Methods of using Bacillus anthracis protective antigen sequences for vaccination

FIELD OF THE INVENTION

This invention relates to improved methods of preparing Bacillus anthracis protective antigen (PA) for use in vaccines.

BACKGROUND

Anthrax, a potentially fatal disease, is caused by Bacillus anthracis. The virulence of this pathogen is mediated by a capsule of a poly-D-.gamma.-glutamic acid and an exotoxin composed of three proteins (14, 16, 17). The three protein components are the protective antigen (PA, 82 KDa), lethal factor (LF, 90.2 KDa) and edema factor (EF, 88.8 KDa) These proteins, non-toxic by themselves, form lethal toxins when combined with an activated PA (16). The genes coding for these three protein components and the capsule are found in the endogenous plasmids pXO1 and pXO2, respectively (29).

The capsule of Bacillus anthracis, composed of poly-D-glutamic acid, serves as one of the principal virulence factors during anthrax infection. By virtue of its negative charge, the capsule is purported to inhibit host defense through inhibition of phagocytosis of the vegetative cells by macrophages. In conjunction with lethal factor (LF) and edema factor (EF), whose target cells include macrophages and neutrophils, respectively, the capsule allows virulent anthrax bacilli to grow virtually unimpeded in the infected host. Spores germinating in the presence of serum and elevated CO.sub.2 release capsule through openings on the spore surface in the form of blebs which may coalesce before sloughing of the exosporium and outgrowth of the fully encapsulated vegetative cell. It has not been established that spore encapsulation plays a role in the early events of anthrax infection. The capsule appears exterior to the S-layer of the vegetative cell and does not require the S-layer for its attachment to the cell surface.

There is only indirect evidence, albeit extensive, identifying the components of vaccine-induced immunity to anthrax and there is evidence that anti-PA neutralizing antibody titers can be a reliable surrogate marker for protective immunity (23). The protective antigen (PA), seems to be an essential component of all vaccines for anthrax (7, 18, 30): both mono and polyclonal antibodies to PA neutralize the anthrax toxin and confer immunity to B. anthracis in animal models. The US licensed vaccine for anthrax "Anthrax Vaccine Adsorbed" (AVA) is produced from the formalin-treated culture supernatant of B. anthracis Sterne strain, V770-NP1-R (pXO1.sup.+, pXO2.sup.-), adsorbed onto aluminum hydroxide (22). Although AVA has been shown to be effective against cutaneous infection in animals and humans and against inhalation anthrax by rhesus monkeys (12), it has several limitations: 1) AVA elicits relatively high degree of local and systemic adverse reactions probably mediated by variable amounts of undefined bacterial products, making standardization difficult; 2) the immunization schedule requires administration of six doses within an eighteen-month period, followed by annual boosters for those at risk; and 3) there is no defined vaccine-induced protective level of serum PA to evaluate new lots of vaccines.

Development of a well characterized, standardized, effective and safe vaccine that would require fewer doses to confer immunity to both inhalational and cutaneous anthrax is needed (9, 30). It has been suggested that a vaccine composed of modified purified recombinant PA would be effective, safer, allow precise standardization, and probably would require fewer injections (27). Such a PA can be designed to be biologically inactive, more stable, and still maintained high immunogenicity.

In the examples herein, we describe the development of a production and purification process for recombinant PA from the non-sporogenic avirulent B. anthracis BH445 (pXO1.sup.-, pXO2.sup.-) strain. Following an 18-hour fermentation and three purification steps, large quantities of protective antigen suitable for vaccine production were obtained. The purified PA was tested in mice and was able to elicit neutralizing antibodies.

BRIEF DESCRIPTION OF THE INVENTION

This invention relates to improved methods of preparing Bacillus anthracis protective antigen (PA).

The invention also relates to PA and/or compositions thereof, which are useful for inducing or eliciting an immunogenic response in mammals, including responses which provide protection against, or reduce the severity of, infections caused by B. anthracis. In particular, the invention relates to methods of using PA, and/or compositions thereof, to induce or elicit serum antibodies which have neutralizing activity against B. anthracis toxin. PA and/or compositions thereof are useful as vaccines to induce serum antibodies which are useful to prevent, treat or reduce the severity of infections caused by B. anthracis, such as inhalation anthrax, cutaneous anthrax and/or gastrointestinal anthrax.

The invention also relates to nucleic acids encoding PA of B. anthracis, and compositions thereof, which produce PA in sufficient amounts to be useful as pharmaceutical compositions or vaccines to induce serum antibodies for preventing and/or treating illnesses caused by B. anthracis. The invention also relates to suitable expression systems, viral particles, vectors, vector systems, and transformed host cells containing those nucleic acids.

The invention also relates to antibodies which immunoreact with the PA of B. anthracis, and/or compositions thereof. Such antibodies may be isolated, or may be provided in the form of serum containing these antibodies.

The invention also relates to pharmaceutical compositions and/or vaccines comprising at least one of the PAs, nucleic acids, viral particles, vectors, vector systems, transformed host cells or antibodies of the invention.

The invention also relates to methods for the prevention or treatment of B. anthracis infection in a mammal, by administration of pharmaceutical or vaccine compositions of the invention.

The invention also provides kits comprising one or more of the agents of the invention which are useful for vaccinating mammals for the treatment or prevention of B. anthracis infection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Production and proteolytic activity of PA-SNKE-.DELTA.FF-E308D (SEQ ID NO: 4) and PA-N657A (SEQ ID NO: 5). (a) PA production (mg/g cells) .circle-solid. SNKE, .box-solid. N657A; proteolytic activity .largecircle. SNKE, N657A; (b) SDS-PAGE analysis of partially purified PA-N657A (SEQ ID No: 5) and PA-SNKE-.DELTA.FF-E308D (SEQ ID NO: 4).

FIG. 2. Effect of EDTA and PMSF on proteolytic activity. Supernatants from two different cultures taken after 24 hours of growth were analyzed without inhibitors (control), with 1 .mu.g/.mu.L PMSF, and with 15 mM EDTA. Fluorescence is proportional to proteolytic activity.

FIG. 3. Fermentation process for the production of PA-SNKE-.DELTA.FF-E308D (SEQ ID NO: 4) from B. anthracis BH445. Acid and base values are cumulative.

FIG. 4. SDS-PAGE analysis of culture supernatants obtained throughout the fermentation. Samples were taken at 13, 14, 16, 18, 22, and 34 hours of growth. Arrow indicates the location of PA(83 KDa) in the gel.

FIG. 5. PA production and proteolytic activity of B. anthracis BH445 [pSY5:SNKE-.DELTA.FF-E308D; SEQ ID NO: 4] in fed-batch cultures supplied with tryptone/yeast extract or glucose. .circle-solid. Specific PA production in tryptone/yeast extract (mg/g cells); v Volumetric PA production in tryptone/yeast extract (mg/liter); .tangle-solidup. Proteolytic activity in tryptone/yeast extract; .largecircle. Specific PA production in glucose (mg/g cells); Volumetric PA production in glucose (mg/liter); .DELTA. Proteolytic activity in glucose.

FIG. 6. SDS-PAGE analysis of purified PA fractions. (a) PA purified by packed bed chromatography; (b) PA after hydrophobic interaction chromatography and gel filtration; (c) PA fraction shown in Lane (b) after 3 months; (d) PA after expanded bed hydrophobic interaction chromatography, anion exchange, and gel filtration. MW indicates molecular weight markers. Arrows indicate the location of PA(83 KDa) in the gel.

SEQUENCE LISTING

SEQ ID NOS: 1 and 2 show an exemplary full-length PA nucleic acid and protein sequence, respectively. The signal sequence of PA is amino acids 1-29, and the mature PA protein is amino acids 30-764.

SEQ ID NO: 3 is a mature wild-type PA protein sequence.

SEQ ID NO: 4 is a PA-SNKE-.DELTA.FF-E308D mutant protein sequence.

SEQ ID NO: 5 is a PA-N657A mutant protein sequence.

SEQ ID NO: 6 is a K397D D425K mutant PA protein sequence.

SEQ ID NO: 7 is a PA-L1 protein sequence, with a MMP cleavage site.

SEQ ID NO: 8 is a PA-L2 protein sequence, which is cleavable with MMP.

SEQ ID NO: 9 is a PrAg-U1 protein sequence, which is cleavable by uPA/tPA.

SEQ ID NO: 10 is a PrAg-U2 protein sequence, which is cleavable by uPA.

SEQ ID NO: 11 is a PrAg-U3 protein sequence, which is cleavable by uPA.

SEQ ID NO: 12 is a PrAg-U4 protein sequence, which is cleavable by tPA.

SEQ ID NO: 13 shows amino acids 175-764 of wild-type PA.

SEQ ID NO: 14 shows the amino acid RKKR which is replaced with SEQ ID NO: 15 in some PA mutants.

SEQ ID NO: 15 shows the amino acid SNKE replaces the native RKKR sequence in some PA mutants.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only, and are not restrictive of the invention, as claimed. The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate an embodiment of the invention and, together with the description, serve to explain the principles of the invention.

The invention relates to methods of producing and recovering PA from a cell or organism, particularly a recombinant cell or microorganism. Exemplified herein is the production and purification of modified PA from a non-sporgenic strain of Bacillus anthracis. As discussed further herein, greater quantities of PA are obtainable from these cells or microorganisms than were obtainable by previously described methods.

The invention also relates to PA, and/or compositions thereof, which are useful for eliciting an immunogenic response in mammals, in particular humans, including responses which provide protection against, or reduce the severity of, infections caused by B. anthracis. The invention also relates to methods of using such PA, and/or compositions thereof, to induce serum antibodies against PA. PA, and/or compositions thereof, are useful as vaccines to induce serum antibodies which are useful to prevent, treat or reduce the severity of infections caused by B. anthracis, such as inhalation anthrax and/or cutaneous anthrax. The PAs of this invention are expected to induce a strong protective IgG antibody response in mammals, including humans.

The invention also relates to nucleic acids encoding PA of this invention. Nucleic acids encoding PA, and compositions thereof, are also useful as pharmaceutical compositions or vaccines to induce serum antibodies which are useful to prevent and/or treat illnesses caused by B. anthracis.

The invention also relates to antibodies which immunoreact with the PA of B. anthracis that are induced by PAs of the invention, and/or compositions thereof. Such antibodies may be isolated, or may be provided in the form of serum containing these antibodies.

The invention also relates to a method for the prevention or treatment of B. anthracis infection in a mammal, by administration of compositions containing one or more of a PA of the invention, nucleic acids encoding a PA if the invention, antibodies and/or serum containing antibodies of the invention.

The invention also provides kits for vaccinating mammals for the treatment or prevention of B. anthracis infection in a mammal comprising one or more of the agents of the invention.

The present invention also encompasses methods of using mixtures of one or more of the PA, nucleic acids, and/or antibodies of the invention, either in a single composition or in multiple compositions containing other immunogens, to form multivalent vaccine for broad coverage against either B. anthracis itself or a combination of B. anthracis and one or more other pathogens, which may also be administered concurrently with other vaccines, such as the DTP vaccine.

Pharmaceutical compositions of this invention are capable, upon injection into a human, of inducing serum antibodies against B. anthracis. The induced anti-PA antibodies have anthrax toxin neutralizing activity which are preferably at least comparable to those induced by the currently licensed anthrax vaccine.

The vaccines of this invention are intended for active immunization for prevention of B. anthracis infection, and for preparation of immune antibodies. The vaccines of this invention are designed to confer specific immunity against infection with B. anthracis, and to induce antibodies specific to B. anthracis PA. The B. anthracis vaccine is composed of non-toxic bacterial components, suitable for infants, children of all ages, and adults.

The methods of using the agents of this invention, and/or compositions thereof will be useful in increasing resistance to, preventing, ameliorating, and/or treating B. anthracis infection in humans.

This invention also provides compositions, including but not limited to, mammalian serum, plasma, and immunoglobulin fractions, which contain antibodies which are immunoreactive with B. anthracis PA. These antibodies and antibody compositions may be useful to prevent, treat, and/or ameliorate infection and disease caused by the microorganism. The invention also provides such antibodies in isolated form.

