); the SV40 early promoter (Benoist et al., Nature (London) 290:304-31, 1981); and the yeast gal4 gene sequence promoter (Johnston et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975, 1982; Silver et al., Proc. Natl. Acad. Sci. (USA) 81 :5951-5955, 1984).]

[00151] Translation of eukaryotic mRNA is initiated at the codon which encodes the first methionine. For this reason, it is preferable to ensure that the linkage between a eukaryotic promoter and a DNA sequence which encodes a chimeric plgR-targeting peptide of the invention (or a functional derivative thereof) does not contain any intervening codons which are capable of encoding a methionine (i.e, AUG). The presence of such codons results either in the formation of a chimera (if the AUG codon is in the same reading frame as the chimeric plgR-targeting peptide of the invention coding sequence) or a frame-shift mutation (if the AUG codon is not in the same reading frame as the chimeric plgR-targeting peptide of the invention coding sequence).

[00152] Protein purification elements also include secretion sequences that direct recombinantly produced proteins out of the host cell and into the cellular media. Secreted proteins can then be separated from the host cells that produce them by simply collecting the media. Examples of secretion elements include those described in U.S. Pat. Nos. 5,846,818; 5,212,070; 5,631,144; 5,629,172; and 6,103,495; and Hardig et al., J. Biol. Chem. 268:3033-3036, 1993; Sizmannetal.,YearImmunol. 7:119-130, 1993; and Power et al., Gene 113:95-99, 1992). Protein purification elements also include sequences that direct a recombinant protein to the periplasmic space of bacteria (Battistoni et al., Protein Expr. Purif. 6:579-587, 1995). Those skilled in the art will be able to determine which purification elements are desired, appropriate or necessary for a given chimera and/or expression system.

[00153] Of particular interest are purification elements that can be used to isolate a chimera from the host cells or media of an expression system. Examples of purification elements include a "His tag" (6 contiguous His residues, a.k.a. 6xHis), which binds to surfaces that have been coated with nickel; streptavidin or avidin, which bind to surfaces that have been coated with biotin or "biotinylated" (see U.S. Pat. No. 4,839,293 and Airenne et al., Protein Expr. Purif. 17:139-145, 1999); and glutathione-s-transferase (GST), which binds glutathione (Kaplan et al., Protein Sci. 6:399-406, 1997; U.S. Pat. No. 5,654,176). Polypeptides that bind to lead ions have also been described (U.S. Pat. No. 6,111,079). "Epitope tags" such as the c-myc epitope or FLAG-tag can be used to purify recombinant proteins via affinity chromatography using antibodies to such epitope tags. [00154] As used herein, the term "protein purification element" also includes elements designed to enhance the solubility and or assist in the proper folding of a protein. Such elements include GST and members of the 14-3-3 family of proteins (U.S. Pat. No. 6,077,689).

5.6 Methods of Using the Chimeras

[00155] The chimeras can be used for any use apparent to those of skill in the art.

For instance, the chimeras can be used in vitro or ex vivo to modulate the activity of, lyse or kill a target cell. The target cell can be any cell that can be modulated, lysed or killed by the enzyme on which the chimera is based. For instance, where the chimera is a chimera of the bacteriophage gamma PIyG, the target cell can be any cell that can be modulated, lysed or killed by PIyG. Such target cells include B. anthracis cells and cells of certain strains of B. cereus, as discussed in the examples below. Further target cells include E. faecalis and E. faecium, for chimeras of PIyV 12, and pneumococcal cells for chimeras of CpI-I. Other target cells include those that can be modulated, lysed or killed by te lytic enzymes such as the autolysins, cell wall hydrolases, bacteriocins and colicins described above. [00156] In other aspects, the present invention provides in vivo use of the chimeras.

In certain embodiments, the chimeras can be used to treat or prevent an infection by one or more target cell. In further embodiments, the chimeras of the invention can be used to modulate, lyse or kill a target cell in vivo. The chimera can be used in any subject, preferably human subjects.

[00157] For use in the methods of the invention, the chimeras can be formulated and administered as described in detail in the sections below.

5.7 Pharmaceutical Compositions and Modes of Administration [00158] The present invention provides pharmaceutical compositions comprising the chimeras of the invention. Generally, the pharmaceutical compositions comprise a chimera of the invention and one or more pharmaceutically acceptable carriers, excipients or diluents. Preferred pharmaceutical compositions are described herein. [00159] According to the invention, a "composition" refers to a mixture comprising at least one carrier, preferably a physiologically acceptable carrier, and one or more compositions or compounds of the invention. The term "carrier" defines a chemical compound that does not inhibit or prevent the incorporation of the compositions or compounds into cells or tissues. A carrier typically is an inert substance that allows an active ingredient to be formulated or compounded into a suitable dosage form (e.g., a pill, a capsule, a gel, a film, a tablet, a microp article (e.g., a micro sphere), a solution; an ointment; a paste, an aerosol, a droplet, a colloid or an emulsion etc.). A "physiologically acceptable carrier" is a carrier suitable for use under physiological conditions that does not abrogate (reduce, inhibit, or prevent) the biological activity and properties of the composition or compound of the invention. For example, dimethyl sulfoxide (DMSO) is a carrier as it facilitates the uptake of many organic compounds into the cells or tissues of an organism. Preferably, the carrier is a physiologically acceptable carrier, preferably a pharmaceutically or veterinarily acceptable carrier, in which the composition or compound of the invention is disposed.

[00160] The chimera is included in the pharmaceutical composition in an amount sufficient to produce the desired effect upon the subject. The pharmaceutical compositions of the invention can further comprise other chemical components, such as diluents and excipients. A "diluent" is a chemical compound diluted in a solvent, preferably an aqueous solvent, that facilitates dissolution of the chimera in the solvent, and it may also serve to stabilize the biologically active form of the chimera or one or more of its components. Salts dissolved in buffered solutions are utilized as diluents in the art. For example, preferred diluents are buffered solutions containing one or more different salts. A preferred buffered solution is phosphate buffered saline (particularly in conjunction with compositions intended for pharmaceutical administration), as it mimics the salt conditions of human blood. Since buffer salts can control the pH of a solution at low concentrations, a buffered diluent rarely modifies the biological activity of a biologically active peptide. [00161] An "excipient" is any more or less inert substance that can be added to a composition in order to confer a suitable property, for example, a suitable consistency or to form a chimera. Suitable excipients and carriers include, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol cellulose preparations such as, for example, maize starch, wheat starch, rice starch, agar, pectin, xanthan gum, guar gum, locust bean gum, hyaluronic acid, casein potato starch, gelatin, gum tragacanth, polyacrylate, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents can also be included, such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Other suitable excipients and carriers include hydrogels, gellable hydrocolloids, and chitosan. Chitosan microspheres and microcapsules can be used as carriers. See WO 98/52547 (which describes microsphere formulations for targeting compounds to the stomach, the formulations comprising an inner core (optionally including a gelled hydrocolloid) containing one or more active ingredients, a membrane comprised of a water insoluble polymer (e.g., ethylcellulose) to control the release rate of the active ingredient(s), and an outer layer comprised of a bioadhesive cationic polymer, for example, a cationic polysaccharide, a cationic protein, and/or a synthetic cationic polymer; U.S. Pat. No. 4,895,724. Typically, chitosan is cross-linked using a suitable agent, for example, glutaraldehyde, glyoxal, epichlorohydrin, and succinaldehyde. Compositions employing chitosan as a carrier can be formulated into a variety of dosage forms, including pills, tablets, microparticles, and microspheres, including those providing for controlled release of the active ingredient(s). Other suitable bioadhesive cationic polymers include acidic gelatin, polygalactosamine, polyamino acids such as polylysine, polyhistidine, polyornithine, polyquaternary compounds, prolamine, polyimine, diethylaminoethyldextran (DEAE), DEAE-imine, DEAE-methacrylate, DEAE-acrylamide, DEAE-dextran, DEAE-cellulose, poly-p-aminostyrene, polyoxethane, copolymethacrylates, polyamidoamines, cationic starches, polyvinylpyridine, and polythiodiethylaminomethylethyl- ene. [00162] The chimeras of the invention can be formulated in any suitable manner. The compositions or compounds may be uniformly (homogeneously) or non-uniformly (heterogenously) dispersed in the carrier. Suitable formulations include dry and liquid formulations. Dry formulations include freeze dried and lyophilized powders, which are particularly well suited for aerosol delivery to the sinuses or lung, or for long term storage followed by reconstitution in a suitable diluent prior to administration. Other preferred dry formulations include those wherein a pharmaceutical composition according to the invention is compressed into tablet or pill form suitable for oral administration or compounded into a sustained release formulation. When the pharmaceutical composition is intended for oral administration but the composition or compound of the invention is to be delivered to epithelium in the intestines, it is preferred that the formulation be encapsulated with an enteric coating to protect the formulation and prevent premature release of the chimeras included therein. As those in the art will appreciate, the pharmaceutical compositions of the invention can be placed into any suitable dosage form. Pills and tablets represent some of such dosage forms. The pharmaceutical compositions can also be encapsulated into any suitable capsule or other coating material, for example, by compression, dipping, pan coating, spray drying, etc. Suitable capsules include those made from gelatin and starch. In turn, such capsules can be coated with one or more additional materials, for example, and enteric coating, if desired. Liquid formulations include aqueous formulations, gels, and emulsions.

[00163] Some preferred embodiments concern compositions that comprise a bioadhesive, preferably a mucoadhesive, coating. A "bioadhesive coating" is a coating that allows a chimera to adhere to a biological surface or substance better than occurs absent the coating. A "mucoadhesive coating" is a preferred bioadhesive coating that allows a substance, for example, a composition according to the invention, to adhere better to mucosa occurs absent the coating. For example, micronized particles (e.g., particles having a mean diameter of about 5, 10, 25, 50, or 100 μm) can be coated with a mucoadhesive. The coated particles can then be assembled into a dosage form suitable for delivery to an organism. Preferably, and depending upon the location where the cell surface transport moiety to be targeted is expressed, the dosage form is then coated with another coating to protect the formulation until it reaches the desired location, where the mucoadhesive enables the formulation to be retained while the compositions or compounds of the invention interact with the target cell surface transport moiety.

