WO2009126355A2

WO2009126355A2 - Methods of increasing recombinant production of bacillus anthracis protective antigen

Info

Publication number: WO2009126355A2
Application number: PCT/US2009/031845
Authority: WO
Inventors: Milan Blake; Mikhail Donets; Karen Long-Rowe
Original assignee: Baxter International, Inc.
Priority date: 2008-01-28
Filing date: 2009-01-23
Publication date: 2009-10-15
Also published as: WO2009126355A3

Abstract

The present invention is directed to methods for the recombinant production of the Protective Antigen protein ("rPA"), or antigenic fragments thereof, of Bacillus anthracis, which methods exhibit improved efficiency relative to current methods known in the art. In particular, the invention provides nucleic acid sequences encoding rPA, or antigenic fragments thereof, that have been optimized for use in standard recombinant production procedures and/or systems, including E coli recombinant systems. The methods of the invention allow for the efficient expression of rPA, improving protein yields and lowering production costs. The invention further provides methods of manufacture of rPA from recombinant systems, unparticular for use in pharmaceutical compositions for inducing a therapeutic or prophylactic, i.e. protective, immune response in a subject. Accordingly, the methods of the invention also encompass the use of pharmaceutical compositions comprising rPA for the prevention or treatment of infection by Bacillus anthracis (i.e., anthrax).

Description

METHODS OF INCREASING RECOMBINANT PRODUCTION OF BACILLUS ANTHRACIS PROTECTIVE ANTIGEN

1. FIELD OF THE INVENTION

[0001] The present invention is directed to methods for the recombinant production of the Protective Antigen protein ("rPA"), or antigenic fragments thereof, of Bacillus anthracis, which methods exhibit improved efficiency relative to current methods known in the art. In particular, the invention provides nucleic acid sequences encoding rPA, or antigenic fragments thereof, that have been optimized for use in standard recombinant production procedures and/or systems, including E coli recombinant systems. The methods of the invention allow for the efficient expression of rPA, improving protein yields and lowering production costs. The invention further provides methods of manufacture of rPA from recombinant systems, in particular for use in pharmaceutical compositions for inducing a therapeutic or prophylactic, i.e. protective, immune response in a subject. Accordingly, the methods of the invention also encompass the use of pharmaceutical compositions comprising rPA for the prevention or treatment of infection by Bacillus anthracis {i.e., anthrax).

2. BACKGROUND OF THE INVENTION

[0002] Bacillus anthracis (B. anthracis) is a gram-positive, spore-forming bacterium that is most widely known as the etio logic agent responsible for anthrax in man and animals. Classically, the population most at risk for development of anthrax was restricted to individuals routinely exposed to infected animals and/or their products: for example, veterinarians, laboratory technicians, ranchers, and employees working with skin or hair of animals. However, anthrax has become recognized as a potential bioweapon, expanding the potential at-risk population and increasing the need for affordable, defined vaccine components.

[0003] Anthrax may present as an intestinal, pulmonary, or cutaneous form, depending on the mode of infection, and, in severe cases, may lead to the death of the infected subject. The pathogenicity of B. anthracis is mediated by three protenaceous agents, together forming "anthrax toxin:" (i) lethal factor ("LF"), thought to kill host cells by disrupting the mito gen-activated protein kinase pathway, (ii) edema factor ("EF"), an adenylyl cyclase causing edema in the infected host and (iii) protective antigen ("PA"), which binds to eukaryotic cell surface receptors and mediates transport of LF or EF into the cell. LF and EF require the presence of PA for activity and none of the toxin components are known

1011497 vl -1- to be toxic alone. The three toxin components, PA, LF and EF, are known in the art (e.g., Fish et al. 1968, J. Bacteriol. 95: 907-17; hereby incorporated by reference in its entirety), as are the combinations of PA and LF and of PA and EF (e.g., Leppla et al., 1982, PNAS-USA 79-3162-66; hereby incoporated by reference in its entirety).

[0004] Currently available anthrax vaccines in the United States and Europe consist of alum precipitated material from cultures of toxic, non-encapsulated strain of B. anthracis. Immunization with these vaccines requires several boosters and occasionally causes local pain and edema. Efforts to better define vaccine components have focused on the Protective Atnigen protein ("PA"), so named because, during initial immunization studies, the protein was discovered to be able to elicit an immune response that afforded protective immunity against subsequent infection by B. anthracis. Both native and recombinant PA preparations have been shown to elicit high antibody response. Thus, PA protein can be used to develop an effective acellular recombinant vaccine against anthrax.