High titer anti-PA sera, or antibodies isolated therefrom, may be used for therapeutic treatment for patients with B. anthracis infection. Antibodies elicited by the agents of this invention may be used for the treatment of established B. anthracis infections, and may also be useful in providing passive protection to an individual exposed to B. anthracis.

The present invention also provides kits comprising vaccines for the prevention and/or treatment of B. anthracis, containing the one or more of the PAs, nucleic acids, viral particles, vectors, vector systems, or transformed host cells or antibodies of the invention and/or compositions thereof. The PAs, nucleic acids viral particles vectors, host cells and/or antibodies of the present invention may be isolated and purified by methods known in the art. Preferably, the PA of the invention is purified by one of the methods exemplified herein.

The vaccines of the invention are intended to be included in the immunization schedule of individuals at risk for B. anthracis infection. They are also planned to be used for intervention in the event of the use of B. anthracis in bioterrorism or biowarfare. For example, it is anticipated that the vaccines of the invention may be provided to the entire U.S. population. Additionally, they may be used as component(s) of a multivalent vaccine for B. anthracis and/or other pathogens.

Definitions

As used herein, unless otherwise specifically noted, "PA" refers to all forms of PA which are useful in the compositions and/or methods of the invention, including unmodified native or recombinant B. anthracis protective antigen (PA), or a modified form (variant) or fragment thereof, for use in vaccines. Variants and fragments of PA must be able to produce an immune response in a mammal to whom they are administered. The immune response is suitably protective against infection by Bacillus anthracis although the protective effect may be seen only after repeated applications, as would be determinable by methods known in the art. Modified PA variants comprise peptides and proteins which resemble PA in their ability to induce or elicit antibodies which bind to native PA, but have different amino acid sequence. For example, variants may be 60% homologous to PA protein, suitably 80% homologous and more particularly at least 90% homologous. Fragments are suitably peptides which contain at least one antigenic determinant of PA.

A modified (variant) PA of the invention includes any substituted analog or chemical derivative of PA, so long as the modified (variant) PA is capable of inducing or eliciting the production of antibodies capable of binding native (or naturally-occurring) PA. Preferably, the antibodies are neutralizing antibodies. PA can be subject to various changes that provide for certain advantages in its use. For example, PA with changes which increase in vitro and/or in vivo stability of PA, while still retaining the desired immunogenic activity, are preferred. In the modified PA used in the examples herein (SEQ ID NO: 4)two regions were altered, i.e., the furin cleavage site region (RKKR.sup.167 to SNKE.sup.167), and the chymotrypsin and thermolysin cleavage site region (two Phe at positions 313-314 were deleted and Glu acid at position 308 was substituted with Asp), resulting in a more stable PA. As used herein, the terms "immunoreact" and "immunoreactivity" refer to specific binding between an antigen or antigenic determinant-containing molecule and a molecule having an antibody combining site, such as a whole antibody molecule or a portion thereof.

As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules. Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and portions of an immunoglobulin molecule, including those portions known in the art as Fab, Fab', F(ab').sub.2 and F(v), as well as chimeric antibody molecules.

As used herein, the term "transduction" generally refers to the transfer of genetic material into the host via infection, e.g., in this case by the lentiviral vector. The term "transfection" generally refers to the transfer of isolated genetic material into cells via the use of specific transfection agents (e.g., calcium phosphate, DEAE Dextran, lipid formulations, gold particles, and other microparticles) that cross the cytoplasmic membrane and deliver some of the genetic material into the cell nucleus.

Monomers, Polymers and Polymeric Carriers

The present invention encompasses monomers of PA, as well as homogeneous or heterogeneous polymers of PA (e.g., concatenated, cross-linked and/or fused identical polypeptide units or concatenated, cross-linked and/or fused diverse peptide units), and mixtures of the polypeptides, polymers, and/or conjugates thereof. The present invention also encompasses PA bound to a non-toxic, preferably non-host, protein carrier to form a conjugate.

Linkers useful in the invention may, for example, be simply peptide bonds, or may comprise amino acids, including amino acids capable of forming disulfide bonds, but may also comprise other molecules such as, for example, polysaccharides or fragments thereof.

The linkers for use with this invention may be chosen so as to contribute their own immunogenic effect which may be either the same, or different, than that elicited by the consensus sequences of the invention. For example, such linkers may be bacterial antigens which also elicit the production of antibodies to infectious bacteria. In such instances, for example, the linker may be a protein or protein fragment of an infectious bacteria.

Carriers are chosen to increase the immunogenicity of the PA and/or to raise antibodies against the carrier which are medically beneficial. Carriers that fulfill these criteria are well known in the art. A polymeric carrier can be a natural or a synthetic material containing one or more functional groups, for example primary and/or secondary amino groups, azido groups, or carboxyl groups. Carriers can be water soluble or insoluble.

Methods for Attaching PA to a Protein Carrier.

PA of the invention may be covalently attached to other proteins, with or without a linker, by methods known in the art, such as via their side chains or via peptide bonds in the primary chain. Cysteine molecules may provide a convenient attachment point through which to chemically conjugate other proteins or non-protein moieties to PA.

Dosage for Vaccination

The pharmaceutical compositions of this invention contain a pharmaceutically and/or therapeutically effective amount of at least one PA, nucleic acid, vector, viral particle, host cell immunogen or antibody of the invention. The effective amount of immunogen per unit dose is an amount sufficient to induce an immune response which is sufficient to prevent, treat or protect against the adverse effects of infection with B. anthracis. The effective amount of immunogen per unit dose depends, among other things, on the species of mammal inoculated, the body weight of the mammal and the chosen inoculation regimen, as is well known in the art.

In such circumstances, inocula for a human or similarly sized mammal typically contain PA concentrations of 0.5 .mu.g to 1 mg per mammal per inoculation dose. Initial tests of the PA vaccine in humans will use approximately 10 .mu.g or 20 .mu.g per dose. Preferably, the route of inoculation of the peptide will be subcutaneous or intramuscular. The dose is administered at least once.

To monitor the antibody response of individuals administered the compositions of the invention, antibody levels may be determined. In most instances it will be sufficient to assess the antibody titer in serum or plasma obtained from such an individual. Decisions as to whether to administer booster inoculations or to change the amount of the composition administered to the individual may be at least partially based on the level.

The level may be based on either an immunobinding assay which measures the concentration of antibodies in the serum which bind to a specific antigen, i.e. PA. The ability to neutralize in vitro and in vivo biological effects of the B. anthracis toxins may also be assessed to determine the effectiveness of the treatment.

The term "unit dose" as it pertains to the inocula refers to physically discrete units suitable as unitary dosages for mammals, each unit containing a predetermined quantity of active material calculated to produce the desired immunogenic effect in association with the required diluent.

Inocula are typically prepared in physiologically and/or pharmaceutically tolerable (acceptable) carrier, and are preferably prepared as solutions in physiologically and/or pharmaceutically acceptable diluents such as water, saline, phosphate-buffered saline, or the like, to form an aqueous pharmaceutical composition. Adjuvants, such as aluminum hydroxide, may also be included in the compositions.

Depending on the intended mode of administration, the compounds of the present invention can be in various pharmaceutical compositions. The compositions will include, as noted above, an effective amount of the selected immunogen and/or antibody of the invention in combination with a pharmaceutically acceptable carrier and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, etc. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the immunogen and/or antibody or other composition without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

The route of inoculation may be intramuscular, subcutaneous or the like, which results in eliciting antibodies protective against B. anthracis. In order to increase the antibody level, a second or booster dose may be administered approximately 4 to 6 weeks after the initial injection. Subsequent doses may be administered as indicated herein, or as desired by the practitioner.

Parenteral administration, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system, such that a constant level of dosage is maintained. See, e.g., U.S. Pat. No. 3,710,795, which is incorporated by reference herein.

Antibodies

An antibody of the present invention in one embodiment is characterized as comprising antibody molecules that immunoreact with B. anthracis PA.

An antibody of the present invention is typically produced by immunizing a mammal with an immunogen or vaccine containing an B. anthracis PA to induce, in the mammal, antibody molecules having immunospecificity for the immunizing PA. Antibody molecules having immunospecificity for the protein carrier will also be produced. The antibody molecules may be collected from the mammal and, optionally, isolated and purified by methods known in the art.

Human or humanized monoclonal antibodies are preferred, including those made by phage display technology, by hybridomas, or by mice with human immune systems. The antibody molecules of the present invention may be polyclonal or monoclonal. Monoclonal antibodies may be produced by methods known in the art. Portions of immunoglobulin molecules, such as Fabs, may also be produced by methods known in the art.

The antibody of the present invention may be contained in blood plasma, serum, hybridoma supernatants and the like. Alternatively, the antibodies of the present invention are isolated to the extent desired by well known techniques such as, for example, ion exchange chromatography, sizing chromatography, or affinity chromatography. The antibodies may be purified so as to obtain specific classes or subclasses of antibody such as IgM, IgG, IgA, IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4 and the like. Antibodies of the IgG class are preferred for purposes of passive protection. The antibodies of the present invention have a number of diagnostic and therapeutic uses. The antibodies can be used as an in vitro diagnostic agents to test for the presence of B. anthracis in biological samples or in meat and meat products, in standard immunoassay protocols. Such assays include, but are not limited to, agglutination assays, radioimmunoassays, enzyme-linked immunosorbent assays, fluorescence assays, Western blots and the like. In one such assay, for example, the biological sample is contacted first with antibodies of the present invention which bind to B. anthracis PA, and then with a labeled second antibody to detect the presence of B. anthracis to which the first antibodies have bound.

Such assays may be, for example, of direct format (where the labeled first antibody is reactive with the antigen), an indirect format (where a labeled second antibody is reactive with the first antibody), a competitive format (such as the addition of a labeled antigen), or a sandwich format (where both labeled and unlabelled antibody are utilized), as well as other formats described in the art. The antibodies of the present invention are also useful in prevention and treatment of infections and diseases caused by B. anthracis.

In providing the antibodies of the present invention to a recipient mammal, preferably a human, the dosage of administered antibodies will vary depending upon such factors as the mammal's age, weight, height, sex, general medical condition, previous medical history and the like.

In general, it is desirable to provide the recipient with a dosage of antibodies which is in the range of from about 1 mg/kg to about 10 mg/kg body weight of the mammal, although a lower or higher dose may be administered. The antibodies of the present invention are intended to be provided to the recipient subject in an amount sufficient to prevent, or lessen or attenuate the severity, extent or duration of the infection by B. anthracis. When proteins of other organisms are used as carriers, antibodies which immunoreact with those proteins are intended to be provided to the recipient subject in an amount sufficient to prevent, lessen or attenuate the severity, extent or duration of an infection by the organisms producing those proteins.

The administration of the agents of the invention may be for either "prophylactic" or "therapeutic" purpose. When provided prophylactically, the agents are provided in advance of any symptom. The prophylactic administration of the agent serves to prevent or ameliorate any subsequent infection. When provided therapeutically, the agent is provided at (or shortly after) the onset of a symptom of infection. The agent of the present invention may, thus, be provided prior to the anticipated exposure to B. anthracis, so as to attenuate the anticipated severity, duration or extent of an infection and disease symptoms, after exposure or suspected exposure to these bacteria, or after the actual initiation of an infection.

For all therapeutic, prophylactic and diagnostic uses, one or more of the PAs or other agents of this invention, as well as antibodies and other necessary reagents and appropriate devices and accessories, may be provided in kit form so as to be readily available and easily used.

Nucleic Acids, Vectors and Hosts

The invention also relates to isolated and purified nucleic acid molecules which code for the PAs of the invention. The encoded PAs may be monomers, polymers or linked to other peptide sequences (e.g., they may be fusion proteins).