[00164] One pharmaceutical a composition of the invention is a pill, e.g., a capsule, tablet, caplet or the like, that is suitable for oral administration. Numerous capsule manufacturing, filling, and sealing systems are well-known in the art. Preferred capsule dosage forms can be prepared from gelatin and starch. Gelatin has been the traditional material, and the dosage forms are generally produced by well known dip molding techniques. After manufacture, gelatin capsules are filled with the desired composition and then sealed. A more recently developed alternative to gelatin dosage forms are capsules produced from starch. Starch capsules (typically made from potato starch) afford several advantages compared to gelatin capsules, including pH-independent dissolution, better suitability for enteric coating, water in the dosage form is tightly bound to the starch (and is thus less likely to migrate into the composition encapsulated in the dosage form), and the absence of animal-derived ingredients (which may be antigenic or contaminated with pathogens). Vilivilam, et al., PSTT 3:64-69, 2000). Starch capsules are odorless and rigid, and exhibit similar dissolution properties as compared to gelatin capsules. [00165] Capsules of any suitable size can be manufactured. Starch capsules are typically made in two pieces, a cap and a body, using injection molding techniques. See Eith et al., Manuf. Chem. 58: 21-25, 1987; Idrissi et al., Pharm. Acta. HeIv. 66: 246-252, 1991; Eith et al., Chimera Dev. Ind. Pharm. 12: 2113-2126, 1986. The two pieces are then sealed together during filling to prevent separation. Sealing can achieved by applying a hydroalcoholic solution to the inner surface of the cap.

[00166] After making the capsule dosage forms, if desired, they can be coated with one or more suitable materials. For example, when it is desired to deliver the encapsulated composition to the intestines, one or enteric coatings may be applied. Traditionally, enteric coatings were used to prevent gastric irritation, nausea, or to prevent the active ingredient from being destroyed by acid or gastric enzymes. However, these coatings can also be used to deliver agents to particular gastrointestinal regions.

[00167] A variety of enteric coatings are known in the art, and any suitable coating, or combinations of coatings, may be employed. Suitable coatings for starch capsules include aqueous dispersions of methacrylic acid copolymers and water-based reconstituted dispersion of cellulose acetate phthalate (CAP). See Brogmann et al., Pharm. Res. 1 :S-167; Vilivalam, et al., Pharm. Res. 14:S-659, 1999; Vilivalam et al., Pharm. Res. 15:S-645, 1998; Bums et al., Int. J. Pharm. 134: 223-230, 1996; Davis et al., Eur. J. Nucl. Med., 19: 971- 986, 1992. Indeed, a variety of coatings can be used to coat encapsulated dosage forms. These coatings include pH-sensitive materials, redox-sensitive materials, and materials that can be broken down by specific enzymes or microorganisms present in the intestine. Watts et al. (1995), WIPO publication WO35 100, reports an enteric -coated starch capsule system for targeting sites in the colon. The pH sensitive enteric coating begins to dissolve when the dosage form enters the small intestine, and coating thickness dictates in which region of the intestine the capsule disintegrates, for example, in the terminal ileum or in the ascending, transverse, or descending colon. Other coatings, or combinations of coatings, can also be used to achieve the same effect.

[00168] Chimeras can be administered parenterally or enterally. Enteral refers to the administration of the chimera into the gastrointestinal tract, preferable via oral administration. Parenteral administration is the administration of the chimera via any other route, e.g., intravenous injection directly into the bloodstream. In either-case, the goal of the chimera administration is to move the chimera from the site of administration to the site in the body where the chimera acts to produce its effect, or to administer a systemic therapeutically effective amount of the chimera.

[00169] Oral administration of chimeras is by far the most common method. When administered orally, chimera absorption usually occurs due to the transport across the membranes of the epithelial cells within the gastrointestinal tract. Absorption after oral administration is confounded by numerous factors that vary along the length of the gastrointestinal (GI) tract, including but not limited to the luminal pH, surface area per luminal volume, perfusion of tissue, bile and mucus flow, and the epithelial barrier. Pulmonary administration of chimeras, i.e., delivery via the respiratory system, is also known.

[00170] Although parenteral administration does provide a method for eliminating a number of the variables that are present with oral administration, parenteral administration is not a preferable route. This is because parenteral administration usually requires the use of medical personnel and is not practical for the administration of many chimeras. Even when required, parenteral administration is not preferred due to concerns such as subject discomfort, risk of infection, etc., as well as the equipment and costs involved. However, in some cases, despite various attempts, certain therapies require parenterally delivered chimeras. Such chimeras include polypeptides and other macromolecules that are degraded in the body, which occurs to a large degree in the GI tract. Despite such obstacles, it is desired to, for example, deliver insulin, growth hormones, interleukins, and monoclonal and other antibodies, by non-parenteral forms of administration. Epithelial barriers must be overcome to achieve non-parenteral routes of administration, such as oral and pulmonary administration.

[00171] In these and other routes of administration, a chimera must traverse several semipermeable cell membranes before reaching general circulation or their targeted site of action. These membranes act as a biological barrier that inhibits the passage of chimera molecules. In many instances, the barrier comprises epithelial cells and is thus an epithelial barrier. Epithelial barriers include, by way of non-limiting example, those that line the lumen of an organ. Epithelial barriers thus include, but are not limited to, surfaces that line the gastrointestinal lumen, the pulmonary lumen, the nasal lumen, the nasopharyngeal lumen, the pharyngeal lumen, the buccal lumen, the sublingual lumen, the vaginal lumen, a urogenital lumen, an ocular lumen, a tympanic lumen, and an ocular surface. [00172] The pharmaceutical compositions of the invention facilitate administration of chimeras to an organism, preferably an animal, preferably a mammal, bird, fish, insect, or arachnid. Preferred mammals include bovine, canine, equine, feline, ovine, and porcine animals, and non-human primates. Humans are particularly preferred. Multiple techniques of administering or delivering a compound exist in the art including, but not limited to, oral, rectal (e.g., an enema or suppository) aerosol (e.g., for nasal or pulmonary delivery), parenteral, and topical administration. Preferably, sufficient quantities of the composition or compound of the invention are delivered to achieve the intended effect. The particular amount of composition or compound to be delivered will depend on many factors, including the effect to be achieved, the type of organism to which the composition is delivered, delivery route, dosage regimen, and the age, health, and sex of the organism. As such, the particular dosage of a composition or compound of the invention included in a given formulation is left to the ordinarily skilled artisan's discretion. [00173] Those skilled in the art will appreciate that when the pharmaceutical compositions of the present invention are administered as agents to achieve a particular desired biological result, which may include a therapeutic or protective effect(s) (including vaccination), it may be necessary to combine the composition or compound of the invention with a suitable pharmaceutical carrier. The choice of pharmaceutical carrier and the preparation of the composition or compound as a therapeutic or protective agent will depend on the intended use and mode of administration. Suitable formulations and methods of administration of therapeutic agents include, but are not limited to, those for oral, pulmonary, nasal, buccal, ocular, dermal, rectal, or vaginal delivery. [00174] Depending on the mode of delivery employed, the context-dependent functional entity can be delivered in a variety of pharmaceutically acceptable forms. For example, the context-dependent functional entity can be delivered in the form of a solid, solution, emulsion, dispersion, micelle, liposome, and the like, incorporated into a pill, capsule, tablet, suppository, areosol, droplet, or spray. Pills, tablets, suppositories, areosols, powders, droplets, and sprays may have complex, multilayer structures and have a large range of sizes. Aerosols, powders, droplets, and sprays may range from small (1 micron) to large (200 micron) in size.

[00175] Pharmaceutical compositions of the present invention can be used in the form of a solid, a lyophilized powder, a solution, an emulsion, a dispersion, a micelle, a liposome, and the like, wherein the resulting composition contains one or more of the compounds of the present invention, as an active ingredient, in admixture with an organic or inorganic carrier or excipient suitable for enteral or parenteral applications. The active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, and any other form suitable for use. The carriers which can be used include glucose, lactose, mannose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea, medium chain length triglycerides, dextrans, and other carriers suitable for use in manufacturing preparations, in solid, semisolid, or liquid form. In addition auxiliary, stabilizing, thickening and coloring agents and perfumes may be used. Examples of a stabilizing dry agent includes triulose, preferably at concentrations of 0.1% or greater (See, e.g., U.S. Pat. No. 5,314,695). [00176] Although individual needs may vary, determination of optimal ranges for effective amounts of pharmaceutical compositions is within the skill of the art. Human doses can be extrapolated from animal studies (Katocs et al., Chapter 27 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990). Generally, the dosage required to provide an effective amount of a pharmaceutical composition, which can be adjusted by one skilled in the art, will vary depending on the age, health, physical condition, weight, type and extent of the disease or disorder of the recipient, frequency of treatment, the nature of concurrent therapy (if any) and the nature and scope of the desired effect(s). See, for example, Nies et al., Chapter 3 In: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al., eds., McGraw-Hill, New York, N. Y., 1996)

[00177] Dosing of therapeutic compositions is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of chimera accumulation in the body of the subject. The term "subject" is intended to encompass animals (e.g., cats, dogs and horses) as well as humans. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual therapeutic agents, and can generally be estimated based on EC50 found to be effective in in vitro and in vivo animal models.

[00178] The range of doses (the amount of chimera administered) is broad, since in general the efficacy of a therapeutic effect for different mammals varies widely with doses typically being 20, 30 or even 40 times smaller (per unit body weight) in man than in the rat. In general, dosage is from 0.01 ug to 100 g per kg of body weight, preferably 0.01 ug to 10 g/kg of body weight, 0.01 ug to 1000 mg/kg of body weight, 0.01 ug to 100 mg/kg of body weight, 0.01 ug to 10 mg/kg of body weight, 0.01 ug to 1 mg/kg of body weight, 0.01 ug to to 100 ug/kg of body weight, 0.01 ug to to 10 ug/kg of body weight, 0.01 ug to 1 ug/kg of body weight, 0.01 ug to 10 ug/kg of body weight, 0.01 ug to 1 ug/kg of body weight, 0.01 ug to 0.1 ug/kg of body weight, and ranges based on the boundaries of the preceding ranges of concentrations. Thus, for example, the preceding description of dosages encompasses dosages within the range of 100 to 1O g per kg of body weight, 10 g to 1000 mg/kg of body weight, 1000 mg to 100 mg, etc.

[00179] Doses may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the chimera in bodily fluids or tissues. Following successful treatment, it may be desirable to have the subject undergo maintenance therapy to prevent the recurrence of the disease state, wherein the therapeutic agent is administered in maintenance doses, ranging from 0.01 ug to 100 g per kg of body weight, once or more daily, to once every 20 years. Some chimeras, such as vaccines, may be administered once in a lifetime, or with booster shots only as circumstances warrant.