[0005] Eschericia coli is well known as an expression system for a range of recombinant protein products, including components of vaccines for human use. Although the successful recombinant expression of PA genes using E. coli has been reported, attempts at high level production have been hindered by low protein yields and proteolytic degradation (e.g., Chauhan et al., 2001, Biochem. and Biophys. Res. Comm. 283:308-315; Singh et al., 1989, J. Biol. Chem. 264:11099-11102; Vodkin et al., 1993, Cell 34:693-697; Sharma et al., 1996, Protein Expr. Purif. 7:33-38; each of which is hereby incorporated by reference in its entirety). However, such poor results may not be protein specific, but instead a factor of the expression system. Constitutive expression systems are often characterized by low protein yield due to any number of conditions, including low protein expression levels, rapid degradation of the protein inside the host cells (due to prolonged exposure to cellular proteases), and/or cell death due to cytotoxicity of the recombinant protein. [0006] One method to improve protein expression in recombinant systems is codon optimization. Codon optimization takes advantage of the redundancy in the genetic code and the fact that differing organisms exhibit differing preferences with respect to specific codon usage. Using codon optimization, the nucleic acid sequence derived from one organism is modified such that the encoded amino acid sequence remains unchanged, but that the codons encoding the individual amino acids correspond to the preferred codons of the host organism. By optimizing codons for a particular expression system, it is generally expected that expression levels of the encoded protein will be enhanced. Indeed, it has been reported that

1011497 vl codon optimization of a recombinant PA gene results in expression of unexpectedly high levels of recombinant PA in E. coli (see, e.g., International Patent Application Publication No.: WO 02/04646, hereby incorporated by reference in its entirety).

3. SUMMARY OF THE INVENTION

[0007] Among the various aspects of the present invention is the provision of an effective acellular recombinant vaccine, and methods of producing such a vaccine, that provides protective immunity from infection by Bacillus anthracis (anthrax). This vaccine comprises any immunogenic protein derived from B. anthracis that confers a prophylactic immune response to infection by the bacteria or a therapeutic effect when administered to one exposed to Anthrax toxin. In certain embodiments, the vaccine comprises a recombinant Protective Antigen protein (rPA) that has been optimized for efficient and economic production through expression in E. coli. In certain embodiments, optimized includes protein modifications to reduce proteolytic degradation and/or cell toxicity from the higher expression levels. The polypeptides of the invention comprise amino acid sequences of B. anthracis proteins, which sequences contain one or more amino acid residue modifications (including, e.g., insertions, substitutions or deletions) relative to the wild-type protein. A polynucleotide encoding a polypeptide of the invention, e.g., rPA, or fragment thereof, may be obtained, and the nucleotide sequence thereof determined, by any method and from any suitable source described herein and/or known in the art. Non-limiting examples of sources of nucleic acids encoding B. anthracis proteins include, nucleic acids isolated from any cell culture, tissue sample, or cell sample comprising B. anthracis {e.g., B. Anthracis cell culture, tissue sample comprising a B. anthracis infection) and cDNA libraries of B. anthracis. [0008] In certain embodiments, the polypeptides of the invention comprise a derivative of LF, EF or PA, or an antigenic fragment thereof. In particular, the invention encompasses B. anthracis proteins comprising one or more amino acid modifications that eliminate one or more protease recognition sequences within said protein, i.e., within said polypeptide chain. The protease recognition sequences may be eliminated from said polypeptide chain by any method described herein or known in the art to disrupt protease recognition of its target sequence. Commonly, protease recognition sequences are eliminated within a polypeptide chain by altering the residues within such sequences. Accordingly, the invention encompasses eliminating a protease recognition sequence within a recombinant B. anthracis protein by modifying the amino acid residues of said sequence by, e.g.,

1011497 vl substitution, insertion or deletion. Nonlimiting examples of protease recognition sequences that may be modified according to the methods of the invention include serine protease (e.g., chemotrypsin, trypsin, elastase, etc.), threonine protease, cysteine protease (e.g., papain, cathepsin, caspase, calpain, etc.), aspartic acid protease (chymosin, renin, cathepsin D, pepsin, plasmepsin, etc.), metalloprotease, glutamic acid protease and/or furin recognition sequences.