Nucleic acids encoding the PAs of the invention can be introduced into a vector such as a plasmid, cosmid, phage, virus, viral particle or mini-chromosome and inserted into a host cell or organism by methods well known in the art. The vectors which can be utilized to clone and/or express these nucleic acids are the vectors which are capable of replicating and/or expressing the nucleic acids in the host cell in which the nucleic acids are desired to be replicated and/or expressed. See, e.g., F. Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Interscience (1992) and Sambrook et al. (1989) for examples of appropriate vectors for various types of host cells. Vectors and compositions for enabling production of the peptides in vivo, i.e., in the individual to be treated or immunized, are also within the scope of this invention. Strong promoters compatible with the host into which the gene is inserted may be used. These promoters may be inducible. The host cells containing these nucleic acids can be used to express large amounts of the protein useful in pharmaceuticals, diagnostic reagents, vaccines and therapeutics. Vectors include retroviral vectors and also include direct injection of DNA into muscle cells or other receptive cells, resulting in the efficient expression of the peptide, using the technology described, for example, in Wolff et al., Science 247:1465-1468 (1990), Wolff et al., Human Molecular Genetics 1(6):363-369 (1992) and Ulmer et al., Science 259:1745-1749 (1993). See also, for example, WO 96/36366 and WO 98/34640.

In general, vectors containing nucleic acids encoding PA can be utilized in any cell, either eukaryotic or prokaryotic, including mammalian cells (e.g., human (e.g., HeLa), monkey (e.g., COS), rabbit (e.g., rabbit reticulocytes), rat, hamster (e.g., CHO and baby hamster kidney cells) or mouse cells (e.g., L cells), plant cells, yeast cells, insect cells or bacterial cells (e.g., E. coli). However, bacterial vectors and host cells are preferred in the present invention.

There are numerous E. coli expression vectors known to one of ordinary skill in the art useful for the expression of PA. Other microbial hosts suitable for use include bacilli, such as B. subtilus, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts one can also make expression vectors, which will typically contain expression control sequences compatible with the host cell (e.g., an origin of replication). In addition, any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (Trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda. The promoters will typically control expression, optionally with an operator sequence, and have ribosome binding site sequences for example, for initiating and completing transcription and translation. If necessary an amino terminal methionine can be provided by insertion of a Met codon 5' and in-frame with the antigen. Also, if desired, the carboxy-terminal or other region of the antigen can be removed using standard oligonucleotide mutagenesis procedures.

The nucleotide (DNA) sequences can be expressed in hosts after the sequences have been operably linked to, i.e., positioned to ensure the functioning of, an expression control sequence. These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors can contain selection markers, e.g., tetracycline resistance or hygromycin resistance, to permit detection and/or selection of those cells transformed with the desired DNA sequences (see, e.g., U.S. Pat. No. 4,704,362).

Host bacterial cells may be chosen that are mutated to be reduced in or free of proteases, so that the proteins produced are not degraded. For bacillus expression systems in which the proteins are secreted into the culture medium, strains are available that are deficient in secreted proteases.

Polynucleotides encoding a variant polypeptide may include sequences that facilitate transcription (expression sequences) and translation of the coding sequences such that the encoded polypeptide product is produced. Construction of such polynucleotides is well known in the art. For example, such polynucleotides can include a promoter, a transcription termination site (polyadenylation site in eukaryotic expression hosts), a ribosome binding site, and, optionally, an enhancer for use in eukaryotic expression hosts, and, optionally, sequences necessary for replication of a vector.

Fermentation and Purification Procedures

This invention relates to improved methods of preparing B. anthracis PA for use in vaccines. Procedures are exemplified herein for purifying modified PA from is a protease-deficient nonsporogenic avirulent strain of B. anthracis. However, it is expected that these procedures will be useful for growing and purifying PA, including natural or recombinant PA, as well as various modified or truncated forms of PA, from other microorganisms, particularly Bacillus. Bacillus strains and/or expression systems which are expected to be suitable include, for example, the B. anthracis strain described in U.S. Pat. No. 5,840,312 (Nov. 24, 1998) and the B. subtilis strain and PA expression system described in U.S. Pat. No. 6,267,966 (Jul. 31, 2001).

In the method of the invention, the culture is preferably maintained at about pH 7 to about pH 8, most preferably about pH 7.5, substantially throughout the fermentation process. It has also been found to be advantageous to add EDTA before separating the culture supernatant from the cells, preferably at or near the end of fermentation, since if it is added during the fermentation stage, it may interfere somewhat with the growth of the cells.

The purification procedure of the invention is preferably essentially a three-step procedure, including (1) hydrophobic interaction chromatography, (2) ion exchange chromatography and (3) gel filtration. While ion exchange chromatography may precede hydrophobic interaction chromatography in the purification process, and still permit obtaining a good yield of PA, it is a less efficient process. Therefore, in view of this, it is preferred that hydrophobic interaction chromatography precede ion exchange chromatography in the purification process. Alternatively, this three step procedure need not be used and an alternative purification scheme may be used.

In addition, the resins used in the exemplified the purification procedure can be substituted. For example, in the hydrophobic interaction chromatography step, phenyl sepharose (Pharmacia) is used as the resin in the example, but any other hydrophobic resin can be used. Likewise, in the ion exchange chromatography step, Q sepharose (Pharmacia) is used as the resin in the example, but any other anion exchanger can be used. Likewise, for the gel filtration step, Superdex (Pharmacia) is the residue used in the example, but it can be replaced by other gel filtration resins. Furthermore, with respect to the fermentation conditions, similar compounds can replace the tryptone and the yeast extract that are obtained from Difco.

The expression and the stability of two recombinant PA variants, PA-SNKE-.DELTA.FF-E 308D (SEQ ID NO: 4) and PA-N657A (SEQ ID NO: 5),were studied. However, the methods of the invention are also expected to be useful for producing and recovering native PA; PA wherein the receptor-binding domain has been altered; PA which cannot be cleaved at the chymotrypsin cleavage site; PA which cannot be cleaved at the furin cleavage site; other PA which cannot be cleaved at either the chymotrypsin or the furin cleavage site in addition to the one exemplified herein (see, e.g., those described in (22)); PA fragments (e.g., a PA fragment having aa 175-764 (36), SEQ ID NO: 13); PA mutants having a strong dominant-negative effect (e.g., PA double mutants K397D and D425K, SEQ ID NO: 6) (37), and PA mutants with substitutions in domain 2 (37)).

In addition, the methods of the invention are also expected to be useful for producing and recovering PA in which the chymotrypsin site, FF, is replaced by a furin site. This may be a suicide protein, getting easily cleaved by furin after binding to receptor. Cleavage at that site inactivates PA.

The methods of the invention are also expected to be useful for producing and recovering PA with a protease cleavage site (thrombin, Factor IV, etc.) at approximately residue 605. PA made in large amounts in the expression system could be cleaved to produce a soluble domain 4, which would compete with PA for receptor, and could be a therapeutic agent.

The methods of the invention are also expected to be useful for producing and recovering PA with matrix metalloprotease or plasminogen activator sites replacing the furin site (38, 39, SEQ ID NO: 7-12).

The methods of the invention are also expected to be useful for producing and recovering other proteins, such as LF. See, e.g., (21), wherein expression system is the same, except the structural gene for PA is replaced by the LF gene. This can be generalized to include LF mutants altered in the catalytic site residues: HEFGH, 686-690. The system may also have utility with EF.

The following examples are exemplary of the present processes and incorporate suitable process parameters for use herein. These parameters may be varied, however, and the following should not be deemed limiting.

EXAMPLE 1

In this example, the expression and the stability of two recombinant PA variants, PA-SNKE-.DELTA.FF-E308D (SEQ ID NO: 4)and PA-N657A (SEQ ID NO: 5), were studied. These proteins were expressed in the non-sporogenic avirulent strain BH445. Initial results indicated that PA-SNKE-.DELTA.FF-E308D (SEQ ID NO: 4),which lacks two proteolysis-sensitive sites, is more stable than PA-N657A (SEQ ID NO: 5). Process development was conducted to establish an efficient production and purification process for PA-SNKE-.DELTA.FF-E308D (SEQ ID NO: 4). Various parameters such as pH, media composition, growth strategy, and protease inhibitors composition were analyzed. The production process chosen was based on batch growth of B. anthracis using tryptone and yeast extract as the only sources of carbon, pH control at 7.5, and antifoam 289. Optimal harvest time was found to be 14-18 hours after inoculation, and EDTA (5 mM) was added upon harvesting for proteolysis control. In one of the processes described herein, recovery of the PA was performed by expanded bed adsorption (EBA) on a hydrophobic interaction resin, eliminating the need for centrifugation, microfiltration, and diafiltration. The EBA step was followed by ion exchange and gel filtration. PA yields before and after purification were 130 mg/L and 90 mg/L, respectively.

Materials and Methods

Strains and Plasmids

The non-sporogenic, protease deficient, avirulent strain B. anthracis BH445 (pXO1.sup.-, pXO2.sup.-, cm.sup.r) was used (17). The Bacillus-E. coli shuttle vector pYS5 (amp.sup.r, kan.sup.r)(26) was used to clone two recombinant forms of the protective antigen: N657A and SNKE-.DELTA.FF-E308D (SEQ ID NO: 4) (28). In the N657A mutant (SEQ ID NO: 5), the receptor-binding domain of PA was altered by substitution of Asn with Ala at position 657 (domain 4). In the SNKE-.DELTA.FF-E308D mutant (SEQ ID NO: 4) two regions were altered, the furin site (RKKR.sup.167 to SNKE.sup.167) and the chymotrypsin site (two Phe at positions 313-314 were deleted and Glu acid at position 308 was substituted with Asp). Both PA constructs contain the DNA sequence encoding the signal peptide of PA.

Culture and Expression Conditions

Modified FA medium (21) containing (per liter) 35 g tryptone (Difco Laboratories, Detroit, Mich.), 5 g yeast extract (Difco Laboratories), and 100 mL of 10.times. salts was used in all experiments. The 10.times. salt solution (per liter) consisted of 60 g Na 2 HPO.sub.4.7H.sub.2O, 10 g KH.sub.2PO.sub.4, 55 g NaCl, 0.4 g L-tryptophan, 0.4 g L-methionine, 0.05 g thiamine, and 0.25 g uracil. It was filter-sterilized and added to the fermentor after cooling. The pH of the medium was adjusted to 7.5; 100 .mu.g/mL kanamycin and 20 .mu.g/mL chloramphenicol were added. Fermentation experiments were performed by inoculating a 12-14 hour-old starter culture grown from a frozen stock. The medium in the fermentor was supplemented with 0.2 mL/L of antifoam 289 (Sigma, St. Louis, Mo.). Three- to ten-liter fermentations were done using B. Braun Biostat MD DCU (Melsungen, Germany), controlling dissolved oxygen (DO) at 30% saturation, temperature at 37.degree. C., and pH at 7.5 with HCl and NH.sub.4OH. At harvest time, 5 mM EDTA and 10 .mu.g/mL PMSF (phenylmethyl sulfonyl fluoride) (in one of the experiments described herein) were added to the culture. Shake flask experiments (100 mL) utilizing modified FA medium were supplemented with glucose, lactose, glycerol, and casitone at a concentration of 10 g/L.

Analytical Methods

Optical density (OD) was measured at 600 nm. Protease analysis was done on supernatant samples collected during growth and stored frozen at -20.degree. C. EDTA was added to supernatant samples used for SDS-PAGE and radial immunodiffusion to a final concentration of 10 mM.

Extracellular protease activity was detected using the EnzChek green fluorescence assay kit (Molecular Probes Eugene, Oreg.). Fluorescence was measured with a LS508 luminescence spectrophotometer (Perkin-Elmer Boston, Mass.). This assay was conducted at pH of 7.5 or 6.0 depending on the experiment. Proteolytic activity is reported as fluorescence change per unit sample.