[00180] The specific dose is calculated according to the approximate body weight or surface area of the subject. Other factors in determining the appropriate dosage can include the disease or condition to be treated or prevented, the severity of the disease, the route of administration, and the age, sex and medical condition of the subject. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely made by those skilled in the art, especially in light of the dosage information and assays disclosed herein. The dosage can also be determined through the use of known assays for determining dosages used in conjunction with appropriate dose-response data. [00181] An individual subject's dosage can be adjusted as the progress of the disease is monitored. Blood levels of the chimera in a subject can be measured to see if the dosage needs to be adjusted to reach or maintain an effective concentration. Pharmacogenomics may be used to determine which chimeras and dosages thereof are most likely to be effective for a given individual (Schmitz et al., Clinica Chimica Acta 308:43-53, 2001; Steimer et al., Clinica Chimica Acta 308:33-41, 2001).

[00182] The pharmaceutical compositions of the invention facilitate administration of biologically active complexes and compounds to an organism, preferably an animal, preferably a mammal, bird, fish, insect, or arachnid. Preferred mammals include bovine, canine, equine, feline, ovine, and porcine animals, and non-human primates. Humans are particularly preferred. Multiple techniques of administering or delivering a pharmaceutical composition exist in the art including, but not limited to, oral, aerosol (e.g., for nasal or pulmonary delivery), parenteral, and topical administration. Preferably, a sufficient quantity of the biologically active complex or compound, or a bioactive portion or metabolite thereof, of the pharmaceutical composition is delivered to achieve the intended effect. The particular amount of the biologically active complex or compound to be delivered will depend on many factors, including the effect to be achieved, the type of organism to which the pharmaceutical composition is delivered, delivery route, dosage regimen, and the age, health, and sex of the organism. As such, the particular dosage of composition or compound of the invention included in a given formulation is left to the ordinarily skilled artisan's discretion.

[00183] The compositions and compounds of the invention are also useful in diagnostic and related applications. One aspect of the invention involves the diagnosis and monitoring of certain diseases, preferably in kit form. This aspect is useful for assaying and monitoring the course of the diagnosis and prognosis of disease, for monitoring the effectiveness and/or distribution of a therapeutic agent or an endogenous compound, in a subject as well as other related functions.

[00184] In this aspect of the invention, it may be desirable to monitor or determine if, or determine the degree to which, a subject's plgR-displaying cells are capable of, or presently are, endocytosing a detectably labeled composition or compound of the invention. Such methods are used in a variety of systems depending on the nature of the plgR-targeting element(s) of a given protein conjugate.

[00185] For example, the degree to which a subject, or a biological sample therefrom, endocytoses a composition or compound that has a plgR-targeting element derived from a bacterial protein that binds plgR is a measure of a subject's susceptibility to infection by bacteria having that element. A higher degree or rate of uptake of the detectable label indicates that the subject is more susceptible to such infection. [00186] As another example, the activity, distribution and/or concentration of endogenous plgR proteins may be altered in various ways during the course of a disease or disorder. The plgR proteins in a subject are measured over the course of a disease for diagnostic and prognostic purposes, as well as over the course of treatment of a disease or disorder, in order to monitor the effects on plgR proteins. Diseases to which this aspect of the invention can be applied include but are not limited to diseases that involve the respiratory system, such as lung cancer and tumors, asthma, pathogenic infections, allergy- related disorders, and the like; the gastrointestinal tract, including cancers, tumors, pathogenic infections, disorders relating to gastroinstestinal hormones, Chron's disease, eating disorders, and the like; and any disease or disorder that is known or suspected to involve plgR-displaying cells.

[00187] Compositions and compounds of the invention may be detectably labeled by virtue of comprising a detectable polypeptide such as, e.g., a green fluorescent protein (GFP) or a derivative thereof. If the protein conjugate comprises an epitope for which antibodies are available (including but not limited to commercially available ones such as c- myc epitope and the FLAG-tag), it may be detected using any of a variety of immunoassays such as enzyme-linked immunosorbent assay (ELISA) or a radioimmunoassay (RIA). [00188] Those skilled in the art are aware of pharmacological properties that influence the efficacy of chimeras, and how to determine parameters that reflect these properties. See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway, N. J., which is hereby incorporated by reference in its entirety. Some of pharmacological properties are as follows.

[00189] The absorption rate constant expresses the speed of chimera absorption.

Chimera absorption refers to the process of chimera movement from the site(s) of administration of the chimera into the body of an animal. Various factors, including the formulation of the chimera, influence the efficacy of rate of absorption of a chimera. For example, most orally administered chimeras are in the form of tablets or capsules, for reasons such as convenience, economy, stability, and subject acceptance and compliance. These capsules or tablets must disintegrate or dissolve before absorption of the chimera can occur. There are a variety of factors capable of varying or retarding disintegration of solid dosage forms, and effecting the dissolution rate, thereby determining the availability of the chimera for absorption.

[00190] The absorption of some chimeras is further influenced by factors that result from the consumption of food. For example, the presence of fiber or other substances in the GI tract may limit the absorption of chimeras, and the secretion of fluids that occur in response to ingestion or during digestion may also impact their absorption. Once such fluid is bile, which enhances absorption of many substances, including some chimeras. The release of digestive enzymes may be induced by ingestion, and these enzymes may effect the rate of dissolution of pills, tablets, and the like, and/or degrade the chimera. [00191] The bioavailability of a chimera is another pharmacological property.

Bioavailability is defined as the rate at which and the extent to which a chimera, or a biologically active metabolite or portion thereof, enters the general circulation and/or its targeted site of action. Bioavailability is influenced by a number of factors, including how the chimera product is designed and manufactured, its physicochemical properties, the rate at which the chimera is eliminated from the body, and factors that relate to the physiology and pathology of the subject. Reactions that compete with absorption can reduce bioavailability. They include complex formation (eg, between tetracycline and polyvalent metal ions), hydrolysis by gastric acid or digestive enzymes (e.g., penicillin and chloramphenicol palmitate hydrolysis), conjugation in the gut wall (e.g., sulfoconjugation of isoproterenol), adsorption to other chimeras (e.g., digoxin and cholestyramine), and metabolism by luminal microflora. Any of these factors can be changed to influence bioavailability, which is a pharmacological property that can be adjusted to achieve or enhance desirable attributes.

[00192] Assessment of bioavailability from plasma concentration-time data usually involves determining the maximum (peak) plasma chimera concentration, the time at which maximum plasma chimera concentration occurs (peak time), and the area under the plasma concentration-time curve (AUC). The plasma chimera concentration increases with the extent of absorption; the peak is reached when the chimera elimination rate equals absorption rate. Bioavailability determinations based on the peak plasma concentration can be misleading, because chimera elimination begins as soon as the chimera enters the bloodstream. The most widely used general index of absorption rate is peak time; the slower the absorption, the later the peak time. However, peak time is often not a good statistical measure because it is a discrete value that depends on frequency of blood sampling and, in the case of relatively flat concentrations near the peak, on assay reproducibility. AUC is a more reliable measure of bioavailability, as it is directly proportional to the total amount of unchanged chimera that reaches the systemic circulation.

[00193] The rate of elimination of a chimera from the body varies and effects its efficacy. A higher rate of elimination corresponds to decreased bioavailability. Thus, lower rates of elimination are generally preferred, although higher rates may be preferable for chimeras having undesirable effects, such as toxicity. One parameter relating elimination rate to plasma concentration is total clearance, which equals renal clearance plus extrarenal (metabolic) clearance. The elimination rate constant is a function of how a chimera is cleared from the blood by the eliminating organs and how the chimera distributes throughout the body. Another factor relating to elimination is the fraction excreted unchanged, which reflects the amount of chimera that is excreted relative to the amount that is metabolized. A low fraction indicates that hepatic metabolism is the likely mechanism of elimination, whereas higher fractions indicate that renal excretion is the predominant form of chimera elimination. [00194] The rate of elimination is desirably increased or decreased depending on the nature and use of the chimera in question. Often, a decreased rate of elimination is desirable, as this increases bioavailability. However, in the case of some agents, an increased rate of elimination may be preferable. For example, not every molecule of a targeted chimera that is introduced will find its intended site of action, and it may be desirable to remove these molecules from the body before they cause an undesirable effect at some other site in the body. Within the scope of the invention are chimeras comprising elements that increase the bioavailability and/or extend the circulating half-life of the chimera.

[00195] Another pharmacological property involves the therapeutic index, which is a measure of the relative desirability of a chimera for the attaining of a particular therapeutic result. The therapeutic index is usually expressed as the ratio of the largest dose producing no toxic symptoms to the smallest dose that results in a desired therapeutic result. Higher therapeutic indicia are preferred and an index of <1 is unacceptable, except in the case of some terminal diseases.

[00196] Following oral administration, many chimeras are absorbed intact from the

GI tract and transported first via the portal system to the liver, where they undergo extensive metabolism. Such metabolism may deactivate or degrade the chimera, thus lowering or eliminating its biological activity, which in turn reduces bioavailability. Such processes, which typically but need not occur in the liver, are called first-pass effects. First-pass effects may so greatly limit the bioavailability of an orally administered chimera that alternative routes of administration must be employed in order to achieve a therapeutically effective dose of the chimera. Chimeras transported through epithelial tissues may bypass first-pass effects, which is a pharmacological property that is a desirable attribute. [00197] The half-life of a chimera is the time required for chimera concentration or the amount of chimera in the body to decrease by 50%. For most chimeras, half-life remains the same regardless of how much chimera is in the body, but there are exceptions (e.g., phenytoin, theophylline, and heparin). Generally, a higher half-life is preferred, as this reduces the amount and lowers the frequency of administration of the chimera necessary to achieve its intended therapeutic effect. However, there are times when a decreased half life is preferred, particularly when the chimera has undesirable side-effects, e.g., toxicity. [00198] After a dosage from is prepared, it is typically packaged in a suitable material. For pill or tablet dosage forms, the dosage forms may be packaged individually or bottled en masse. An example of individual packaging PVC-PVdC-AIu, where aluminum blisters are covered with PVC (polyvinyl chloride) coated with PVdC (polyvinylidene chloride) to improve water vapor and oxygen protection. Suitable bottling materials include tinted, transluscent, or opaque high density polyethylene.

[00199] Those skilled in the art will be able to use the preceding information to prepare appropriate formulations for the gastrointestinal delivery of the chimeras of the invention. Other related information is known in the art and may be utilized to prepare appropriate formulations for gastrointestinal delivery of the chimeras.

5.7.1 Pharmaceutical Compositions for Pulmonary Administration [00200] One aspect of the invention relates to an aerosol inhaler, or other medical device, for delivery of a monoclonal antibody. Such devices are useful for inhalation therapies based on the compositions and compounds of the invention. The term "inhalation therapy" refers to the delivery of a therapeutic agent, such as a chimera or a chimera of the invention, in an aerosol form to the respiratory tract (i.e, pulmonary delivery). For reviews, see Gonda (J. Pharm. 89:940-945, 2000); Byron et al. (J. Aerosol Med. 7:49-75, 1994; and Niven (Crit. Rev. Ther. Chimera Carrier Syst. 12:151-231, 1995). [00201] The compositions and compounds of the invention are formulated for pulmonary delivery, and incorporated into medical devices such as inhalers, according to the following considerations and criteria, as well as other considerations and criteria known to those skilled in the art. A practicioner of the art will be able to use the following information to prepare appropriate formulations and medical devices for pulmonary delivery of the compositions and compounds of the invention.