[0009] In specific embodiments, the invention encompasses a recombinant B. anthracis PA protein comprising one or more amino acid modifications that eliminate one or more protease recognition sequences within said protein. In specific embodiments, the invention encompasses a modified PA, or antigenic fragment thereof, comprising one or more amino acid modifications relative to wild-type PA, wherein said one or more modifications is at one or more positions corresponding to position 1, 164, 165, 167, 266, 285, 308, 313 or 314 of SEQ ID NO:2. In certain embodiments, the wild-type PA that is modified comprises SEQ ID NO:2.

[0010] The invention encompasses a modified PA polypeptide comprising one or more amino acid modifications that alter one or more chymotrypsin recognition sequences within the polypeptide. In a specific example in accordance with this embodiment, the one or more modifications comprises a modification at position 285. In a preferred embodiment, the modification at position 285 is a substitution at position 285. In a more preferred embodiment, the substitution at position 285 is a substitution with glutamic acid. In another specific embodiment, the invention encompasses a modified PA polypeptide comprising one or more amino acid modifications that alter one or more chymotrypsin recognition sequences within the polypeptide, which one or more modifications comprises a modification at position 308. In a preferred example in accordance with this embodiment, the modification at position 308 is a substitution at position 308. In a more preferred embodiment, the substitution at position 308 is a substitution with aspartic acid. In still another specific embodiment, the invention encompasses a modified PA polypeptide comprising one or more amino acid modifications that alter one or more chymotrypsin recognition sequences within the polypeptide, which one or more modifications comprises a modification at position 313. In a preferred example in accordance with this embodiment, the modification at position 313 is a deletion of residue 313. In yet another specific embodiment, the invention encompasses a modified PA polypeptide comprising one or more amino acid modifications that alter one or more chymotrypsin recognition sequences within the polypeptide, which one or more

1011497 vl modifications comprises a modification at position 314. In a preferred embodiment, the modification at position 314 is a deletion of residue 314.

[0011] In yet other embodiments, the invention encompasses a modified PA polypeptide comprising one or more amino acid modifications that alter one or more furin recognition sequences within the polypeptide. In a specific embodiment, the invention encompasses a modified PA polypeptide, or antigenic fragment thereof, comprising one or more amino acid modifications that alter one or more furin recognition sequences, wherein said one or more furin recognition sequences comprises the furin recognition sequence spanning residues 164-167 of SEQ ID NO:2, i.e., SEQ ID NO:3. In yet other embodiments, the one or more modified furin recognition sequence does not span residues 164-167 of the PA polypeptide. The furin recognition sequence, SEQ ID NO:3 may be modified by modification at one or more of residues 1, 2, 3, or 4, e.g.., corresponding to residues 164, 165, 166, or 167 of SEQ ID NO:2, respectively. In a more preferred embodiment, the furin recognition sequence SEQ ID NO:3 is modified at one or more of residues 1, 2 and 4. In a still more preferred embodiment, the furin recognition sequence SEQ ID NO: 3 is modified at each of residues 1, 2 and 4 by substitution at 1, 2 and 4. In a specific example in accordance with this embodiment, SEQ ID NO: 3 is modified by substitution at positions 1, 2 and 4 to produce SEQ ID NO:4.

[0012] In specific embodiments, the invention encompasses polypeptides, or fragments thereof, comprising one or more of the aforementioned amino acid modifications, wherein said polypeptides comprise SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:16.

[0013] In related non limiting embodiments, the invention encompasses isolated nucleotide sequences encoding one or more of the aforementioned polypeptides. In a specific example in accordance with this embodiment, the isolated nucleic acids encoding the polypeptides comprising SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO: 14, or SEQ ID NO: 16 comprise SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 15, respectively.