Protein was determined using BCA assay (Pierce Rockford, Ill.). PA expression was quantified by SDS-PAGE (Invitrogen/Novex, Carlsbad, Calif.) gel analysis and by the Mancini immunodiffusion assay (19) using agarose plates containing polyclonal PA antibody. Pure PA was used as the standard, both polyclonal PA antibodies and pure PA were supplied by S. Leppla

Purification

a. Packed Bed Hydrophobic Interaction Chromatography

The cell suspension containing 5 mM EDTA was centrifuged and the supernatant passed through a 0.2 .mu.m hollow fiber filter (AGT, Needham, Mass.). The filtered broth was then concentrated 20.times. using a 10K membrane in a Pellicon-2 (Millipore, Bedford, Mass.). 200 g (NH.sub.4).sub.2SO.sub.4 per liter (1.5 M) were added to the concentrated supernatant. The small amount of precipitate produced after addition of (NH.sub.4).sub.2SO.sub.4 was eliminated with centrifugation and filtration. Phenyl Sepharose Fast Flow (Amersham Pharmacia Biotech) was equilibrated with buffer containing 1.5 M (NH.sub.4).sub.2SO.sub.4/10 mM HEPES/5 mM EDTA pH=7.0 (equilibration buffer) at a flow rate of 15 cm/h. After sample loading, the column was washed with 10 column volumes (CV) of equilibration buffer and PA was eluted with a 30 CV linear gradient from 1.5 M to 0 M (NH.sub.4).sub.2SO.sub.4 in 10 mM HEPES/5 mM EDTA; pH=7.0. Fractions were analyzed by SDS-PAGE and the PA-containing samples were pooled for further purification.

b. Expanded Bed Hydrophobic Interaction Chromatography

The cell suspension containing 5 mM EDTA was diluted 1:1 with buffer containing 3.0 M (NH.sub.4).sub.2SO.sub.4/20 mM HEPES/5 mM EDTA and 0.005% Pluronic F-68 (Life Technologies, Inc. Gaithersburg, Md.). STREAMLINE.TM. Phenyl adsorbent. (Amersham Pharmacia Biotech) was expanded in a streamline column in equilibration buffer. The diluted cell suspension was loaded upward at 300 cm/h. The column was washed in expanded mode (2) with 10 CV of equilibration buffer containing 0.005% pluronic F-68. Elution was performed in packed bed mode with 8 CV of elution buffer at 100 cm/h. The eluent was analyzed by SDS-PAGE and radial immunodifussion.

c. Anion Exchange Chromatography

Fractions from HIC were dialyzed against 20 mM Tris pH=8.9 and loaded on a Q Sepharose Fast Flow (Amersham Pharmacia Biotech) column equilibrated with 20 mM Tris pH=8.9 at 15 cm/h. The protein was eluted using a 20 CV linear gradient from 0 to 0.5 M NaCl in the same buffer. PA containing fractions were concentrated and dialyzed against PBS.

d. Gel Filtration

The pooled PA was further purified using a Superdex 75 column (Amersham Pharmacia Biotech) in PBS/5 mM EDTA pH=7.4 at 12 cm/h.

Results and Discussion

a. Expression of Two Recombinant PA: PA-N657A and PA-SNKE-AFF-E308D

The expression of two recombinant versions of PA and the extracellular proteolytic activity of the culture were analyzed (FIG. 1). Production of PA-SNKE-.DELTA.FF-E308D (SEQ ID NO: 4)the protein lacking the furin and chymotrypsin cleavage sites, was nearly 60% higher than that of PA-N657A (SEQ ID NO: 5),the protein containing a mutation in the receptor-binding domain (FIG. 1a). The extracellular proteolytic activity (fluorescence/OD) of both cultures was similar. SDS-PAGE analysis of partially purified PA recovered from these cultures shows higher concentration of smaller fragments in the sample from PA-N657A (SEQ ID NO: 5)compared to the sample from PA-SNKE-.DELTA.FF-E308D (FIG. 1b; SEQ ID NO: 4). Western blot analysis with polyclonal PA antibody confirmed that the smaller fragments were reactive against PA (data not shown). As indicated in FIG. 1a, the proteolytic activity was similar in both strains. Therefore, it was apparent that PA-SNKE-.DELTA.FF-E308D (SEQ ID NO: 4) is a better candidate, due to its stability, and it was selected for further studies.

b. pH Effect

Based on previous information (5, 21), initial production studies with PA-SNKE-.DELTA.FF-E308D (SEQ ID NO: 4) were done by controlling pH with NH.sub.4OH only, which resulted in pH 8.7 at the end of the fermentation. When pH was controlled at 7.4 during the entire fermentation, the PA production was 30 mg per g cell and the proteolytic activity per OD unit was 8, compared to values of 20 mg PA per g cells and proteolytic activity per OD of 30 when the pH control was done only by NH.sub.4OH. When the process was performed at a lower pH, both PA production and protease activity were lower. At pH 6.1 production declined nearly six times and protease activity two times compared to what was found at pH 7.4. Possibly, intracellular expression is lower or secretion is inhibited at low pH. From the above information it is obvious that pH significantly affects the proteolytic activity and the PA expression. Controlling pH throughout the fermentation process resulted in a 30% increase in PA yield, compared to previously reported strategies.

c. Effect of Various Carbon Sources and Protease Inhibitors

Attempts to increase PA expression by supplementing the basic growth medium with different carbon sources is summarized in Table 1.

TABLE-US-00001 TABLE 1 Effect of various carbon sources on PA production. PA production Medium mg PA/g cell mg PA/L culture Basic medium 31.3 129.5 Glycerol + basic medium 23.7 117.3 Glucose + basic medium 25.3 113.3 Lactose + basic medium 33.9 116.0 Casitone + basic medium 28.3 135.1

Neither the volumetric production nor the production per gram cells could be enhanced with the addition of various carbon sources. The effect of PMSF and EDTA on extracellular proteolysis was also examined. As shown in FIG. 2, addition of EDTA (15 mM) significantly reduced proteolytic activity whereas the proteolytic activity of the PMSF-containing fraction (1 g/mL) was similar to that of the control. Based on this information, EDTA was added at the end of the fermentation, before the protein was processed.

d. Growth and Production Conditions

Based on the parameters determined previously, a production process for the recombinant PA-SNKE-.DELTA.FF-E308D (SEQ ID NO: 4)from B. anthracis BH445 was established. The process is based on growth in a batch fermentation controlled at pH 7.5 with NH.sub.4OH/HCl and at 30% dissolved oxygen saturation for a period of 18 hours. A typical fermentation is seen in FIG. 3.

In general, the final OD.sub.600 values fluctuated between 16 to 20. During the first five hours, growth was exponential and the pH was controlled by base addition. Later in the fermentation the pH was controlled by acid addition. Accumulation of PA occurred mostly during the stationary phase and reached a final concentration of 160 mg per liter. The results shown in FIG. 4 indicate that PA degraded if the fermentation was extended for more than 18 hours, therefore, a harvest time between 14 and 18 hours was selected.

Attempts to increase the PA production by implementing a fed-batch growth strategy were conducted. The addition of 10.times. tryptone/yeast extract/salts or 50% glucose/10.times. salts resulted in a 50% increase in cell density but not an increase in protein production (FIG. 5). The observations that PA production was not improved by the implementation of a fed batch growth strategy or by the addition of various carbon sources such as casein, glucose, glycerol or lactose is an indication that perhaps a specific nutritional factor is missing. It is also important to mention that the specific proteolytic activity was almost five times lower when glucose was added to the tryptone/yeast extract media (FIG. 6). This was expected since glucose is known to be a repressor of proteases in Bacillus (10, 25).

e. Purification

The purification protocol developed for PA (Materials and Methods) consisted of hydrophobic interaction chromatography (Phenyl Sepharose) followed by anion exchange (Q Sepharose) and gel filtration (Superdex 75). Replacing the initial capturing step with expanded bed chromatography (2) can simplify and shorten the recovery process since it eliminates the clarification steps. Therefore, the use of expanded bed adsorption (EBA) was investigated by substituting the traditional packed-bed resin (Phenyl Sepharose) with the expanded bed hydrophobic resin STREAMLINE.TM. Phenyl. The static binding capacity for STREAMLINE.TM. Phenyl was approximately 15 mg protein/mL of resin which is comparable to the capacity of Phenyl Sepharose. Optimal binding of PA to S STREAMLINE.TM. Phenyl adsorbent occurred at 1.5 M (NH.sub.4).sub.2SO.sub.4.

Preliminary experiments performed with cell-containing broth in expanded mode resulted in the formation of aggregates and eventual collapse of the bed. It was possible to stabilize the expanded column only after the addition of a detergent which probably altered some of the hydrophobic interactions but did not prevent PA from binding. Pluronic F-68 was chosen due its non-toxicity in humans. The static binding capacities of STREAMLINE.TM. Phenyl adsorbent were 15, 11, and 5 mg protein/mL resin with 0%, 0.005%, and 0.01% pluronic F-68, respectively. Successful operation of the HIC EBA column occurred when using a load concentration of 15 g wet cells/L, 0.8 mL resin/g wet cells, and 0.005% pluronic F-68 in the load as well as the wash buffer. Under these conditions some signs of aggregation appeared at the end of the loading phase but cell debris was eliminated in the washing phase. A 70% recovery was obtained.

PA purity after hydrophobic interaction chromatography was higher than 80%. Further purification was achieved by adding gel filtration step (FIG. 6, Lane b). However, this material was not stable when stored at 4.degree. C. for three months (FIG. 6, Lane c). In contrast, pure and stable PA was obtained after hydrophobic interaction chromatography on expanded bed, followed by anion exchange and gel filtration (FIG. 6, Lane d). Similar results to the expanded bed process were obtained when packed bed hydrophobic interaction chromatography was followed by ion exchange and gel filtration (FIG. 6, Lane a).

Replacing the packed-bed capturing step with expanded bed adsorption proved to be more efficient since it eliminated the centrifugation and filtration steps, however, twenty times more (NH.sub.4).sub.2SO.sub.4 and three times more resin were required to process the same amount of culture (Table 2).

TABLE-US-00002 TABLE 2 Comparison of packed bed and expanded bed absorption as capturing processes for PA Packed Bed Expanded Bed Adsorption 1. Total processing time 15.5 h 1. Total processing time: 8 h a) downstream processing: 6 h a) downstream processing: 1 h (4 unit operations) (1 unit operation) b) loading: 2 h b) loading: 4 h c) column wash: 3.5 h c) column wash: 1.5 h d) elution: 4 h d) elution: 1.5 h 2. 400 g (NH.sub.4).sub.2SO.sub.4 needed 2. 8000 g (NH.sub.4).sub.2SO.sub.4 needed 3. 100 mL resin needed 3. 300 mL resin needed 4. Load/wash steps require little attention 4. Load/wash steps cannot be left unattended 5. 82% recovery 5. 70% recovery

Initial work with hydrophobic interaction chromatography using expanded bed ad sorption to capture PA resulted in bed collapse. This was avoided after the addition of a surfactant (pluronic F-68). These results suggest that the characteristics of the cell membrane were most likely the cause of cell aggregation. Since no polyglutamic acid capsule is present in the recombinant strain, the two hydrophobic membrane proteins forming the S-layer (4, 6) may be responsible for associating with neighboring cell membranes and the resin. After evaluating the possible interactions affecting the system, it was found that successful operation of the expanded bed was possible by carefully adjusting the cell concentration of the load, increasing the adsorbent-to-cell ratio, and choosing the appropriate detergent type and concentration. The expanded bed approach was more efficient in spite of the slightly lower yield (70% vs. 82%) and the higher amount of (NH.sub.4).sub.2SO.sub.4 and resin needed since it eliminated the need for centrifugation and filtration. To obtain stable and highly purified protein, anion exchange and gel filtration steps were added.

Conclusions

Once the gene encoding PA (pagA) was cloned (31) and sequenced (32), several researchers have reported on the expression of PA in hosts like B. subtilis (1, 13, 20, 26), E. coli (8, 24, 31), Salmonella typhimurium (3), viruses (11), and avirulant B. anthracis (5, 15). From these reports, the highest PA yield achieved has been in the order of 50 mg/L in B. anthracis (15). In this work, a scalable fermentation and purification process suitable for vaccine development which produced almost three times more product than what have been reported earlier, is presented. This was accomplished by using a biologically inactive protease-resistant PA variant in a protease-deficient nonsporogenic avirulent strain of B. anthracis.