[00202] Inhalers comprising bioactive, particulary therapeutic, chimeras complexes and compoundds may be used to deliver them quickly, and via self-administration. Such medical devices can be used to treat chronic or acute disorders or disease where it is desired to deliver a chimera via an inhalation route and in a short period of time. Chronic attacks of a disorder or disease include, for example, asthma attacks. A non-limiting example of a chimera useful for treating asthma is the monoclonal antibody CDP 835. Other Mab's that may desirably be delivered via inhalation include without limitation BEC2, ABX-EGF, E25, Palivixumab, and the like.

[00203] Compositions and compounds that are intended to be used in inhalation therapy must be formulated into a composition that is appropriate for delivery via inhalation. Two formulations of therapeutic agents that are useful for inhalation therapy include those in the form of liquid particles and solid particles. The liquid formulations are generated by nebulizing solutions of the therapeutic agent. Solid particle formulations are either in the form of a powder suspended in a propellant which is administered from a metered dose inhaler, or simply as a powder that is administered from a dry powder inhaler. In the case of polypeptide therapeutic agents, solid particle aerosols can be made by lyophilizing the polypeptide from solution and then milling or grinding the lyophilized chimera to the desired particle size for pulmonary administration. [00204] Non-limiting examples of formulations of therapeutic agents, including proteins, for inhalation therapy are described in Bittner et al. (J. Microencapsul. 16:325-341, 1999; Flament et al. (Int. J. Pharm. 178:101-109, 1999); and Langenback et al. (Pediatr. Pulmonol. 27:124-129, 1999), and references cited therein. Non-limiting examples of inhalation formulations of proteins are described in U.S. Pat. Nos. 5,230,884; 5,354,562; 5,457,044; 5,888,477; 5,952,008; 5,970,973; 6,000,574; 6,051,551; 6,060,069; 6,085,753; and 6,121,247.

[00205] An "aerosol inhaler" or "inhaler" is a device by which a subject can actively breathe in a given dose of a therapeutic agent. A typical application for such a medical device is for the treatment of an acute asthma attack. Delivery of chimeras via inhalation, however, can be used for many other treatments including those described herein. For example, chimeras administered by inhalation may be taken up by cells lining the interior of the pulmonary system and be delivered into the body therefrom. In the present invention, chimeras that comprise a biologically active polypeptide and an appropriate plgR targeting polypeptide and, as a result of reverse transcytosis, will be delivered into the circulatory system of a subject.

[00206] Inhalers have long been used to deliver chimeras into a subject's lungs.

Typically, an inhaler provides a mixture of therapeutic agents and air or some other type of propellant gas. The formulation of the therapeutic agent is delivered into the subject when he or she inhales from a mouthpiece on the inhaler. In general aerosol delivery systems rely on a mixture of the therapeutic agent with one or more propellants, and optional inactive ingredients, to increase dispersion and stability of the therapeutic agent. Inhalation of the formulation can be by either the nose or mouth and often is self-administered. Because of the small volume of each dosage, the propellant generally evaporates simultaneously or shortly after delivery of the therapeutic agent.

[00207] Correct inhalation of an aerosol formulation may require good hand-breath coordination. In the case of some inhalers, delivery ideally proceeds in such a manner that a subject first exhales and then applies the device to his mouth and as he begins to inhale, triggers the action of the inhaler by activating an actuating element thereof. Upon such activation, the aerosol formulation consisting of a propellant and therapeutic agent present in the said propellant and distributed therein, passes from the inhaler through a nozzle into the respiratory system of the subject. Inhalation of the therapeutic formulation into the respiratory system can be via the nasal cavity, the bucal cavity, or both. As the subject actively inhales gases from these cavities the aerosol formulation is delivered to the lungs. Atomization and dispersion of the therapeutic formulation in an inhaler can be triggered electronically or mechanically.

[00208] In general, there are three types of inhalers that are used to deliver therapeutic agents during inhalation therapy: nebulizers, metered dose inhalers (MDIs) and dry powder inhalers (DPIs). Each of these types of inhaler may be used to deliver the chimeras of the invention.

[00209] Nebulizers are electrical devices that send a therapeutic composition directly into a subject's mouth by tube or, in children, by clear mask. Nebulizers require no hand- breath coordination. The prescribed amount of medicine is placed in the device, a tube in inserted into the mouth (or, in the case of children, a mask is placed the child's nose and mouth), and breathing commences normally until the therapeutic composition is depleted. [00210] Measured-dose inhalers (MDIs, a.k.a. metered dose inhaler) send a measured dose of a therapeutic composition into the mouth using a small amount of pressurized gas. In MDIs, a "spacer" may be placed between the chimera reservoir and the mouth to control the amount inhaled in a single application. The therapeutic composition into the spacer, which is then squeezed by the subject as he quickly inhales the composition. MDIs have recently fallen out of favor because the common MDI propellant chlorofluorocarbon (CFC) has been found to deplete the atmosphere's ozone layer, and there are international agreements to phase out the production and use of CFC.

[00211] Dry-powder inhalers (DPIs) provide a popular alternative to aerosol-based inhalers. DPIs have the advantage of not requiring a propellant. However, because they have no propellant, PDIs depend on the force of inhalation to get the therapeutic composition into the lungs. Children, people with severe asthma, and people suffering acute attacks may be unable to produce enough airflow to use dry-powder inhalers successfully. Nonetheless, DPIs are used in inhalation therapies involving the chimeras of the invention. [00212] Various types of inhalers for delivering therapeutic agents are known. By way of non-limiting examples, see U.S. Pat. Nos. 3,938,516; 4,627,432; 5,941,240; 6,116,239; 6,119,688; and 6,1 19,684. One example of a dry powder inhaler that is within the scope of the invention is the Diskhaler, which is described in U.S. Pat. No. 4,627,432. The Spiros inhaler, described in U.S. Pat. No. 5,921,237, is another dry powder inhaler that is within the scope of the invention. Other dry powder inhalers that are within the scope of the invention include but are not limited to those described in U.S. Pat. Nos. 6,012,454; 6,045,828; 6,055,980; 6,056,169; 6,116,237; and 6,116,238.

[00213] Those skilled in the art will be able to use the preceding information to prepare appropriate formulations and medical devices for pulmonary delivery of the molecules of the invention. Other necessary information is known in the art and may be utilized to prepare appropriate formulations and medical devices.

6. EXAMPLES

6.1 Example 1: Exemplary Chimeras of the Invention

[00214] Exemplary chimeras of the invention comprise a lytic enzyme, a targeting element and an optional linker linking the lytic enzyme to the targeting element. Preferred components of the chimeras are in the tables that follow. [00215] Preferred lytic enzymes include the lysin PIyG (SEQ ID NO: 1):

PIVG (SEO ID NO: !)

MEIQKKL VDPSKYGTKCPYTMKPKYITVHNTYNDAP AENEVSYMISNNNEVSFHIAVDDKKAIQGIPLERNAW ACGDGNGSGNRQSISVEICYSKSGGDRYYKAEDNAVD WRQLMSMYNIPIENVRTHQSWSGKYCPHRMLAEGR WGAFIQKVKNGNVATTSPTKQNIIQSGAFSPYETPDVMGALTSLKMTADFILQSDGLTYFISKPTSDAQLKAM KEYLDRKGWWYEVK

[00216] Preferred targeting elements include peptides, antibodies and fragments such as diabodies and single chain antibodies that recognize the stalk of plgR. Examples include the following peptides and single chain antibodies to the stalk of plgR:

sFyl (SEO ID NO^

QVQLQQSGGGWQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRD NAKNSLYLQMNSLRAEDTAVYYCARDTRGYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSSELT QDPAMSVALGQTVRITCQGDSLRKYHASWYQQKPGQAPVLVIYGKNERPSGIPERFSGSTSGDTASLTISGLQ AEDEADYYCHSRDSNADLWFGGGTKVTVLG sFv2 (SEO ID NO:4)

QVQLQQSGGGWQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRD NAKNSLYLQMNSLRAEDTAVYYCARDTRGYFDLWGRGTLVTVSSGGGGSSELTQDPAMSVALGQTVRITCQGD SLRKYHASWYQQKPGQAPVLVIYGKNERPSGIPERFSGSTSGDTASLTISGLQAEDEADYYCHSRDSNADLW FGGGTKVTVLG

PlgR Stalk Binding Peptide (SEO ID NO:5)

AGGWFCEDGYECGHMGT

sFvl is derived from the variable portions of a heavy chain and a light chain from an antibody to plgR linked by a 25 amino acid linker (underlined) having 5 repeats of the amino acid sequence GGGGS. sFv2 is derived from the variable portions of a heavy chain and a light chain from an antibody to plgR linked by a 5 amino acid linker (underlined) having the amino acid sequence GGGGS. The plgR stalk binding peptide is a peptide that is capable of binding the stalk of the polymeric immunoglobulin receptor, as discussed in the sections above.

[00217] Useful linkers for linking a lytic enzyme to a targeting element are described extensively in the sections above. The examples below use three relatively stable linkers and two linkers that can be cleaved by the enzyme elastase having the following sequences. Also useful in the chimeras of the invention are the following linkers that are sensitive to B. anthracis lethal factor:

Linker 1 (SEQ ID NO:6)

GGPR

Linker 2 (SEO ID NO:7)

GGGGS

Linker 3 (SEQ ID NO:8)

GGPPPPPPGG

Elastase Linker 1 (SEQ ID NO:9)

GAAPVG

Elastase Linker 2 (SEO ID NO: 10)

GAAPVGAAPVGGGGSG

Lethal Factor Linker 1 (SEQ ID NO:11)

MPKKKPTP IQLNPAPD

Lethal Factor Linker 2 (SEQ ID NO: 12)

ARRKPVLP ALTINPTI

Lethal Factor Linker 3 (SEQ ID NO: 13)

SKRKKDVR ISCMSKPP

Lethal Factor Linker 4 (SEQ ID NO: 14)

KKRNPGLK IPKEAFEQ

Lethal Factor Linker 5 (SEO ID NO: 15)

QGKRKALK LNFANPPF Lethal Factor Linker 6 CSEO ID NO: 16)

PPFKSTAR FTLNPNPT

Lethal Factor Linker 7 TSEO ID NO: 17)

QRPRPTLQ LPLANDGG

Lethal Factor Linker 8 (SEO ID NO: 18)

ARPRHMLG LPSTLFTP

Lethal Factor Linker 9 (SEO ID NO: 19)

XBBBXHX HXXXXXXX

Lethal Factor Linker 10 (SEO ID NO:20)

NIeKKKKVLP IQLNAATD

Lethal Factor Linker 11 (SEO ID NO:21)

NIePKKKPTP IQLNPAPD

Lethal Factor Linker 12 (SEQ ID NO:22)

MPKKKPTP IQLNPAPDGG

Lethal Factor Linker 13 (SEO ID NO:23)

NIeKKKKVLP IQLNLAATDKGG

Lethal Factor Linker 14 (SEQ ID NO:24)

NIeKKKKVLP IQLNAATDKGG

Cathepsin Linker 1 (SEQ ID NO:25)

TPFSALQ

[00218] In the tables above, the term "NIe" refers to the amino acid norleucine.