[0014] The amino acid sequence of the polypeptide of the invention may also be designed to reduce or eliminate formation of secondary transcription products. For example, in some cases, a nucleic acid sequence may, once transcribed to RNA, comprise favorable locations for the initiation of translation other than at the expected translation initiation site, i.e., traditionally at or near the 5' end of the gene (e.g., translation from an internal ribosome

1011497 vl recognition/entry site; or from an internal codon encoding methionine (i.e., an internal methionine) etc.). Such sites are known as internal entry sites and lead to parallel translation from a single mRNA. Parallel translation can result in decreased efficiency of recombinant production systems, for example, by directing the production of non- functional truncations of a polypeptide of interest and/or the use of cellular resources that would otherwise be used for the production of the full-length polypeptide of interest. Accordingly, identifying and eliminating the internal translation ignition sites can lead to improved recombinant production, e.g. , improved efficiency and/or improved yield. Accordingly, in certain embodiments, the invention encompasses a modified B. anthracis polypeptide comprising one or more amino acid modification relative to a wild type polypeptide to alter an internal ribosyme entry site or to modify an internal methionine residue. In a specific embodiment, the invention encompasses a modified PA comprising a modification to an internal methionine residue, which methionine residue corresponds to M266 of SEQ ID NO: 2. In a specific example in accordance with this embodiment, the modification to M266 is a substitution. In a preferred embodiment, the substitution at M226 is M266L. In another specific embodiment, the invention encompasses a modified PA comprising a modification to an internal methionine residue, which methionine residue corresponds to M350 of SEQ ID NO: 2. In a specific example, the modification to M350 is a substitution. In a preferred embodiment, the substitution at M350 is M266L or M350I. In specific embodiments, the invention encompasses polypeptides, or fragments thereof, comprising one or more of the aforementioned modifications, wherein said polypeptides comprise SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, or SEQ ID NO:16. In related non limiting embodiments, the invention encompasses isolated nucleotide sequences encoding one or more of the aforementioned polypeptides. In a specific example in accordance with this embodiment, the isolated nucleic acids encoding the polypeptides comprising SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, or SEQ ID NO:16 comprise SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO: 15, respectively.

[0015] The invention thus encompasses isolated nucleic acid sequences encoding B. anthracis proteins, their derivatives, and/or fragments thereof, that have been codon optimized for expression in a particular recombinant system. In certain embodiments, the recombinant expression system is E. coli. In specific embodiments, the invention encompasses an isolated nucleic acid sequence encoding PA that has been codon optimized for recombinant expression in E. coli, wherein said sequence is SEQ ID NO: 19. In another

1011497 vl embodiment, the invention encompasses an isolated nucleic acid sequence encoding an antigenic fragment of PA that has been codon optimized for recombinant expression in E. coli, wherein said sequence is SEQ ID NO: 17 or SEQ ID NO: 15. In still another specific embodiment, the invention encompasses an isolated nucleic acid sequence encoding a derivative of PA, i.e., rPA, that has been codon optimized for expression in E. coli, wherein said sequence is SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO:14.

[0016] The present invention is also directed to a method for producing a recombinant

Bacillus anthracis protein, e.g., a recombinant protective antigen (rPA), which may be used in pharmaceutical compositions to stimulate an immune response protective against Bacillus anthracis infection and/or as a therapeutic against Anthrax toxin exposure. This method comprises transforming a host cell with a nucleotide sequence encoding a Bacillus anthracis protein, e.g., protective antigen (rPA), culturing the transformed host cell, and recovering the rPA therefrom. The invention encompasses the use of any recombinant host system known in the art and or described herein. In certain embodiments, the recombinant host cell is E. coli. The invention encompasses the addition of any additives and or media alterations known in the art to stimulate or improve recombinant protein production and/or recovery from a host system. In certain embodiments, the invention encompasses the addition of rifampicin to the culture systems described herein. In specific embodiments the concentration of rifampicin in the culture media is about 0.25 to about 100 μg/ml. In other embodiments the concentration of rifampicin in the culture medium is about 0.25 to about 90 μg/ml, 0.25 to about 80 μg/ml, 0.25 to about 70 μg/ml, 0.25 to about 60 μg/ml, 0.25 to about 50 μg/ml, 0.25 to about 40 μg/ml, 0.25 to about 30 μg/ml, 0.25 to about 20 μg/ml, 0.25 to about 10 μg/ml, 0.25 to about 5 μg/ml, 0.25 to about 2 μg/ml, 0.25 to about 1.5 μg/ml, 0.25 to about 1 μg/ml, 0.5 to about 1 μg/ml, 0.75 to about 1 μg/ml, 0.8 to about 1 μg/ml, or 0.9 to about 1 μg/ml. In preferred embodiments, the concentration of rifampicin in the culture medium is about 0.9 to about 1 μg/ml.