EXAMPLE 2

Composition of the Vaccines

Four combinations of the recombinant (modified) protective antigen ("rPA") were made: (1) rPA in PBS ("phosphate buffered saline"), (2) rPA in formalin, (3) rPA in aluminum hydroxide and (4) rPA in formalin and aluminum hydroxide. Another formulation of succinylated rPA was prepared and tested (data not shown).

EXAMPLE 3

Immunogenicity in Mice

The four formulations described above were immunogenic in mice, and induced antibody levels comparable to those induced by the currently licensed anthrax vaccine. The induced antibodies had anthrax toxin neutralizing activity. It is planned to evaluate these formulations in humans, and to chose the best one for use as a vaccine.

The data from the mice experiments are set forth in the tables 3 to 5 below:

TABLE-US-00003 TABLE 3 Number of Mice and Immunogen Group Number Number of Mice Immunogen 1056 11 PA (2.5 .mu.g)-Untreated 1057 11 PA (12.5 .mu.g)-Untreated 1058 11 PA (2.5 .mu.g) + Alum 1059 10 PA.sub.SUCC 10:1.25 (2.5 .mu.g) 1060 10 PA.sub.SUCC 10:1.25 (12.5 .mu.g) 1061 10 PA.sub.SUCC 10:3 (2.5 .mu.g) 1062 10 PA.sub.SUCC 10:3 (12.5 .mu.g) 1063 10 PA-Formalin 0.3 (2.5 .mu.g) 1064 10 PA-Formalin 0.3 (12.5 .mu.g) 1065 10 PA-Formalin 3.0 (2.5 .mu.g) 1066 10 PA-Formalin 3.0 (12.5 .mu.g) 1067 10 PA-Formalin 7.12 (2.5 .mu.g) 1068 10 PA-Formalin 7.12 (12.5 .mu.g) 1069 11 Anthrax Vaccine 0.1 ml 1070 10 Control

TABLE-US-00004 TABLE 4 Antibody Levels and Neutralization Titers Mice .mu.g/ml Neutral, Titer 1056A 130.64 4000 1056B 11.24 200 1056K 21.3 1000 1057A 146.65 3000 1057I 490.14 7000 1058A 725.31 8000 E 710.46 7000 J 513.46 4000 1059A 53.89 1500 1060A 125.92 850 1061A 97.1 1500 C 21.2 200 E 54.22 700 1062A 24.9 1500 J 14.35 2000 1063A 68.31 1500 C 179.16 2000 H 564.94 2000 1064A 581.34 10,000 1064D 204.56 8000 E 742.21 11,000 F 418.95 7000 G 814.91 10,000 1065A 77.73 1250 E 214.37 5000 1066C 65.47 4000 D 513.32 10,000 E 248.91 4000 F 260.36 8000 J 1041.65 10,000 1067A 261.54 3000 G 415 5000 1068A 512.99 10,000 I 414.82 5000 1069A 339.18 3000 1069J 879.65 3000 1070E <.05 20

5-6 weeks old female general purpose mice were injected subcutaneously with 0.1 mL of the immunogens depicted in Table 3, 2 or 3 times 2 weeks apart. The mice were exsanguinated one week after the last injection and their sera assayed for IgG anti PA and anthrax toxin neutralization. Antibodies measured by Elisa were related to a standard containing 1.8 mg/ml of anti-PA monoclonal antibody.

TABLE-US-00005 TABLE 5 IgG anti PA levels induced in mice by various rPA formulations dose .times. number PA lot formulation of injections .mu.g/ml 0 PA 2.5.mu. .times. 2 1.3 0 PA 2.5.mu. .times. 3 109.1 2 PA 2.5.mu. .times. 3 24.9 2 PA 12.5.mu. .times. 3 226 0 PA/Al (OH).sub.3 2.5.mu. .times. 2 86.1 0 PA/Al (OH).sub.3 2.5.mu. .times. 3 312. 2 PA/Al (OH).sub.3 2.5.mu. .times. 3 435. 2 PA formalin 0.3 2.5.mu. .times. 3 182 2 PA formalin 0.3 12.5.mu. .times. 3 350. 0 PA formalin 3.0 2.5.mu. .times. 2 2.79 0 PA formalin 3.0 2.5.mu. .times. 3 136.4 0 PA formalin 3.0 5.0.mu. .times. 2 1.98 2 PA formalin 3.0 2.5.mu. .times. 3 220 2 PA formalin 3.0 12.5.mu. .times. 3 270 0 PA formalin 7.12 2.5.mu. .times. 3 266 0 PA formalin 7.12 12.5.mu. .times. 3 229 Anthrax Vaccine 1/10 human dose .times. 2 43.15 1/10 human dose .times. 3 297 PBS control .times.2 <.05 .times.3 <.05

5-6 weeks old female mice, 10 per group, were injected subcutaneously with the listed formulations, 2 or 3 times, two weeks apart and exsanguinated one week after the last injection. Antibodies were measured by Elisa, calculated relative to a standard containing 1.8 mg/ml of anti-PA monoclonal antibody, and expressed as geometric means of the groups.

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The disclosures of all the references cited hereinabove are incorporated by reference herein.

Modifications of the above described modes for carrying out the invention that are obvious to those of skill in the fields of immunology, protein chemistry, microbiology, medicine, and related fields are intended to be within the scope of the following claims.