[00219] Also found in the examples below is a Saccharomyces cerevisiae alpha factor secretion signal peptide useful for secreting chimeras from yeast such as S. cerevisiae and Pichia pastoris. The signal peptide is below.

Signal Peptide (SEO ID NO:26)

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASI AAKEEGVSLEKRE [00220] In this example, the present invention provides chimeras comprising one of the targeting elements above linked to the lytic enzyme PIyG. The targeting element is linked to the amino terminus of PIyG either directly or by way of one of the linkers above. [00221] The chimera is produced from an expression vector encoding the chimera in an appropriate host cell. For secretion, the chimera can comprise the signal peptide, again linked directly or via one of the linkers above.

6.2 Example 2: A Chimera Comprising a Single Chain Antibody Directed to the Stalk of plgR and PIyG Lyse a Model for B. anthracis

[00222] This example provides a chimera comprising the lysin PIyG (SEQ ID NO: 1) having an sFv (SEQ ID NO:3) at its amino terminus linked via a linker.

[00223] The chimera was expressed from a vector comprising SEQ ID NO:27.

Nucleic Acid Encoding Chimera 2-1 CSEO ID NO:27)

GTTAAA

Chimera 2-1 (SEO ID NO:28)

QVQLQQSGGGWQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTI SRDNAKNSLYLQMNSLRAEDTAVYYCARDTRGYFDLWGRGTL VTVSSGGGGSGGGGSGGGGSGGGGSGGG GSSELTQDPAMSVALGQTVRITCQGDSLRKYHASWYQQKPGQAPVLVIYGKNERPSGIPERFSGSTSGDT ASLTISGLQAEDEAD YYCHSRDSNADLWFGGGTKVTVLGGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS

ME IQKKLVDPS KYGTKCPYTMKPKYI TVHNTYNDAPAENE VS YMI SNNNE VS FH I AVDDKKAIQGI PLER NAWACGDGNGSGNRQSISVEICYSKSGGDRYYKAEDNAVD WRQLMSMYNIPIENVRTHQSWSGKYCPHR MLAEGRWGAFIQKVKNGNVATTSPTKQNIIQSGAFSPYETPDVMGALTSLKMTADFILQSDGLTYFISKP TSDAQLKAMKEYLDRKGWWYEVK

[00224] The chimera 2-1 comprises a linker (underlined) having six repeats of the amino acid sequence GGGGS. [00225] Pichia cells are transformed with the above vectors according to standard techniques, and the chimeras were expressed with the EasySelect™ Pichia Expression Kit (Invitrogen catalog no. K 1740-01). Chimeras are purified by protein A chromatography and/or by ion exchange chromatography. Cellular supernatant is harvested and filtered and then applied to a Protein A column at room temperature. The column is washed with approximately five times the column bed volume of PBS and 10% glycerol to remove any nonspecifically bound proteins. A 20% linear gradient (four times bed volume) of PBS, 3M MgCl₂ and 10% glycerol is applied followed by a step gradient (five times bed volume) to 100% PBS, 3M MgCl₂ and 10% glycerol to elute the chimera. Volume = 5X bed volume. Fractions with the chimera are identified by SDS-PAGE and pooled. Buffer is exchanged in PBS, 10% glycerol to reduce volume and remove salt. Protein is quantitated by Coomassie method and verified by SDS-PAGE. Some chimeras are stored at -80° C prior to assays. [00226] The chimeras were assayed for the ability to lyse strain RSVFl of B. cereus used as a model for B. anthracis {see, e.g., Schuch et al, supra). A single colony of B. cereus RSFVl was grown in 3-5 mL BHI media at 30° C, 300 RPM overnight . The culture was then diluted by about 100 fold into about 7.5 mL BHI and grown for about 3 hours at 30° C, 300 RPM. Cells were harvested by centrifugation, washed with PBS and resuspended to an OD₆₀O of about 1.0 in 1.0 mL PBS.

[00227] About 100 μL of the cell suspension was added to about 100 μL of each solution of lysin or chimera in a 96 well plate. In some assays, serial dilutions of lysin or chimera were evaluated. The OD₆₅₀ of the wells of each plates were measured immediately (Spectramax Plus 384, Molecular Devices, at 37° C) and then every fifteen seconds over fifteen minutes with agitation between each reading. Units were defined as the inverse of the enzyme dilution that yields 50% OD loss over the 15 minute assay. Positive controls included 1 μg lysin in about 100 μL PBS.

[00228] 30 μg of chimera 2-1 produced nearly equivalent lytic activity to 0.5 - 1.0 μg of PIyG (FIG. 2A). Because of molecular weight differences between the chimera and PIyG lysin, 30 μg of the chimera is equivalent to about 15 μg of PIyG lysin. It is believed that some free lysin was in the sample of chimera 2-1, but not an amount sufficient to be responsible for a significant amount of the activity in FIG. 2A. Lytic activity was dependent on the amount of the chimera (5, 10, or 20 μg) in the assay as shown in FIG. 2B. Under the conditions of the assay, saturation with respect to the enzyme was achieved as there was essentially no difference in the rate of lysis using 2.5 or 5.0 μg of PIyG lysin. 6.3 Example 3: Chimeras Comprising a Single Chain Antibody Directed to the Stalk of plgR and PIyG via an Elastase Sensitive Linker Lyses a Model for B. anthracis

[00229] This example provides chimeras of the invention that were prepared, purified and activated by elastase to release active PIyG lysin. The chimeras comprised an sFv (SEQ ID NO:3) directed to the stalk of plgR linked to PIyG (SEQ ID NO:1). The linker comprised two or three elastase sensitive sites .

[00230] The chimera was prepared by recombinant expression from a vector comprising the nucleic acid sequences SEQ ID NOS:29 and 31.

Nucleic Acid Encoding Chimera 3-1 (SEQ ID NO:29)

CTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTC AGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGAC AACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGA

GTCCGGAATCCCAGAGCGATTCTCTGGGTCCACTAGTGGAGACACAGCTTCCTTGACCATCAGTGGGCTCCAG GCGGAAGATGAGGCTGACTATTACTGTCACTCCCGAGACTCTAATGCTGATCTTGTGGTGTTCGGCGGAGGGA CCAAGGTCACCGTCCTAGGTGGTGCTGCCCCAGTTGGTGCTGCCCCTGTTGGTATGGAAATCCAAAAAAAATT

TATAATGATGCTCCAGCTGAAAATGAAGTGAGTTACATGATTAGTAACAATAATGAGGTGTCGTTTCATATTG CAGTAGATGACAAGAAAGCGATTCAAGGTATTCCGTTGGAACGTAATGCATGGGCTTGCGGAGACGGCAATGG

ATCAATCCTGGTCAGGTAAATATTGTCCGCATAGAATGTTAGCTGAGGGAAGGTGGGGAGCATTCATTCAGAA GGTTAAGAATGGGAATGTGGCGACTACTAGTCCAACAAAACAAAACATCATCCAATCCGGAGCTTTCTCACCG

AGGTTGGTGGTATGAAGTTAAA

Chimera 3-1 (SEO ID NO:3(n

QVQLQQSGGGWQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTI SRDNAKNSLYLQMNSLRAEDTA WYCARDTRGYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGG GΞSELTQDPAMSVALGQTVRITCQGDSLRKYHASWYQQKPGQAPVLVIYGKNERPSGIPERFSGSTSGDT ASLTISGLQAEDEAD YYCHSRDSNADLWFGGGTKVTVLGGAAPVGAAPVGMEIQKKL VDPSKYGTKCPY TMKPKYITVHNTYNDAP AENEVSYMISNNNEVSFHIAVDDKKAIQGIPLERNAWACGDGNGSGNRQSISV EICYSKSGGDRYYKAEDNAVD WRQLMSMYNIPIENVRTHQSWSGKYCPHRMLAEGRWGAFIQKVKNGNV ATTSPTKQNIIQSGAFSPYETPDVMGALTSLKMTADFILQSDGLTYFISKPTSDAQLKAMKEYLDRKGWW YEVK

Nucleic Acid Encoding Chimera 3-2 TSEO ID NO:3n

CAGGTGCAGCTGCAGCAATCAGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCT CTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTC AGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGAC

CGGAGGCGGAGGGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGTGGCGGAGGATCCTCTGAGCTGACT GTCCGGAATCCCAGAGCGATTCTCTGGGTCCACTAGTGGAGACACAGCTTCCTTGACCATCAGTGGGCTCCAG GCGGAAGATGAGGCTGACTATTACTGTCACTCCCGAGACTCTAATGCTGATCTTGTGGTGTTCGGCGGAGGGA CCAAGGTCACCGTCCTAGGTGGTGCTGCCCCAGTTGGTGCTGCCCCTGTTGGTGCTGCCCCTGTTGGTATGGA AATCCAAAAAAAATTAGTTGATCCAAGTAAGTATGGTACCAAGTGTCCGTATACAATGAAGCCTAAATATATC ACTGTTCACAACACATATAATGATGCTCCAGCTGAAAATGAAGTGAGTTACATGATTAGTAACAATAATGAGG TGTCGTTTCATATTGCAGTAGATGACAAGAAAGCGATTCAAGGTATTCCGTTGGAACGTAATGCATGGGCTTG CGGAGACGGCAATGGTTCGGGGAATCGTCAATCCATTTCTGTAGAAATCTGTTATTCAAAATCAGGAGGAGAT AGATACTATAAAGCTGAGGATAATGCTGTTGATGTTGTACGACAACTTATGTCTATGTACAATATTCCGATTG AAAATGTTCGAACTCATCAATCCTGGTCAGGTAAATATTGTCCGCATAGAATGTTAGCTGAGGGAAGGTGGGG

ATACCTTGACCGTAAAGGTTGGTGGTATGAAGTTAAA

Chimera 3-2 (SEQ ID NO:32)

QVQLQQSGGGWQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTI SRDNAKNSLYLQMNSLRAEDTAVYYCARDTRGYFDLWGRGTL VTVSSGGGGSGGGGSGGGGSGGGGSGGG GSSELTQDPAMSVALGQTVRITCQGDSLRKYHASWYQQKPGQAP VLVIYGKNERPSGIPERFSGSTSGDT ASLTISGLQAEDEAD YYCHSRDSNADLWFGGGTKVTVLGGAAPVGAAPVGAAPVGMEIQKKL VDPSKYG TKCPYTMKPKYITVHNTYNDAP AENEVSYMISNNNEVSFHIAVDDKKAIQGIPLERNAWACGDGNGSGNR QSISVEICYSKΞGGDRYYKAEDNAVD VWQLMSMYNIPIENVRTHQSWSGKYCPHRMLAEGRWGAFIQKV KNGNVATTSPTKQNIIQSGAFSPYETPDVMGALTSLKMTADFILQSDGLTYFISKPTSDAQLKAMKEYLD RKGWWYEVK

[00231] The chimeras comprise linkers (underlined) between an sFv that binds polymeric immunoglobulin receptor stalk and PIyG. The linkers included two or three peptide sequences sensitive to human neutrophil elastase, an enzyme present in the lung. The orientation, therefore, was from amino terminus to carboxyl terminus sFv - elastase sensitive linker - lysin. The elastase sensitive sites were positioned so as to leave less than 4 residues at the amino terminus of lysin after elastase digestion. [00232] Chimeras 3-1 and 3-2 were expressed and purified by the ion exchange method according to the methods in the example above. Chimeras 3-1 and 3-2 were activated with human neutrophil elastase prior to assaying for activity against a model for B. anthracis. For activation, each chimera was contacted with 20- to 1000- fold human neutrophil elastase at 0.5 μg/μL and incubated at 37° for 10 minutes to 3 hours in a volume of about 50 μL 5OmM Tris, pH 5.5 20OmM NaCl. The chimeras were assayed before and after elastase activation for lysis of B. cereus strain RSFVl, used as a model for B. anthracis, according to the methods of Example 2.

[00233] The intact chimera did not lyse a significant number of B. cereus cells.

However, when digested with elastase and assayed for lytic activity, strong lytic activity was observed. FIG. 3 A illustrates activation of chimera by elastase. The lytic activity of the chimera was measured against B. cereus as shown in FIG. 3B. Because the plates were not shaken, the absorbance of the controls drifts down over the 20 minute assay. The activity of the intact chimera is less than an equivalent amount of PIyG lysin. When the chimera was treated briefly with human neutrophil elastase, its lytic activity was indistinguishable from an equivalent molar quantity of PIyG lysin or PIyG lysin treated with elastase. Within 3 minutes, base line was reached with all three of these incubations. [00234] Bacterial infections are known to recruit neutrophils to the site of the infection. Neutrophils release elastase at that site and elastase is present during inflammation of the lung. This example demonstrates that the chimeras of the invention can be activated by the human neutrophil elastase that is known to be present at the site of bacterial infections. Thus, the chimeras have utility as therapeutics that can be delivered, for example by inhalation, to the lung could as potent therapeutics for inhaled anthrax.

6.4 Example 4: Further Chimeras of PIyG

[00235] This example provides further chimeric Plyg molecules. The chimeras comprise one or more targeting elements linked to the amino terminus of PIyG as follows.

Nucleic Acid Encoding Chimera 4-1 (SEO ID NO:33)

AGTTAAA

Chimera 4-1 TSEO ID NO:34)

QVQLQQSGGGWQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTI SRDNAKNSLYLQMNSLRAEDTA WYCARDTRGYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGG GSSELTQDPAMSVALGQTVRITCQGDSLRKYHAS WYQQKPGQAPVLVIYGKNERPSGIPERFSGSTSGDT ASLTISGLQAEDEADYYCHSRDSNADLWFGGGTKVTVLGGAAPVGMEIQKKL VDPSKYGTKCPYTMKPK YITVHNTYND AP AENEVSYMISNNNEVSFHIAVDDKKAIQGIPLERNAWACGDGNGSGNRQSISVEICYS KSGGDRYYKAEDNAVD WRQLMSMYNIPIENVRTHQSWSGKYCPHRMLAEGRWGAFIQKVKNGNVATTSP TKQNIIQSGAFSPYETPDVMGALTSLKMTADFILQSDGLTYFISKPTSDAQLKAMKEYLDRKGWWYEVK

Nucleic Acid Encoding Chimera 4-2 CSEO ID NO: 35)

CAGGTGCAGCTGCAGCAATCAGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCT CTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTC AGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGAC AACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGA GAGATACCCGAGGGTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACCGTGAGCTCAGGAGGCGGAGGGTC CGGAGGCGGAGGGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGTGGCGGAGGATCCTCTGAGCTGACT CAGGACCCTGCTATGTCTGTGGCCTTGGGACAGACAGTCAGAATCACCTGTCAAGGGGACAGTCTCAGAAAGT ATCATGCAAGCTGGTATCAGCAGAAGCCAGGGCAGGCCCCTGTTCTTGTCATCTATGGTAAGAATGAACGGCC

CCAAGGTCACCGTCCTAGGTGGTGCTGCCCCAGTTGGTGCTGCCCCTGTTGGTGGCGGAGGGTCAGGTATGGA

TGTCGTTTCATATTGCAGTAGATGACAAGAAAGCGATTCAAGGTATTCCGTTGGAACGTAATGCATGGGCTTG

ATACCTTGACCGTAAAGGTTGGTGGTATGAAGTTAAA

Chimera4-2(SEQIDNO:36)

QVQLQQSGGGWQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTI SRDNAKNSLYLQMNSLRAEDTAVYYCARDTRGYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGG GSSELTQDPAMSVALGQTVRITCQGDSLRKYHASWYQQKPGQAPVLVIYGKNERPSGIPERFSGSTSGDT ASLTISGLQAEDEADYYCHSRDSNADLWFGGGTKVTVLGGAAPVGAAPVGGGGSGMEIQKKLVDPSKYG TKCPYTMKPKYITVHNTYNDAPAENEVSYMISNNNEVSFHIAVDDKKAIQGIPLERNAWACGDGNGSGNR QSISVEICYSKSGGDRYYKAEDNAVDWRQLMSMYNIPIENVRTHQSWSGKYCPHRMLAEGRWGAFIQKV KNGNVATTSPTKQNIIQSGAFSPYETPDVMGALTSLKMTADFILQSDGLTYFISKPTΞDAQLKAMKEYLD RKGWWYEVK

Nucleic Acid Encoding Chimera 4-3 CSEO ID NO: 37)

ACCGTAAAGGTTGGTGGTATGAAGTTAAA

Chimera4-3(SEQIDNO:38)

AGGWFCEDGYECGHMGTGGASEIQKKLVDPSKYGTKCPYTMKPKYITVHNTYNDAPAENEVSYMISNNNE VSFHIAVDDKKAIQGIPLERNAWACGDGNGSGNRQSISVEICYSKSGGDRYYKAEDNAVDWRQLMSMYN IPIENVRTHQSWSGKYCPHRMLAEGRWGAFIQKVKNGNVATTSPTKQNIIQSGAFSPYETPDVMGALTSL KMTADFILQSDGLTYFISKPTSDAQLKAMKEYLDRKGWWYEVK

Nucleic Acid Encoding Chimera 4-4 CSEO ID NO:39)

TAAGAATGGGAATGTGGCGACTACTAGTCCAACAAAACAAAACATCATCCAATCCGGAGCTTTCTCACCGTAT GAAACCCCTGATGTTATGGGAGCATTAACGTCACTTAAAATGACAGCTGATTTTATCTTACAATCGGATGGAT TAACTTATTTTATTTCCAAACCGACTTCAGATGCACAACTAAAAGCAATGAAAGAATACCTTGACCGTAAAGG TTGGTGGTATGAAGTTAAA

Chimera 4-4 fSEO ID NO -.40)

AGGWFCEDGYECGHMGTGGARAGGWFCEDGYECGHMGTGGASEIQKKLVDPSKYGTKCPYTMKPKYITVH NTYNDAPAENEVSYMISNNNEVSFHIAVDDKKAIQGIPLERNAWACGDGNGSGNRQSISVEICYSKSGGD RYYKAEDNAVD WRQLMSMYNIPIENVRTHQSWSGKYCPHRMLAEGRWGAFIQKVKNGNVATTSPTKQNI IQSGAFSPYETPDVMGALTSLKMTADFILQSDGLTYFISKPTSDAQLKAMKEYLDRKGWWYEVK

Nucleic Acid Encoding Chimera 4-5 fSEO ID N0:4n

GAAGTTAAA

Chimera 4-5 (SEQ ID NO:42)

AGGWFCEDGYECGHMGTGGARAGGWFCEDGYECGHMGTGGARAGGWFCEDGYECGHMGTGGASEIQKKLV DPSKYGTKCPYTMKPKYITVHNTYNDAPAENEVSYMISNNNEVSFHIAVDDKKAIQGIPLERNAWACGDG NGSGNRQSISVEICYSKSGGDRYYKAEDNAVDWRQLMSMYNIPIENVRTHQSWSGKYCPHRMLAEGRWG AFIQKVKNGNVATTSPTKQNIIQSGAFSPYETPDVMGALTSLKMTADFILQSDGLTYFISKPTSDAQLKA MKEYLDRKGWWYEVK

Nucleic Acid Encoding Chimera 4-6 CSEO ID NO:43)

Chimera 4-6 fSEO ID NO:44)

AGGWFCEDGYECGHMGTGGARAGGWFCEDGYECGHMGTGGARAGGWFCEDGYECGHMGTGGARAGGWFCE DGYECGHMGTGGASEIQKKLVDPSKYGTKCPYTMKPKYITVHNTYNDAPAENEVSYMISNNNEVSFHIAV DDKKAIQGIPLERNAWACGDGNGSGNRQSISVEICYSKSGGDRYYKAEDNAVDWRQLMSMYNIPIENVR THQSWSGKYCPHRMLAEGRWGAFIQKVKNGNVATTSPTKQNIIQSGAFSPYETPDVMGALTSLKMTADFI LQSDGLTYFISKPTSDAQLKAMKEYLDRKGWWYEVK

[00236] The above chimeras were prepared according to the methods of the previous examples. Chimera 4-1 comprises an sFv according to the previous examples linked to PIyG via an elastase sensitive linker (underlined). Chimera 4-2 comprises the sFv linked to PIyG via a longer linker comprising two elastase sensitive sites (underlined). [00237] Chimeras 4-3, 4-4, 4-5 and 4-6 comprise a peptide capable of binding the stalk of the polymeric immunoglobulin linker. The peptide has the sequence AGGWFCEDGYECGHMGT (SEQ ID NO:5), and is present in the chimeras in one, two, three or four copies, all linked by linking peptides GGAR or GGAS (underlined).