[0017] The invention also encompasses a novel isolated nucleic acid sequence encoding one or more derivatives of one or more B. anthracis proteins, which sequences have been optimized for recombinant production in E. coli, wherein the derivative comprises an amino acid substitution at one or more residues to a more positionally preferred residue. In certain embodiments, the amino acid residue that is substituted is that immediately following the initial methionine. In certain embodiments, the amino acid residue immediately

1011497 vl following the initial methionine corresponds to amino acid residue 1 of SEQ ID NO:2 or SEQ ID NO:6. In a specific embodiment, the invention encompasses a modified nucleic acid sequence for optimized expression in E. coli, which sequence encodes a derivative of PA, e.g., an rPA, comprising a modification of a codon relative to the wild type sequence, wherein said codon encodes a positionally preferred amino acid residue. In a specific example in accordance with this embodiment, the positionally preferred codon is the codon encoding the residue at position 1 of the encoded rPA. In specific embodiments of this example, the nucleotide sequence of the codon encoding the residue at position 1 is GGT (glycine) or GAC (aspartic acid). In another specific example in accordance with this embodiment, the nucleotide sequence of the codon encoding the residue at position 1 of the encoded rPA is GGT (glycine) and the optimized sequence encoding rPA is SEQ ID NO: 12. In another specific example in accordance with this embodiment, the nucleotide sequence of the codon encoding the residue at position 1 of the encoded rPA is GAC (aspartic acid) and the optimized sequence encoding rPA is SEQ ID NO: 10, SEQ ID or SEQ ID: 14. [0018] A further aspect of the invention is directed to a method of recovering and/or purifying a Bacillus anthracis protein, e.g., protective antigen (rPA), from a transformed host cell. In specific embodiments, the transformed host cell is E. coli. This method involves disrupting cells of the host, isolating inclusion bodies that contain the Bacillus anthracis protein, e.g., protective antigen (rPA), from the host cells, solubilizing the inclusion bodies, and processing the inclusion bodies to refold the protein. In specific embodiments, the inclusion bodies are solubilized in urea. Processing of the inclusion bodies may comprise one or more of: adding buffer to decrease the urea concentration of the solubilized inclusion body sample, recycling the buffered sample through a column, e.g., Heparin and/or Sepharose column, washing the loaded columns, and eluting the sample form the loaded columns. In certain embodiments, the column used for sample purification is a tandem column connected in series. In a specific embodiment, the column used for sample purification is a Heparin column in tandem with a Blue Sepharose column. The processing may also comprise one or more steps of collection and purification.

[0019] The present invention also encompasses the use of compositions, in particular pharmaceutical compositions, comprising one or more polypeptides of the invention at therapeutically effective concentrations for inducing an immune response in a subject. Induction of an immune response to the polypeptide of the invention is useful for the treatment and/or prevention of an infection by B. anthracis and/or treatment of a subject

1011497 vl exposed to Anthrax toxin. Accordingly, in certain embodiments, the polypeptides of the invention can be used as a vaccine for prophylaxis or as a therapeutic. In certain embodiments, an immunogenic composition of the invention comprises a plurality of polypeptides derived from more than one type or strain of B. anthracis, thereby inducing an immune response against more that one species/type or strain of bacteria and/or protein derived therefrom.

[0020] Other objects and features will be in part apparent and in part pointed out hereinafter.

3.1. TERMINOLOGY

[0021] As used herein, the term "about" or "approximately" when used in conjunction with a number refers to any number within 1, 5 or 10% of the referenced number or within the experimental error typical of standard methods used for the measurement and/or determination of said number.

[0022] As used herein, the term "amino acid" is used herein in its broadest sense and includes naturally occurring amino acids as well as non-naturally occurring amino acids, amino acid analogs and derivatives. Accordingly, reference herein to an amino acid includes, for example, naturally occurring proteogenic L-amino acids; D-amino acids; chemically modified amino acids such as amino acid analogs and derivatives; naturally occurring non- proteogenic amino acids, and chemically synthesized compounds having properties known in the art to be characteristic of amino acids.