SEQUENCE LISTINGS

1

1514235DNABacillus anthracisCDS(1804)..(4098) 1aagcttctgt cattcgtaaa tttcaaatag aacgtaaatt tagacttctc atcattaaaa 60atgaaaaatc ttatcttttt gattctattg tatattttta ttaaggtgtt taatagttag 120aaaagacagt tgatgctatt actccagata aaatatagct aaccataaat ttattaaaga 180aaccttgttg ttctaaataa tgattttgtg gattccggaa tagatactgg tgagttagct 240ctaattttat agtgatttaa ctaacaattt ataaagcagc ataattcaaa ttttttaatt 300gatttttcct gaagcatagt ataaaagagt caaggtcttc tagacttgac tcttggaatc 360attaggaatt aacaatatat ataatgcgct agacagaatc aaattaaatg caaaaatgaa 420tattttagta agagatccat atcattatga taataacggt aatattgtag gggttgatga 480ttcatattta aaaaacgcat ataagcaaat acttaattgg tcaagcgatg gagtttcttt 540aaatctagat gaagatgtaa atcaagcact atctggatat atgcttcaaa taaaaaaacc 600ttcaaaccac ctaacaaaca gcccagttac aattacatta gcaggcaagg acagtggtgt 660tggagaattg tatagagtat tatcagatgg agcaggattc ctggatttca ataagtttga 720tgaaaattgg cgatcattag tagatcctgg tgatgatgtt tatgtgtatg ctgttactaa 780agaagatttt aatgcagtta ctcgagatga aaatggtaat atagcgaata aattaaaaaa 840caccttagtt ttatcgggta aaataaaaga aataaacata aaaactacaa atattaatat 900atttgtagtt tttatgttta ttatatacct cctattttat attattagta gcacagtttt 960tgcaaatcat gtaattgtat acttatctat gtagaggtat cacaacttat gaatagtgta 1020ttttattgaa cgttggttag cttggacagt tgtatggata tgcatacttt ataacgtata 1080aaatttcacg caccacaata aaactaattt aacaaaaaca aaaacacacc taagatcatt 1140cagttctttt aataaggagc tgcccaccaa gctaaaccta aataatcttt gtttcacata 1200aggttttttt ctaaatatac agtgtaagtt attgtgaatt taaccagtat atattaaaaa 1260tgttttatgt taacaaatta aattgtaaaa cccctcttaa gcatagttaa gaggggtagg 1320ttttaaattt tttgttgaaa ttagaaaaaa taataaaaaa acaaacctat tttctttcag 1380gttgtttttg ggttacaaaa caaaaagaaa acatgtttca aggtacaata attatggttc 1440tttagctttc tgtaaaacag ccttaatagt tggatttatg actattaaag ttagtataca 1500gcatacacaa tctattgaag gatatttata atgcaattcc ctaaaaatag ttttgtataa 1560ccagttcttt tatccgaact gatacacgta ttttagcata atttttaatg tatcttcaaa 1620aacagcttct gtgtcctttt ctattaaaca tataaattct tttttatgtt atatatttat 1680aaaagttctg tttaaaaagc caaaaataaa taattatctc tttttattta tattatattg 1740aaactaaagt ttattaattt caatataata taaatttaat tttatacaaa aaggagaacg 1800tat atg aaa aaa cga aaa gtg tta ata cca tta atg gca ttg tct acg 1848 Met Lys Lys Arg Lys Val Leu Ile Pro Leu Met Ala Leu Ser Thr 1 5 10 15ata tta gtt tca agc aca ggt aat tta gag gtg att cag gca gaa gtt 1896Ile Leu Val Ser Ser Thr Gly Asn Leu Glu Val Ile Gln Ala Glu Val 20 25 30aaa cag gag aac cgg tta tta aat gaa tca gaa tca agt tcc cag ggg 1944Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser Gln Gly 35 40 45tta cta gga tac tat ttt agt gat ttg aat ttt caa gca ccc atg gtg 1992Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro Met Val 50 55 60gtt acc tct tct act aca ggg gat tta tct att cct agt tct gag tta 2040Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser Glu Leu 65 70 75gaa aat att cca tcg gaa aac caa tat ttt caa tct gct att tgg tca 2088Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile Trp Ser80 85 90 95gga ttt atc aaa gtt aag aag agt gat gaa tat aca ttt gct act tcc 2136Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala Thr Ser 100 105 110gct gat aat cat gta aca atg tgg gta gat gac caa gaa gtg att aat 2184Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val Ile Asn 115 120 125aaa gct tct aat tct aac aaa atc aga tta gaa aaa gga aga tta tat 2232Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg Leu Tyr 130 135 140caa ata aaa att caa tat caa cga gaa aat cct act gaa aaa gga ttg 2280Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys Gly Leu 145 150 155gat ttc aag ttg tac tgg acc gat tct caa aat aaa aaa gaa gtg att 2328Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu Val Ile160 165 170 175tct agt gat aac tta caa ttg cca gaa tta aaa caa aaa tct tcg aac 2376Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser Ser Asn 180 185 190tca aga aaa aag cga agt aca agt gct gga cct acg gtt cca gac cgt 2424Ser Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro Asp Arg 195 200 205gac aat gat gga atc cct gat tca tta gag gta gaa gga tat acg gtt 2472Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr Thr Val 210 215 220gat gtc aaa aat aaa aga act ttt ctt tca cca tgg att tct aat att 2520Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser Asn Ile 225 230 235cat gaa aag aaa gga tta acc aaa tat aaa tca tct cct gaa aaa tgg 2568His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu Lys Trp240 245 250 255agc acg gct tct gat ccg tac agt gat ttc gaa aag gtt aca gga cgg 2616Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr Gly Arg 260 265 270att gat aag aat gta tca cca gag gca aga cac ccc ctt gtg gca gct 2664Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val Ala Ala 275 280 285tat ccg att gta cat gta gat atg gag aat att att ctc tca aaa aat 2712Tyr Pro Ile Val His Val Asp Met Glu Asn Ile Ile Leu Ser Lys Asn 290 295 300gag gat caa tcc aca cag aat act gat agt gaa acg aga aca ata agt 2760Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Glu Thr Arg Thr Ile Ser 305 310 315aaa aat act tct aca agt agg aca cat act agt gaa gta cat gga aat 2808Lys Asn Thr Ser Thr Ser Arg Thr His Thr Ser Glu Val His Gly Asn320 325 330 335gca gaa gtg cat gcg tcg ttc ttt gat att ggt ggg agt gta tct gca 2856Ala Glu Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser Val Ser Ala 340 345 350gga ttt agt aat tcg aat tca agt acg gtc gca att gat cat tca cta 2904Gly Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His Ser Leu 355 360 365tct cta gca ggg gaa aga act tgg gct gaa aca atg ggt tta aat acc 2952Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr Met Gly Leu Asn Thr 370 375 380gct gat aca gca aga tta aat gcc aat att aga tat gta aat act ggg 3000Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn Thr Gly 385 390 395acg gct cca atc tac aac gtg tta cca acg act tcg tta gtg tta gga 3048Thr Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val Leu Gly400 405 410 415aaa aat caa aca ctc gcg aca att aaa gct aag gaa aac caa tta agt 3096Lys Asn Gln Thr Leu Ala Thr Ile Lys Ala Lys Glu Asn Gln Leu Ser 420 425 430caa ata ctt gca cct aat aat tat tat cct tct aaa aac ttg gcg cca 3144Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu Ala Pro 435 440 445atc gca tta aat gca caa gac gat ttc agt tct act cca att aca atg 3192Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile Thr Met 450 455 460aat tac aat caa ttt ctt gag tta gaa aaa acg aaa caa tta aga tta 3240Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu Arg Leu 465 470 475gat acg gat caa gta tat ggg aat ata gca aca tac aat ttt gaa aat 3288Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe Glu Asn480 485 490 495gga aga gtg agg gtg gat aca ggc tcg aac tgg agt gaa gtg tta ccg 3336Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser Glu Val Leu Pro 500 505 510caa att caa gaa aca act gca cgt atc att ttt aat gga aaa gat tta 3384Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe Asn Gly Lys Asp Leu 515 520 525aat ctg gta gaa agg cgg ata gcg gcg gtt aat cct agt gat cca tta 3432Asn Leu Val Glu Arg Arg Ile Ala Ala Val Asn Pro Ser Asp Pro Leu 530 535 540gaa acg act aaa ccg gat atg aca tta aaa gaa gcc ctt aaa ata gca 3480Glu Thr Thr Lys Pro Asp Met Thr Leu Lys Glu Ala Leu Lys Ile Ala 545 550 555ttt gga ttt aac gaa ccg aat gga aac tta caa tat caa ggg aaa gac 3528Phe Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly Lys Asp560 565 570 575ata acc gaa ttt gat ttt aat ttc gat caa caa aca tct caa aat atc 3576Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln Asn Ile 580 585 590aag aat cag tta gcg gaa tta aac gca act aac ata tat act gta tta 3624Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn Ile Tyr Thr Val Leu 595 600 605gat aaa atc aaa tta aat gca aaa atg aat att tta ata aga gat aaa 3672Asp Lys Ile Lys Leu Asn Ala Lys Met Asn Ile Leu Ile Arg Asp Lys 610 615 620cgt ttt cat tat gat aga aat aac ata gca gtt ggg gcg gat gag tca 3720Arg Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp Glu Ser 625 630 635gta gtt aag gag gct cat aga gaa gta att aat tcg tca aca gag gga 3768Val Val Lys Glu Ala His Arg Glu Val Ile Asn Ser Ser Thr Glu Gly640 645 650 655tta ttg tta aat att gat aag gat ata aga aaa ata tta tca ggt tat 3816Leu Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser Gly Tyr 660 665 670att gta gaa att gaa gat act gaa ggg ctt aaa gaa gtt ata aat gac 3864Ile Val Glu Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile Asn Asp 675 680 685aga tat gat atg ttg aat att tct agt tta cgg caa gat gga aaa aca 3912Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly Lys Thr 690 695 700ttt ata gat ttt aaa aaa tat aat gat aaa tta ccg tta tat ata agt 3960Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr Ile Ser 705 710 715aat ccc aat tat aag gta aat gta tat gct gtt act aaa gaa aac act 4008Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu Asn Thr720 725 730 735att att aat cct agt gag aat ggg gat act agt acc aac ggg atc aag 4056Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly Ile Lys 740 745 750aaa att tta atc ttt tct aaa aaa ggc tat gag ata gga taa 4098Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly 755 760ggtaattcta ggtgattttt aaattatcta aaaaacagta aaattaaaac atactctttt 4158tgtaagaaat acaaggagag tatgttttaa acagtaatct aaatcatcat aatcctttga 4218gattgtttgt aggatcc 42352764PRTBacillus anthracis 2Met Lys Lys Arg Lys Val Leu Ile Pro Leu Met Ala Leu Ser Thr Ile1 5 10 15Leu Val Ser Ser Thr Gly Asn Leu Glu Val Ile Gln Ala Glu Val Lys 20 25 30Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser Gln Gly Leu 35 40 45Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro Met Val Val 50 55 60Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser Glu Leu Glu65 70 75 80Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile Trp Ser Gly 85 90 95Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala Thr Ser Ala 100 105 110Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val Ile Asn Lys 115 120 125Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg Leu Tyr Gln 130 135 140Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys Gly Leu Asp145 150 155 160Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu Val Ile Ser 165 170 175Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser Ser Asn Ser 180 185 190Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro Asp Arg Asp 195 200 205Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr Thr Val Asp 210 215 220Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser Asn Ile His225 230 235 240Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu Lys Trp Ser 245 250 255Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr Gly Arg Ile 260 265 270Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val Ala Ala Tyr 275 280 285Pro Ile Val His Val Asp Met Glu Asn Ile Ile Leu Ser Lys Asn Glu 290 295 300Asp Gln Ser Thr Gln Asn Thr Asp Ser Glu Thr Arg Thr Ile Ser Lys305 310 315 320Asn Thr Ser Thr Ser Arg Thr His Thr Ser Glu Val His Gly Asn Ala 325 330 335Glu Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser Val Ser Ala Gly 340 345 350Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His Ser Leu Ser 355 360 365Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr Met Gly Leu Asn Thr Ala 370 375 380Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn Thr Gly Thr385 390 395 400Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val Leu Gly Lys 405 410 415Asn Gln Thr Leu Ala Thr Ile Lys Ala Lys Glu Asn Gln Leu Ser Gln 420 425 430Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu Ala Pro Ile 435 440 445Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile Thr Met Asn 450 455 460Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu Arg Leu Asp465 470 475 480Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe Glu Asn Gly 485 490 495Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser Glu Val Leu Pro Gln 500 505 510Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe Asn Gly Lys Asp Leu Asn 515 520 525Leu Val Glu Arg Arg Ile Ala Ala Val Asn Pro Ser Asp Pro Leu Glu 530 535 540Thr Thr Lys Pro Asp Met Thr Leu Lys Glu Ala Leu Lys Ile Ala Phe545 550 555 560Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly Lys Asp Ile 565 570 575Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln Asn Ile Lys 580 585 590Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn Ile Tyr Thr Val Leu Asp 595 600 605Lys Ile Lys Leu Asn Ala Lys Met Asn Ile Leu Ile Arg Asp Lys Arg 610 615 620Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp Glu Ser Val625 630 635 640Val Lys Glu Ala His Arg Glu Val Ile Asn Ser Ser Thr Glu Gly Leu 645 650 655Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser Gly Tyr Ile 660 665 670Val Glu Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile Asn Asp Arg 675 680 685Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly Lys Thr Phe 690 695 700Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr Ile Ser Asn705 710 715 720Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu Asn Thr Ile 725 730 735Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly Ile Lys Lys 740 745 750Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly 755 7603735PRTBacillus anthracis 3Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser1 5 10 15Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro 20 25 30Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser 35 40 45Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile 50 55 60Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala65 70 75 80Thr Ser Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val 85 90 95Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg 100 105 110Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys 115 120 125Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu 130 135 140Val Ile Ser Ser Asp Asn Leu

Gln Leu Pro Glu Leu Lys Gln Lys Ser145 150 155 160Ser Asn Ser Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro 165 170 175Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr 180 185 190Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser 195 200 205Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu 210 215 220Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr225 230 235 240Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val 245 250 255Ala Ala Tyr Pro Ile Val His Val Asp Met Glu Asn Ile Ile Leu Ser 260 265 270Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Glu Thr Arg Thr 275 280 285Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr Ser Glu Val His 290 295 300Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser Val305 310 315 320Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His 325 330 335Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr Met Gly Leu 340 345 350Asn Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn 355 360 365Thr Gly Thr Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val 370 375 380Leu Gly Lys Asn Gln Thr Leu Ala Thr Ile Lys Ala Lys Glu Asn Gln385 390 395 400Leu Ser Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu 405 410 415Ala Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile 420 425 430Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu 435 440 445Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe 450 455 460Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser Glu Val465 470 475 480Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe Asn Gly Lys 485 490 495Asp Leu Asn Leu Val Glu Arg Arg Ile Ala Ala Val Asn Pro Ser Asp 500 505 510Pro Leu Glu Thr Thr Lys Pro Asp Met Thr Leu Lys Glu Ala Leu Lys 515 520 525Ile Ala Phe Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly 530 535 540Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln545 550 555 560Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn Ile Tyr Thr 565 570 575Val Leu Asp Lys Ile Lys Leu Asn Ala Lys Met Asn Ile Leu Ile Arg 580 585 590Asp Lys Arg Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp 595 600 605Glu Ser Val Val Lys Glu Ala His Arg Glu Val Ile Asn Ser Ser Thr 610 615 620Glu Gly Leu Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser625 630 635 640Gly Tyr Ile Val Glu Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile 645 650 655Asn Asp Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly 660 665 670Lys Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr 675 680 685Ile Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu 690 695 700Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly705 710 715 720Ile Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly 725 730 7354733PRTArtificialPA-SNKE-deltaFF-E308D 4Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser1 5 10 15Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro 20 25 30Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser 35 40 45Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile 50 55 60Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala65 70 75 80Thr Ser Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val 85 90 95Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg 100 105 110Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys 115 120 125Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu 130 135 140Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser145 150 155 160Ser Asn Ser Ser Asn Lys Glu Ser Thr Ser Ala Gly Pro Thr Val Pro 165 170 175Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr 180 185 190Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser 195 200 205Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu 210 215 220Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr225 230 235 240Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val 245 250 255Ala Ala Tyr Pro Ile Val His Val Asp Met Glu Asn Ile Ile Leu Ser 260 265 270Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Glu Thr Arg Thr 275 280 285Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr Ser Glu Val His 290 295 300Gly Asn Ala Asp Val His Ala Ser Asp Ile Gly Gly Ser Val Ser Ala305 310 315 320Gly Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His Ser Leu 325 330 335Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr Met Gly Leu Asn Thr 340 345 350Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn Thr Gly 355 360 365Thr Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val Leu Gly 370 375 380Lys Asn Gln Thr Leu Ala Thr Ile Lys Ala Lys Glu Asn Gln Leu Ser385 390 395 400Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu Ala Pro 405 410 415Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile Thr Met 420 425 430Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu Arg Leu 435 440 445Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe Glu Asn 450 455 460Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser Glu Val Leu Pro465 470 475 480Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe Asn Gly Lys Asp Leu 485 490 495Asn Leu Val Glu Arg Arg Ile Ala Ala Val Asn Pro Ser Asp Pro Leu 500 505 510Glu Thr Thr Lys Pro Asp Met Thr Leu Lys Glu Ala Leu Lys Ile Ala 515 520 525Phe Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly Lys Asp 530 535 540Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln Asn Ile545 550 555 560Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn Ile Tyr Thr Val Leu 565 570 575Asp Lys Ile Lys Leu Asn Ala Lys Met Asn Ile Leu Ile Arg Asp Lys 580 585 590Arg Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp Glu Ser 595 600 605Val Val Lys Glu Ala His Arg Glu Val Ile Asn Ser Ser Thr Glu Gly 610 615 620Leu Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser Gly Tyr625 630 635 640Ile Val Glu Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile Asn Asp 645 650 655Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly Lys Thr 660 665 670Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr Ile Ser 675 680 685Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu Asn Thr 690 695 700Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly Ile Lys705 710 715 720Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly 725 7305735PRTArtificialPA-N657A 5Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser1 5 10 15Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro 20 25 30Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser 35 40 45Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile 50 55 60Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala65 70 75 80Thr Ser Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val 85 90 95Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg 100 105 110Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys 115 120 125Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu 130 135 140Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser145 150 155 160Ser Asn Ser Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro 165 170 175Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr 180 185 190Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser 195 200 205Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu 210 215 220Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr225 230 235 240Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val 245 250 255Ala Ala Tyr Pro Ile Val His Val Asp Met Glu Asn Ile Ile Leu Ser 260 265 270Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Glu Thr Arg Thr 275 280 285Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr Ser Glu Val His 290 295 300Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser Val305 310 315 320Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His 325 330 335Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr Met Gly Leu 340 345 350Asn Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn 355 360 365Thr Gly Thr Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val 370 375 380Leu Gly Lys Asn Gln Thr Leu Ala Thr Ile Lys Ala Lys Glu Asn Gln385 390 395 400Leu Ser Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu 405 410 415Ala Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile 420 425 430Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu 435 440 445Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe 450 455 460Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser Glu Val465 470 475 480Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe Asn Gly Lys 485 490 495Asp Leu Asn Leu Val Glu Arg Arg Ile Ala Ala Val Asn Pro Ser Asp 500 505 510Pro Leu Glu Thr Thr Lys Pro Asp Met Thr Leu Lys Glu Ala Leu Lys 515 520 525Ile Ala Phe Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly 530 535 540Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln545 550 555 560Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn Ile Tyr Thr 565 570 575Val Leu Asp Lys Ile Lys Leu Asn Ala Lys Met Asn Ile Leu Ile Arg 580 585 590Asp Lys Arg Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp 595 600 605Glu Ser Val Val Lys Glu Ala His Arg Glu Val Ile Asn Ser Ser Thr 610 615 620Glu Gly Leu Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser625 630 635 640Gly Tyr Ile Val Glu Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile 645 650 655Ala Asp Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly 660 665 670Lys Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr 675 680 685Ile Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu 690 695 700Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly705 710 715 720Ile Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly 725 730 7356735PRTArtificialK397D D425K mutant 6Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser1 5 10 15Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro 20 25 30Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser 35 40 45Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile 50 55 60Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala65 70 75 80Thr Ser Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val 85 90 95Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg 100 105 110Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys 115 120 125Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu 130 135 140Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser145 150 155 160Ser Asn Ser Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro 165 170 175Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr 180 185 190Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser 195 200 205Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu 210 215 220Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr225 230 235 240Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val 245 250 255Ala Ala Tyr Pro Ile Val His Val Asp Met Glu Asn Ile Ile Leu Ser 260 265 270Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Glu Thr Arg Thr 275 280 285Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr Ser Glu Val His 290 295 300Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser Val305 310 315 320Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His 325 330 335Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr Met Gly Leu 340 345 350Asn Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn 355 360 365Thr Gly Thr Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val 370 375 380Leu Gly Lys Asn Gln Thr Leu Ala Thr Ile Lys Ala Asp Glu Asn Gln385 390 395