6.5 Example 5: Split Chimeras of PIyG

[00238] This example demonstrates exemplary split chimeras of the invention. These exemplary split chimeras retain the ability to lyse Bacillus cells while having one or more linkers between the domains of PIyG.

[00239] PIyG lysin comprises two domains, a catalytic or enzymatic domain and a cell wall binding domain. The two domains follow.

Catalytic Domain (SEO ID NO:45)

MEIQKKL VDPSKYGTKCPYTMKPKYITVHNTYNDAP AENEVSYMISNNNEVSFHIAVDDKKAIQGIPLERNAW ACGDGNGSGNRQSISVEICYSKSGGDRYYKAEDNAVD WRQLMSMYNIPIENVRTHQSWSGKYCPHRMLAEGR WGAFIQKVKNGNVATTS PT

Cell Wall Binding Domain (SEQ ID NO:46)

KQNIIQSGAFSPYETPDVMGALTSLKMTADFILQSDGLTYFISKPTSDAQLKAMKEYLDRKGWWYEVK

[00240] As discussed in the sections above, the present invention provides split chimeras wherein a moiety is inserted between the domains of a lytic enzyme such as PIyG. The moiety can be any moiety, including the linkers in the examples above.

6.6 Example 5: Split Chimeras of PIyG

[00241] This example demonstrates that the domains of a lytic enzyme such as PIyG can be split by a polypeptide moiety.

[00242] In this example, split chimera 5-1 was prepared from a vector comprising the following nucleic acid.

Nucleic Acid Encoding Chimera 5-1 (SEQ ID NO:47)

GCTTGCGGAGACGGCAATGGTTCGGGGAATCGTCAATCCATTTCTGTAGAAATCTGTTATTCAAAATCAGGAG

AA Chimera 5-1 fSEO ID NO:48)

MEIQKKLVDPSKYGTKCPYTMKPKYITVHNTYNDAPAENEVSYMISNNNEVSFHIAVDDKKAIQGIPLER NAWACGDGNGSGNRQSISVEICYSKSGGDRYYKAEDNAVDWRQLMSMYNIPIENVRTHQSWSGKYCPHR MLAEGRWGAFIQKVKNGNVATTTSPTGGPPPPPPGGKQNIIQSGAFSPYETPDVMGALTSLKMTADFILQ SDGLTYFISKPTSDAQLKAMKEYLDRKGWWYEVK

[00243] Split chimera 3-1 comprises a "rigid rod" of six proline residues between the enzymatic and cell wall binding domains to demonstrate that the two domains can be split without affecting lytic activity. The sequence of six consecutive prolines (hexaproline) forms a beta helix that mimics a rigid rod.

[00244] Insertion of the hexaproline rigid rod into the interdomain region did not change the lytic activity of the hexaproline split lysin. The lytic activity of PIyG and the modified lysin were essentially identical.

6.7 Example 6: Split Chimeras of PIyG with Lytic Activity

[00245] These exemplary split chimeras retain the ability to lyse Bacillus cells while having one or more linkers between the domains of PIyG. Some of these split chimeras retained activity comparable to native PIyG, while others showed reduced lytic activity.

Nucleic Acid Encoding Chimera 6-1 (SEQ ID NO:49)

TGAGGTGTCGTTTCATATTGCAGTAGATGACAAGAAAGCGATTCAAGGTATTCCGTTGGAACGTAATGCATGG GCTTGCGGAGACGGCAATGGTTCGGGGAATCGTCAATCCATTTCTGTAGAAATCTGTTATTCAAAATCAGGAG

GAGGTTGGTTTTGCGAAGATGGATACGAATGCGGACATATGGGTACAGGTGGAGGTGCTAGCAAACAAAATAT

CAATGAAAGAATACTTGGATAGAAAAGGCTGGTGGTACGAAGTAAAA

Chimera6-1 (SEOIDNO:5(T)

MEIQKKLVDPSKYGTKCPYTMKPKYITVHNTYNDAPAENEVSYMISNNNEVSFHIAVDDKKAIQGIPLER NAWACGDGNGSGNRQSISVEICYSKSGGDRYYKAEDNAVDWRQLMSMYNIPIENVRTHQSWSGKYCPHR MLAEGRWGAFIQKVKNGNVATTSPTGGPRAGGWFCEDGYECGHMGTGGGASKQNIIQSGAFSPYETPDVM GALTSLKMTADFILQSDGLTYFISKPTSDAQLKAMKEYLDRKGWWYEVK

Nucleic Acid Encoding Chimera 6-2 (SEQ ID NO:51)

TGAGGTGTCGTTTCATATTGCAGTAGATGACAAGAAAGCGATTCAAGGTATTCCGTTGGAACGTAATGCATGG GCTTGCGGAGACGGCAATGGTTCGGGGAATCGTCAATCCATTTCTGTAGAAATCTGTTATTCAAAATCAGGAG GAGATAGATACTATAAAGCTGAGGATAATGCTGTTGATGTTGTACGACAACTTATGTCTATGTACAATATTCC GATTGAAAATGTTCGAACACATCAATCATGGTCAGGAAAATATTGTCCACATAGAATGCTGGCTGAAGGAAGA TGGGGGGCTTTTATTCAAAAGGTAAAAAATGGTAACGTGGCTACTACTAGTCCAACAGGTGGACCTAGGGCAG

GTTTTGCGAAGATGGATACGAATGCGGACATATGGGTACAGGTGGAGGTGCTAGCAAACAAAATATAATACAA AGAATACTTGGATAGAAAAGGCTGGTGGTACGAAGTAAAA

Chimera 6-2 (SEO ID NO:52^

MEIQKKLVDPSKYGTKCPYTMKPKYITVHNTYNDAPAENEVSYMISNNNEVSFHIAVDDKKAIQGIPLER NAWACGDGNGSGNRQSISVEICYSKSGGDRYYKAEDNAVDWRQLMSMYNIPIENVRTHQSWSGKYCPHR MLAEGRWGAFIQKVKNGNVATTSPTGGPRAGGWFCEDGYECGHMGTGGGARAGGWFCEDGYECGHMGTGG GASKQNIIQSGAFSPYETPDVMGALTSLKMTADFILQSDGLTYFISKPTSDAQLKAMKEYLDRKGWWYEV K

Nucleic Acid Encoding Chimera 6-3 (SEQ ID NO:53)

TTGGATAGAAAAGGCTGGTGGTACGAAGTAAAA

Chimera 6-3 (SEQ ID NO:54)

MEIQKKLVDPSKYGTKCPYTMKPKYITVHNTYNDAPAENEVSYMISNNNEVSFHIAVDDKKAIQGIPLER NAWACGDGNGSGNRQSISVEICYSKSGGDRYYKAEDNAVDWRQLMSMYNIPIENVRTHQSWSGKYCPHR MLAEGRWGAFIQKVKNGNVATTSPTGGPRAGGWFCEDGYECGHMGTGGGARAGGWFCEDGYECGHMGTGG GARAGGWFCEDGYECGHMGTGGGASKQNIIQSGAFSPYETPDVMGALTSLKMTADFILQSDGLTYFISKP TSDAQLKAMKEYLDRKGWWYEVK

Nucleic Acid Encoding Chimera 6-4 (SEQ ID NO: 55)

GAAAAGGCTGGTGGTACGAAGTAAAA

Chimera6-4(SEOIDNO:56)

MEIQKKLVDPSKYGTKCPYTMKPKYITVHNTYNDAPAENEVSYMISNNNEVSFHIAVDDKKAIQGIPLER NAWACGDGNGSGNRQSISVEICYSKSGGDRYYKAEDNAVDWRQLMSMYNIPIENVRTHQSWSGKYCPHR MLAEGRWGAFIQKVKNGNVATTSPTGGPRAGGWFCEDGYECGHMGTGGGARAGGWFCEDGYECGHMGTGG GARAGGWFCEDGYECGHMGTGGGARAGGWFCEDGYECGHMGTGGGASKQNIIQSGAFSPYETPDVMGALT SLKMTADFILQSDGLTYFISKPTSDAQLKAMKEYLDRKGWWYEVK

NucleicAcidEncodingChimera6-5 (SEOIDNO:57) TGAGGTGTCGTTTCATATTGCAGTAGATGACAAGAAAGCGATTCAAGGTATTCCGTTGGAACGTAATGCATGG

TGAAGTTAAA

Chimera 6-5 (SEO ID NO:58)

MEIQKKLVDPSKYGTKCPYTMKPKYITVHNTYNDAPAENEVSYMISNNNEVSFHIAVDDKKAIQGIPLER NAWACGDGNGSGNRQSISVEICYSKSGGDRYYKAEDNAVDWRQLMSMYNIPIENVRTHQSWSGKYCPHR MLAEGRWGAFIQKVKNGNVATTSPTGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS GGGGSQVQLQQSGGGWQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVSAISGSGGSTYYADSVK GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDTRGYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSGGG GSGGGGSSELTQDPAMSVALGQTVRITCQGDSLRKYHASWYQQKPGQAPVLVIYGKNERPSGIPERFSGS TSGDTASLTISGLQAEDEADYYCHSRDSNADLWFGGGTKVTVLGGGGGSGGGGSGGGGSGGGGSGGGGS GGGGSKQNIIQSGAFSPYETPDVMGALTSLKMTADFILQSDGLTYFISKPTSDAQLKAMKEYLDRKGWWY EVK

Nucleic Acid Encoding Chimera 6-6 CSEO ID NO:59)

GACTTCAGATGCACAACTAAAAGCAATGAAAGAATACCTTGACCGTAAAGGTTGGTGGTATGAAGTTAAA Chimera6-6 (SEQ IDNO:60)

MEIQKKLVDPSKYGTKCPYTMKPKYITVHNTYNDAPAENEVSYMISNNNEVSFHIAVDDKKAIQGIPLER NAWACGDGNGSGNRQSISVEICYSKSGGDRYYKAEDNAVDWRQLMSMYNIPIENVRTHQSWSGKYCPHR MLAEGRWGAFIQKVKNGNVATTSPTGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS GGGGSQVQLQQSGGGWQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVSAISGSGGSTYYADSVK GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDTRGYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSGGG GSGGGGSSELTQDPAMSVALGQTVRITCQGDSLRKYHASWYQQKPGQAPVLVIYGKNERPSGIPERFSGS TSGDTASLTISGLQAEDEADYYCHSRDSNADLWFGGGTKVTVLGGGGGSGGGGSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGSKQNIIQSGAFSPYETPDVMGALTSLKMTADFILQSDGLTYFISKP TSDAQLKAMKEYLDRKGWWYEVK