[0023] As used herein, the term "analog" in the context of proteinaceous agents (e.g., proteins, polypeptides) refers to a proteinaceous agent that possesses a similar or identical function as a second proteinaceous agent but does not necessarily comprise a similar or identical amino acid sequence of the second proteinaceous agent, or possess a similar or identical structure of the second proteinaceous agent. A proteinaceous agent that has a similar amino acid sequence refers to a second proteinaceous agent that satisfies at least one of the following: (a) a proteinaceous agent having an amino acid sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequence of a second proteinaceous agent; (b) a proteinaceous agent encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence encoding a second proteinaceous agent of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15

1011497 vl contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, or at least 150 contiguous amino acid residues; and (c) a proteinaceous agent encoded by a nucleotide sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the nucleotide sequence encoding a second proteinaceous agent. A proteinaceous agent with similar structure to a second proteinaceous agent refers to a proteinaceous agent that has a similar secondary, tertiary or quaternary structure to the second proteinaceous agent. The structure of a polypeptide can be determined by methods known to those skilled in the art, including but not limited to, peptide sequencing, X-ray crystallography, nuclear magnetic resonance, circular dichroism, and crystallographic electron microscopy.

[0024] To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical overlapping positions/total number of positions.times.100%). In certain embodiments, the two sequences are the same length.

[0025] The alignment of and determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the alignment and/or comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877; each of which is hereby incorporated by reference in its entirety. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al, 1990, J. MoI. Biol.

1011497 vl 10 215:403 (by incorporated by reference in its entirety). BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the present invention. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score-50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule of the present invention. The BLAST and XBLAST programs can also be used to align nucleotide and/or polypeptide sequences, respectively, according to the methods of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402 (hereby incorporated by reference in its entirety). Alternatively, PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., the NCBI website). Another preferred non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17 (hereby incorporated by reference in its entirety). Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing and/or aligning amino acid sequences, e.g., a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0026] The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0027] Unless otherwise indicated, the numbering of amino acid residues of the polypeptides of the invention is made relative to the amino acid sequence of a native PA as shown in SEQ ID NO:2. The amino acid sequence of the invention is aligned with that of the reference sequence, i.e. native PA (SEQ ID NO:2), in accordance with methods well known in the art. Residues of the aligned amino acid sequences of the invention are assigned the numbering of the residue of the reference sequence at the same position in the alignment. As commonly understood in the art, residues that occupy the same position in the aligned sequences are referred to as "corresponding" residues. Because of the use of gaps to achieve optimal alignments, residues of one sequence may or may not have corresponding residues in the second sequence.

1011497 vl 11 [0028] As used herein, the term "derivative," in the context of polypeptides or proteins, refers to a polypeptide or protein that comprises an amino acid sequence that has been altered by the introduction of one or more amino acid residue modifications (including, e.g., substitutions, deletions and/or additions). The term derivative as used herein may also refer to a protein or polypeptide having one or more residues chemically derivatized by reaction of a functional side group provided that the requisite activity is retained. Such derivatized molecules include for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O- alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine. Also included as derivatives are those peptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example : 4-hydroxyproline may be substituted for serine; and ornithine may be substituted for lysine. The term "derivative" as used herein may also refer to a polypeptide or protein which has been modified, i.e., by the covalent attachment of any type of molecule to the polypeptide or protein. For example, but not by way of limitation, an recombinant polypeptide of the invention may be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. A derivative polypeptide or protein may be produced by chemical modifications using techniques known to those of skill in the art, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Further, a derivative polypeptide or protein derivative possesses a similar or identical function as the polypeptide or protein from which it was derived. [0029] As used herein, the term "fragment" refers to a peptide or polypeptide comprising an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous

1011497 vl 12 amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues of the amino acid sequence of another polypeptide. In a specific embodiment, a fragment of a polypeptide retains at least one function of the polypeptide. Preferably, fragments of the rPA proteins of the invention are immunogenic and/or antigenic.