400Leu Ser Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu 405 410 415Ala Pro Ile Ala Leu Asn Ala Gln Lys Asp Phe Ser Ser Thr Pro Ile 420 425 430Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu 435 440 445Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe 450 455 460Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser Glu Val465 470 475 480Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe Asn Gly Lys 485 490 495Asp Leu Asn Leu Val Glu Arg Arg Ile Ala Ala Val Asn Pro Ser Asp 500 505 510Pro Leu Glu Thr Thr Lys Pro Asp Met Thr Leu Lys Glu Ala Leu Lys 515 520 525Ile Ala Phe Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly 530 535 540Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln545 550 555 560Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn Ile Tyr Thr 565 570 575Val Leu Asp Lys Ile Lys Leu Asn Ala Lys Met Asn Ile Leu Ile Arg 580 585 590Asp Lys Arg Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp 595 600 605Glu Ser Val Val Lys Glu Ala His Arg Glu Val Ile Asn Ser Ser Thr 610 615 620Glu Gly Leu Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser625 630 635 640Gly Tyr Ile Val Glu Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile 645 650 655Asn Asp Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly 660 665 670Lys Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr 675 680 685Ile Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu 690 695 700Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly705 710 715 720Ile Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly 725 730 7357739PRTArtificialPA-L1 with MMP cleavage site 7Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser1 5 10 15Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro 20 25 30Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser 35 40 45Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile 50 55 60Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala65 70 75 80Thr Ser Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val 85 90 95Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg 100 105 110Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys 115 120 125Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu 130 135 140Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser145 150 155 160Ser Asn Ser Gly Pro Leu Gly Met Leu Ser Gln Ser Thr Ser Ala Gly 165 170 175Pro Thr Val Pro Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu 180 185 190Val Glu Gly Tyr Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser 195 200 205Pro Trp Ile Ser Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys 210 215 220Ser Ser Pro Glu Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe225 230 235 240Glu Lys Val Thr Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg 245 250 255His Pro Leu Val Ala Ala Tyr Pro Ile Val His Val Asp Met Glu Asn 260 265 270Ile Ile Leu Ser Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser 275 280 285Glu Thr Arg Thr Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr 290 295 300Ser Glu Val His Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp Ile305 310 315 320Gly Gly Ser Val Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr Val 325 330 335Ala Ile Asp His Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu 340 345 350Thr Met Gly Leu Asn Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile 355 360 365Arg Tyr Val Asn Thr Gly Thr Ala Pro Ile Tyr Asn Val Leu Pro Thr 370 375 380Thr Ser Leu Val Leu Gly Lys Asn Gln Thr Leu Ala Thr Ile Lys Ala385 390 395 400Lys Glu Asn Gln Leu Ser Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro 405 410 415Ser Lys Asn Leu Ala Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser 420 425 430Ser Thr Pro Ile Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys 435 440 445Thr Lys Gln Leu Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala 450 455 460Thr Tyr Asn Phe Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn465 470 475 480Trp Ser Glu Val Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile 485 490 495Phe Asn Gly Lys Asp Leu Asn Leu Val Glu Arg Arg Ile Ala Ala Val 500 505 510Asn Pro Ser Asp Pro Leu Glu Thr Thr Lys Pro Asp Met Thr Leu Lys 515 520 525Glu Ala Leu Lys Ile Ala Phe Gly Phe Asn Glu Pro Asn Gly Asn Leu 530 535 540Gln Tyr Gln Gly Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln545 550 555 560Gln Thr Ser Gln Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr 565 570 575Asn Ile Tyr Thr Val Leu Asp Lys Ile Lys Leu Asn Ala Lys Met Asn 580 585 590Ile Leu Ile Arg Asp Lys Arg Phe His Tyr Asp Arg Asn Asn Ile Ala 595 600 605Val Gly Ala Asp Glu Ser Val Val Lys Glu Ala His Arg Glu Val Ile 610 615 620Asn Ser Ser Thr Glu Gly Leu Leu Leu Asn Ile Asp Lys Asp Ile Arg625 630 635 640Lys Ile Leu Ser Gly Tyr Ile Val Glu Ile Glu Asp Thr Glu Gly Leu 645 650 655Lys Glu Val Ile Asn Asp Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu 660 665 670Arg Gln Asp Gly Lys Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys 675 680 685Leu Pro Leu Tyr Ile Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala 690 695 700Val Thr Lys Glu Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr705 710 715 720Ser Thr Asn Gly Ile Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr 725 730 735Glu Ile Gly8739PRTArtificialPA-L2 cleavable with MMP 8Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser1 5 10 15Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro 20 25 30Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser 35 40 45Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile 50 55 60Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala65 70 75 80Thr Ser Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val 85 90 95Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg 100 105 110Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys 115 120 125Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu 130 135 140Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser145 150 155 160Ser Asn Ser Gly Pro Leu Gly Leu Trp Ala Gln Ser Thr Ser Ala Gly 165 170 175Pro Thr Val Pro Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu 180 185 190Val Glu Gly Tyr Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser 195 200 205Pro Trp Ile Ser Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys 210 215 220Ser Ser Pro Glu Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe225 230 235 240Glu Lys Val Thr Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg 245 250 255His Pro Leu Val Ala Ala Tyr Pro Ile Val His Val Asp Met Glu Asn 260 265 270Ile Ile Leu Ser Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser 275 280 285Glu Thr Arg Thr Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr 290 295 300Ser Glu Val His Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp Ile305 310 315 320Gly Gly Ser Val Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr Val 325 330 335Ala Ile Asp His Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu 340 345 350Thr Met Gly Leu Asn Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile 355 360 365Arg Tyr Val Asn Thr Gly Thr Ala Pro Ile Tyr Asn Val Leu Pro Thr 370 375 380Thr Ser Leu Val Leu Gly Lys Asn Gln Thr Leu Ala Thr Ile Lys Ala385 390 395 400Lys Glu Asn Gln Leu Ser Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro 405 410 415Ser Lys Asn Leu Ala Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser 420 425 430Ser Thr Pro Ile Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys 435 440 445Thr Lys Gln Leu Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala 450 455 460Thr Tyr Asn Phe Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn465 470 475 480Trp Ser Glu Val Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile 485 490 495Phe Asn Gly Lys Asp Leu Asn Leu Val Glu Arg Arg Ile Ala Ala Val 500 505 510Asn Pro Ser Asp Pro Leu Glu Thr Thr Lys Pro Asp Met Thr Leu Lys 515 520 525Glu Ala Leu Lys Ile Ala Phe Gly Phe Asn Glu Pro Asn Gly Asn Leu 530 535 540Gln Tyr Gln Gly Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln545 550 555 560Gln Thr Ser Gln Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr 565 570 575Asn Ile Tyr Thr Val Leu Asp Lys Ile Lys Leu Asn Ala Lys Met Asn 580 585 590Ile Leu Ile Arg Asp Lys Arg Phe His Tyr Asp Arg Asn Asn Ile Ala 595 600 605Val Gly Ala Asp Glu Ser Val Val Lys Glu Ala His Arg Glu Val Ile 610 615 620Asn Ser Ser Thr Glu Gly Leu Leu Leu Asn Ile Asp Lys Asp Ile Arg625 630 635 640Lys Ile Leu Ser Gly Tyr Ile Val Glu Ile Glu Asp Thr Glu Gly Leu 645 650 655Lys Glu Val Ile Asn Asp Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu 660 665 670Arg Gln Asp Gly Lys Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys 675 680 685Leu Pro Leu Tyr Ile Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala 690 695 700Val Thr Lys Glu Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr705 710 715 720Ser Thr Asn Gly Ile Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr 725 730 735Glu Ile Gly9740PRTArtificialPrAg-U1 cleavable by uPA/tPA 9Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser1 5 10 15Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro 20 25 30Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser 35 40 45Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile 50 55 60Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala65 70 75 80Thr Ser Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val 85 90 95Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg 100 105 110Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys 115 120 125Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu 130 135 140Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser145 150 155 160Ser Asn Ser Pro Cys Pro Gly Arg Val Val Gly Gly Ser Thr Ser Ala 165 170 175Gly Pro Thr Val Pro Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu 180 185 190Glu Val Glu Gly Tyr Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu 195 200 205Ser Pro Trp Ile Ser Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr 210 215 220Lys Ser Ser Pro Glu Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp225 230 235 240Phe Glu Lys Val Thr Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala 245 250 255Arg His Pro Leu Val Ala Ala Tyr Pro Ile Val His Val Asp Met Glu 260 265 270Asn Ile Ile Leu Ser Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp 275 280 285Ser Glu Thr Arg Thr Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His 290 295 300Thr Ser Glu Val His Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp305 310 315 320Ile Gly Gly Ser Val Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr 325 330 335Val Ala Ile Asp His Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala 340 345 350Glu Thr Met Gly Leu Asn Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn 355 360 365Ile Arg Tyr Val Asn Thr Gly Thr Ala Pro Ile Tyr Asn Val Leu Pro 370 375 380Thr Thr Ser Leu Val Leu Gly Lys Asn Gln Thr Leu Ala Thr Ile Lys385 390 395 400Ala Lys Glu Asn Gln Leu Ser Gln Ile Leu Ala Pro Asn Asn Tyr Tyr 405 410 415Pro Ser Lys Asn Leu Ala Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe 420 425 430Ser Ser Thr Pro Ile Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu 435 440 445Lys Thr Lys Gln Leu Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile 450 455 460Ala Thr Tyr Asn Phe Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser465 470 475 480Asn Trp Ser Glu Val Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile 485 490 495Ile Phe Asn Gly Lys Asp Leu Asn Leu Val Glu Arg Arg Ile Ala Ala 500 505 510Val Asn Pro Ser Asp Pro Leu Glu Thr Thr Lys Pro Asp Met Thr Leu 515 520 525Lys Glu Ala Leu Lys Ile Ala Phe Gly Phe Asn Glu Pro Asn Gly Asn 530 535 540Leu Gln Tyr Gln Gly Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp545 550 555 560Gln Gln Thr Ser Gln Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala 565 570 575Thr Asn Ile Tyr Thr Val Leu Asp Lys Ile Lys Leu Asn Ala Lys Met 580 585 590Asn Ile Leu Ile Arg Asp Lys Arg Phe His Tyr Asp Arg Asn Asn Ile 595 600 605Ala Val Gly Ala Asp Glu Ser Val Val Lys Glu Ala His Arg Glu Val 610 615 620Ile Asn Ser Ser Thr Glu Gly Leu Leu Leu Asn Ile Asp Lys Asp Ile625 630 635 640Arg Lys Ile Leu Ser Gly Tyr