NucleicAcidEncodingChimera6-7 (SEO IDNO:6n

TGAAGTTAAA

Chimera 6-7 (SEQ ID NO:62)

MEIQKKL VDPSKYGTKCPYTMKPKYITVHNTYNDAP AENEVSYMISNNNEVSFHIAVDDKKAIQGIPLER NAWACGDGNGSGNRQSISVEICYSKSGGDRYYKAEDNAVD WRQLMSMYNIPIENVRTHQSWSGKYCPHR MLAEGRWGAFIQKVKNGNVATTSPTGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSQVQLQQSGGGWQPG RSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNAKNSLYLQMNS LRAEDTAVYYCARDTRGYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSSELTQDPAMSVAL GQTVRITCQGDSLRKYHAS WYQQKPGQAPVLVIYGKNERPSGIPERFSGSTSGDTASLTISGLQAEDEAD YYCHSRDSNADLWFGGGTKVTVLGGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS GGGGSKQNIIQSGAFSPYETPDVMGALTSLKMTADFILQSDGLTYFISKPTSDAQLKAMKEYLDRKGWWY EVK

Nucleic Acid Encoding Chimera 6-8 (SEQ ID NO^)

GAGATAGATACTATAAAGCTGAGGATAATGCTGTTGATGTTGTACGACAACTTATGTCTATGTACAATATTCC GATTGAAAATGTTCGAACTCATCAATCCTGGTCAGGTAAATATTGTCCGCATAGAATGTTAGCTGAGGGAAGG TGGGGAGCATTCATTCAGAAGGTTAAGAATGGGAATGTGGCGACTACTAGTCCAACAGGCGGAGGCGGCAGCG GCGGAGGCGGCAGCGGAGGCGGAGGGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGTGGCGGAGGATC CCAGGTGCAGCTGCAGCAATCAGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCC TCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCT CAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGA CAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCG AGAGATACCCGAGGGTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACCGTGAGCTCAGGAGGCGGAGGGT CCGGAGGCGGAGGGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGTGGCGGAGGATCCTCTGAGCTGAC TCAGGACCCTGCTATGTCTGTGGCCTTGGGACAGACAGTCAGAATCACCTGTCAAGGGGACAGTCTCAGAAAG TATCATGCAAGCTGGTATCAGCAGAAGCCAGGGCAGGCCCCTGTTCTTGTCATCTATGGTAAGAATGAACGGC CGTCCGGAATCCCAGAGCGATTCTCTGGGTCCACTAGTGGAGACACAGCTTCCTTGACCATCAGTGGGCTCCA GGCGGAAGATGAGGCTGACTATTACTGTCACTCCCGAGACTCTAATGCTGATCTTGTGGTGTTCGGCGGAGGG ACCAAGGTCACCGTCCTAGGTGGTGGCGGAGGGTCAGGTGGCGGAGGGTCAGGTGGCGGAGGGTCAGGTGGCG GAGGGTCAGGTGGCGGAGGGTCAGGTGGCGGAGGGTCAAAACAAAACATCATCCAATCCGGAGCTTTCTCACC GTATGAAACCCCTGATGTTATGGGAGCATTAACGTCACTTAAAATGACAGCTGATTTTATCTTACAATCGGAT

AAGGTTGGTGGTATGAAGTTAAA

Chimera 6-8 (SEQ ID NO:64)

MEIQKKL VDPSKYGTKCPYTMKPKYITVHNTYNDAP AENEVSYMISNNNEVSFHIAVDDKKAIQGIPLER NAWACGDGNGSGNRQSISVEICYSKSGGDRYYKAEDNAVD WRQLMSMYNIPIENVRTHQSWSGKYCPHR MLAEGRWGAF I QKVKNGNVATTSPTGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSQVQLQQSGGGWQPG

RSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNAKNSLYLQMNS LRAEDTAVYYCARDTRGYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSSELTQDPAMSVAL GQTVRITCQGDSLRKYHASWYQQKPGQAPVLVIYGKNERPSGIPERFSGSTSGDTASLTISGLQAEDEAD YYCHSRDSNADLWFGGGTKVTVLGGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSKQNI IQSGAFSPYET PDVMGALTSLKMTADFILQSDGLTYFISKPTSDAQLKAMKEYLDRKGWWYEVK

[00246] Chimera 6-1 features a plgR binding peptide between the catalytic domain and the cell wall binding domain of PIyG. The binding peptide, flanked by two linkers is underlined. Chimera 6-2 features two such binding peptides and three linkers (all underlined) between the two domains. Chimera 6-3 features three binding peptides and four linkers (all underlined), and chimera 6-4 features four binding peptides and five linkers (all underlined).

[00247] Chimera 6-5 features an entire sFv molecule inserted between the two domains of PIyG. In the underlined portion of the sequence, the sFv molecule is flanked by a (GGGGS)io linker and a (GGGGS)₆ linker. In addition, the sFv molecule features a

(GGGGS)₅ linker between its heavy and light chains. Chimera 6-6 features the same sFv molecule flanked by two (GGGGS)ιo linkers while chimera 6-7 features a (GGGGS)₆ linker and a (GGGGS) i₀ linker. Chimera 6-8 features two (GGGGS)₆ linkers.

[00248] The chimeras were expressed and purified according to the methods of

Example 2 to obtain greater than 95% homogeneity.

[00249] Surprisingly, PIyG tolerated large inserts between its functional domains.

Chimeras 6-1, 6-2, 6-3 and 6-4 showed lytic activity that was comparable to PIyG lysin expressed in Pichia. Furthermore, they also showed the ability to bind plgR. Significantly, chimeras 6-5, 6-6, 6-7 and 6-8 showed reduced, but measurable lytic activity while maintaining sFv activity.

[00250] The split chimeras of these examples demonstrate that significant insertions can be made between the domains of PIyG while maintaining lytic activity. With this combination of activities, the split chimeras can be useful for the treatment or prevention of infection by B. anthracis in infected tissue of a subject.

[00251] From the foregoing description, various modifications and changes in the compositions and methods of this invention will occur to those skilled in the art. All such modifications coming within the scope of the appended claims are intended to be included therein. All references cited herein are hereby incorporated by reference in their entireties.

Claims

WHAT IS CLAIMED IS:

1. A chimera comprising PIyG and a targeting element that is capable of binding plgR.

2. The chimera of Claim 1 wherein the PIyG is according to SEQ ID NO: 1.

3. The chimera of Claim 1 wherein the targeting element comprises a peptide capable of binding plgR.

4. The chimera of Claim 1 wherein the targeting element comprises an antibody or an antibody fragment capable of binding plgR.

5. The chimera of Claim 1 wherein the targeting element comprises an antibody or antibody fragment capable of binding the stalk of plgR.

6. The chimera of Claim 5 wherein the antibody or antibody fragment is a single chain antibody or a diabody.

7. The chimera of Claim 1 wherein the targeting element comprises SEQ ID NO:2.

8. The chimera of Claim 1 wherein the PIyG and targeting element are linked by a covalent bond.

9. The chimera of Claim 1 wherein the PIyG and targeting element are linked by an amide bond.

10. The chimera of Claim 1 wherein the PIyG and targeting element are linked by a linker.

11. The chimera of Claim 1 wherein the PIyG and targeting element are linked by peptide or polypeptide.

12. The chimera of Claim 1 wherein the PIyG and targeting element are linked by a peptide or polypeptide of at least 4, 5, 6, 7, 8, 9 or 10 amino acids.

13. The chimera of Claim 8 wherein the targeting element is linked to the amino terminus of PIyG.

14. The chimera of Claim 11 wherein the linker comprises a protease sensitive site.

15. The chimera of Claim 14 that wherein the protease sensitive site is sensitive to elastase or a cathepsin.

16. A split chimera comprising an insertion between the PIyG enzymatic domain and the PIyG cell wall binding domain of the PIyG.

17. The split chimera of Claim 16 wherein the PIyG enzymatic domain comprises residues 1 to 165 of SEQ ID NO:1.

18. The split chimera of Claim 16 wherein the PIyG cell wall binding domain comprises residues 166 to 233 of SEQ ID NO:1.

19. The split chimera of Claim 17 wherein the PIyG cell wall binding domain comprises residues 166 to 233 of SEQ ID NO:1.

20. The split chimera of Claim 16 wherein the insertion is at a site within 20, 15, 10, 5, 4, 3, 2, or 1 residue of boundary between the cell wall binding domain and the enzymatic domain.

21. The split chimera of Claim 19 wherein the insertion is at a site within 20, 15, 10, 5, 4, 3, 2, or 1 residue of boundary between the cell wall binding domain and the enzymatic domain.

22. The split chimera of Claim 16 wherein the insertion comprises a peptide or a polypeptide.

23. The split chimera of Claim 16 wherein the insertion comprises a targeting element that is capable of binding plgR.

24. The split chimera of Claim 16 wherein the insertion comprises a targeting element that is capable of binding the stalk of plgR.

25. The split chimera of Claim 16 wherein the insertion comprises a peptide capable of binding plgR.

26. The split chimera of Claim 16 wherein the insertion comprises an antibody or an antibody fragment capable of binding plgR.

27. The split chimera of Claim 16 wherein the insertion comprises an antibody or antibody fragment capable of binding the stalk of plgR.

28. The split chimera of Claim 16 wherein the insertion comprises a single chain antibody capable of binding plgR.

29. The split chimera of Claim 28 wherein the insertion comprises SEQ ID NO:2.

30. A nucleic acid encoding the chimera of any of the preceding claims.

31. An expression vector comprising the nucleic acid of Claim 30.

32. A host cell comprising the expression vector of Claim 31.

33. A method of making a chimera comprising incubating the host cell of Claim 32 under conditions suitable for expression of the chimera.

34. A method of making an isolated chimera comprising making the chimera according to Claim 33 and isolating the chimera.

35. A pharmaceutical composition comprising the chimera of Claim 1 or 16 and a pharmaceutically acceptable carrier, excipient or diluent.

36. The pharmaceutical composition of Claim 35 wherein the pharmaceutically acceptable carrier, excipient or diluent is an aerosol.

37. A method of lysing Bacillus anthracis comprising contacting the Bacillus anthracis with the chimera of Claim 1 or 16.

38. A method of treating a Bacillus anthracis infection comprising administering the chimera of Claim 1 or 16 to a subject in need thereof.

39. The method of Claim 38 wherein the chimera is administered by pulmonary administration.

40. The method of Claim 38 wherein the chimera is in the form of a pharmaceutical composition.