Ile Val Glu Ile Glu Asp Thr Glu Gly 645 650 655Leu Lys Glu Val Ile Asn Asp Arg Tyr Asp Met Leu Asn Ile Ser Ser 660 665 670Leu Arg Gln Asp Gly Lys Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp 675 680 685Lys Leu Pro Leu Tyr Ile Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr 690 695 700Ala Val Thr Lys Glu Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp705 710 715 720Thr Ser Thr Asn Gly Ile Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly 725 730 735Tyr Glu Ile Gly 74010738PRTArtificialPrAg-U2 cleavable by uPA 10Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser1 5 10 15Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro 20 25 30Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser 35 40 45Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile 50 55 60Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala65 70 75 80Thr Ser Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val 85 90 95Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg 100 105 110Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys 115 120 125Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu 130 135 140Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser145 150 155 160Ser Asn Ser Pro Gly Ser Gly Arg Ser Ala Ser Thr Ser Ala Gly Pro 165 170 175Thr Val Pro Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val 180 185 190Glu Gly Tyr Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro 195 200 205Trp Ile Ser Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser 210 215 220Ser Pro Glu Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu225 230 235 240Lys Val Thr Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His 245 250 255Pro Leu Val Ala Ala Tyr Pro Ile Val His Val Asp Met Glu Asn Ile 260 265 270Ile Leu Ser Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Glu 275 280 285Thr Arg Thr Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr Ser 290 295 300Glu Val His Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp Ile Gly305 310 315 320Gly Ser Val Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr Val Ala 325 330 335Ile Asp His Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr 340 345 350Met Gly Leu Asn Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg 355 360 365Tyr Val Asn Thr Gly Thr Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr 370 375 380Ser Leu Val Leu Gly Lys Asn Gln Thr Leu Ala Thr Ile Lys Ala Lys385 390 395 400Glu Asn Gln Leu Ser Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser 405 410 415Lys Asn Leu Ala Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser 420 425 430Thr Pro Ile Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr 435 440 445Lys Gln Leu Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr 450 455 460Tyr Asn Phe Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp465 470 475 480Ser Glu Val Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe 485 490 495Asn Gly Lys Asp Leu Asn Leu Val Glu Arg Arg Ile Ala Ala Val Asn 500 505 510Pro Ser Asp Pro Leu Glu Thr Thr Lys Pro Asp Met Thr Leu Lys Glu 515 520 525Ala Leu Lys Ile Ala Phe Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln 530 535 540Tyr Gln Gly Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln545 550 555 560Thr Ser Gln Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn 565 570 575Ile Tyr Thr Val Leu Asp Lys Ile Lys Leu Asn Ala Lys Met Asn Ile 580 585 590Leu Ile Arg Asp Lys Arg Phe His Tyr Asp Arg Asn Asn Ile Ala Val 595 600 605Gly Ala Asp Glu Ser Val Val Lys Glu Ala His Arg Glu Val Ile Asn 610 615 620Ser Ser Thr Glu Gly Leu Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys625 630 635 640Ile Leu Ser Gly Tyr Ile Val Glu Ile Glu Asp Thr Glu Gly Leu Lys 645 650 655Glu Val Ile Asn Asp Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg 660 665 670Gln Asp Gly Lys Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu 675 680 685Pro Leu Tyr Ile Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val 690 695 700Thr Lys Glu Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser705 710 715 720Thr Asn Gly Ile Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu 725 730 735Ile Gly 11738PRTArtificialPrAg-U3 cleavable by uPA 11Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser1 5 10 15Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro 20 25 30Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser 35 40 45Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile 50 55 60Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala65 70 75 80Thr Ser Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val 85 90 95Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg 100 105 110Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys 115 120 125Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu 130 135 140Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser145 150 155 160Ser Asn Ser Pro Gly Ser Gly Lys Ser Ala Ser Thr Ser Ala Gly Pro 165 170 175Thr Val Pro Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val 180 185 190Glu Gly Tyr Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro 195 200 205Trp Ile Ser Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser 210 215 220Ser Pro Glu Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu225 230 235 240Lys Val Thr Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His 245 250 255Pro Leu Val Ala Ala Tyr Pro Ile Val His Val Asp Met Glu Asn Ile 260 265 270Ile Leu Ser Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Glu 275 280 285Thr Arg Thr Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr Ser 290 295 300Glu Val His Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp Ile Gly305 310 315 320Gly Ser Val Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr Val Ala 325 330 335Ile Asp His Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr 340 345 350Met Gly Leu Asn Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg 355 360 365Tyr Val Asn Thr Gly Thr Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr 370 375 380Ser Leu Val Leu Gly Lys Asn Gln Thr Leu Ala Thr Ile Lys Ala Lys385 390 395 400Glu Asn Gln Leu Ser Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser 405 410 415Lys Asn Leu Ala Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser 420 425 430Thr Pro Ile Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr 435 440 445Lys Gln Leu Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr 450 455 460Tyr Asn Phe Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp465 470 475 480Ser Glu Val Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe 485 490 495Asn Gly Lys Asp Leu Asn Leu Val Glu Arg Arg Ile Ala Ala Val Asn 500 505 510Pro Ser Asp Pro Leu Glu Thr Thr Lys Pro Asp Met Thr Leu Lys Glu 515 520 525Ala Leu Lys Ile Ala Phe Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln 530 535 540Tyr Gln Gly Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln545 550 555 560Thr Ser Gln Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn 565 570 575Ile Tyr Thr Val Leu Asp Lys Ile Lys Leu Asn Ala Lys Met Asn Ile 580 585 590Leu Ile Arg Asp Lys Arg Phe His Tyr Asp Arg Asn Asn Ile Ala Val 595 600 605Gly Ala Asp Glu Ser Val Val Lys Glu Ala His Arg Glu Val Ile Asn 610 615 620Ser Ser Thr Glu Gly Leu Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys625 630 635 640Ile Leu Ser Gly Tyr Ile Val Glu Ile Glu Asp Thr Glu Gly Leu Lys 645 650 655Glu Val Ile Asn Asp Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg 660 665 670Gln Asp Gly Lys Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu 675 680 685Pro Leu Tyr Ile Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val 690 695 700Thr Lys Glu Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser705 710 715 720Thr Asn Gly Ile Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu 725 730 735Ile Gly12738PRTArtificialPrAg-U4 cleavable by tPA 12Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser1 5 10 15Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro 20 25 30Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser 35 40 45Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile 50 55 60Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala65 70 75 80Thr Ser Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val 85 90 95Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg 100 105 110Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys 115 120 125Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu 130 135 140Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser145 150 155 160Ser Asn Ser Pro Gln Arg Gly Arg Ser Ala Ser Thr Ser Ala Gly Pro 165 170 175Thr Val Pro Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val 180 185 190Glu Gly Tyr Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro 195 200 205Trp Ile Ser Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser 210 215 220Ser Pro Glu Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu225 230 235 240Lys Val Thr Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His 245 250 255Pro Leu Val Ala Ala Tyr Pro Ile Val His Val Asp Met Glu Asn Ile 260 265 270Ile Leu Ser Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Glu 275 280 285Thr Arg Thr Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr Ser 290 295 300Glu Val His Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp Ile Gly305 310 315 320Gly Ser Val Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr Val Ala 325 330 335Ile Asp His Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr 340 345 350Met Gly Leu Asn Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg 355 360 365Tyr Val Asn Thr Gly Thr Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr 370 375 380Ser Leu Val Leu Gly Lys Asn Gln Thr Leu Ala Thr Ile Lys Ala Lys385 390 395 400Glu Asn Gln Leu Ser Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser 405 410 415Lys Asn Leu Ala Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser 420 425 430Thr Pro Ile Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr 435 440 445Lys Gln Leu Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr 450 455 460Tyr Asn Phe Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp465 470 475 480Ser Glu Val Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe 485 490 495Asn Gly Lys Asp Leu Asn Leu Val Glu Arg Arg Ile Ala Ala Val Asn 500 505 510Pro Ser Asp Pro Leu Glu Thr Thr Lys Pro Asp Met Thr Leu Lys Glu 515 520 525Ala Leu Lys Ile Ala Phe Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln 530 535 540Tyr Gln Gly Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln545 550 555 560Thr Ser Gln Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn 565 570 575Ile Tyr Thr Val Leu Asp Lys Ile Lys Leu Asn Ala Lys Met Asn Ile 580 585 590Leu Ile Arg Asp Lys Arg Phe His Tyr Asp Arg Asn Asn Ile Ala Val 595 600 605Gly Ala Asp Glu Ser Val Val Lys Glu Ala His Arg Glu Val Ile Asn 610 615 620Ser Ser Thr Glu Gly Leu Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys625 630 635 640Ile Leu Ser Gly Tyr Ile Val Glu Ile Glu Asp Thr Glu Gly Leu Lys 645 650 655Glu Val Ile Asn Asp Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg 660 665 670Gln Asp Gly Lys Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu 675 680 685Pro Leu Tyr Ile Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val 690 695 700Thr Lys Glu Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser705 710 715 720Thr Asn Gly Ile Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu 725 730 735Ile Gly13590PRTArtificialamino acids 175-764 of PA 13Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser Ser1 5 10 15Asn Ser Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro Asp 20 25 30Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr Thr 35 40 45Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser Asn 50 55 60Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu Lys65 70 75 80Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr Gly 85 90 95Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val Ala 100 105 110Ala Tyr Pro Ile Val His Val Asp Met Glu Asn Ile Ile Leu Ser Lys 115 120 125Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Glu Thr Arg Thr Ile

130 135 140Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr Ser Glu Val His Gly145 150 155 160Asn Ala Glu Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser Val Ser 165 170 175Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His Ser 180 185 190Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr Met Gly Leu Asn 195 200 205Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn Thr 210 215 220Gly Thr Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val Leu225 230 235 240Gly Lys Asn Gln Thr Leu Ala Thr Ile Lys Ala Lys Glu Asn Gln Leu 245 250 255Ser Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu Ala 260 265 270Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile Thr 275 280 285Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu Arg 290 295 300Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe Glu305 310 315 320Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser Glu Val Leu 325 330 335Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe Asn Gly Lys Asp 340 345 350Leu Asn Leu Val Glu Arg Arg Ile Ala Ala Val Asn Pro Ser Asp Pro 355 360 365Leu Glu Thr Thr Lys Pro Asp Met Thr Leu Lys Glu Ala Leu Lys Ile 370 375 380Ala Phe Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly Lys385 390 395 400Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln Asn 405 410 415Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn Ile Tyr Thr Val 420 425 430Leu Asp Lys Ile Lys Leu Asn Ala Lys Met Asn Ile Leu Ile Arg Asp 435 440 445Lys Arg Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp Glu 450 455 460Ser Val Val Lys Glu Ala His Arg Glu Val Ile Asn Ser Ser Thr Glu465 470 475 480Gly Leu Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser Gly 485 490 495Tyr Ile Val Glu Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile Asn 500 505 510Asp Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly Lys 515 520 525Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr Ile 530 535 540Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu Asn545 550 555 560Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly Ile 565 570 575Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly 580 585 590144PRTBacillus anthracis 14Arg Lys Lys Arg1154PRTArtificialSNKE mutation 15Ser Asn Lys Glu1