[0030] As used herein, the terms "immunogenic" and "antigenic" in the context of a polypeptide or protein refer to the ability of the polypeptide or protein to induce or stimulate an immune response in a subject. The immune response may be a cell or antibody mediated response (see e.g., Roitt, Essential Immunology (8^th Edition); hereby incorporated by reference in its entirety). Normally, increased concentration or activity of antibody specific for PA or a fragment thereof, will be an indication of an induced and/or stimulated immune response to the polypeptides of the invention. The immunogenicity of the molecules of the invention may be assessed by any method known in the art either in vivo or in vitro. For example, serum may be obtained from a subject immunized according to the methods of the invention and the presence of anti-PA antibodies assessed by methods commonly known in the art, e.g., immunoassays (including ELISA), BIAcore, or other assays known in the art. In preferred embodiments the rPA and or antigenic fragments thereof, when administered to a patient in a therapeutic dose elicits an immune response against one or more types or strains of B. anthracis and/or against one or more components of Anthrax toxin. [0031] As used herein, the terms "manage," "managing," and "management" refer to the beneficial effects that a subject derives from a therapy (e.g., a prophylactic or therapeutic agent), which does not result in a cure of the disease, e.g., Anthrax. In certain embodiments, a subject is administered one or more therapies (e.g., prophylactic or therapeutic agents, such as a composition of the invention) to "manage" an infectious disease, or a condition or symptom associated therewith, so as to prevent the progression or worsening of disease/disorder.

[0032] As used herein, the terms "prevent", "preventing" and "prevention" refer to the prevention of onset of, the recurrence of, or a reduction in one or more symptoms of a disease/disorder (e.g., infection by a bacterium) in a subject as result of the administration of a therapy (e.g., a prophylactic or therapeutic composition). As used herein, "prevention" also encompasses prevention of infection by one or more types and/or strains of Anthrax causing bacteria in connection with the use of the compositions of the invention as a vaccine.

1011497 vl 13 [0033] As used herein, "polynucleotide" and "oligonucleotide" are used interchangeably and mean a polymer of at least 2 nucleotides joined together by phosphodiester bonds. A polynucleotide or oligonucleotide may consist of either ribonucleotides or deoxyribonucleotides.

[0034] The term "polypeptide" when used herein refers to two or more amino acids that are linked by peptide bond(s), regardless of length, functionality, environment, or associated molecule(s). Typically, a polypeptide is at least four amino acid residues in length and can range in size, up to and including a full-length protein. Accordingly, as used herein, "polypeptide," "peptide," and "protein" are used interchangeably.

[0035] As used herein, the terms "subject" or "patient" are used interchangeably. As used herein, the terms "subject" and "subjects" refers to an animal (e.g., mammals). In some embodiments, the subject is a mammal, including non-primates (e.g., camels, donkeys, zebras, cows, sheep, goats, horses, cats, dogs, rats, and mice) and primates (e.g., monkeys, chimpanzees, and humans). In some embodiments, the subject is a non-human mammal. In other embodiments the subject is a human.

[0036] The term "therapeutic immune response," as used herein, refers to an increase in humoral and/or cellular immunity, as measured by standard techniques, which is directed toward the polypeptides of the invention. Preferably, but not by way of limitation, the induced level of humoral immunity directed toward one or more polypeptides of the invention is at least four- fold, eight-fold, or ten- fold, preferably at least 16-fold, greater than that prior to the administration of the compositions of this invention to the subject. The immune response may also be measured qualitatively, by means of a suitable in vitro or in vivo assay, wherein an arrest in progression or a remission of an infectious disease, e.g., Anthrax, or symptoms thereof, in the subject is considered to indicate the induction of a therapeutic immune response.

[0037] As used herein, the terms "therapies" and "therapy" can refer to any protocol(s), method(s), and/or agent(s) that can be used in the prevention, treatment, management, or amelioration of a disease/disorder (e.g., bacterial infection or a condition or symptom associated therewith). In certain embodiments, the terms "therapies" and "therapy" refer to biological therapy, supportive therapy, and/or other therapies useful in treatment, management, prevention, or amelioration of a disease or condition or symptom(s) associated therewith, an infection or a condition or symptom associated therewith, known to one of skill in the art.

1011497 vl 14 [0038] As used herein, the terms "therapeutic agent" and "therapeutic agents" refer to any agent(s) that can be used in the prevention, treatment, management, or amelioration of a disease (e.g. bacterial infection or a condition or symptom associated therewith). Preferably, a therapeutic agent is an agent which is known to be useful for, or has been or is currently being used for the prevention, treatment, management, or amelioration of a disease or symptom associated therewith (e.g. , a bacterial infection or a condition or symptom associated therewith).

[0039] As used herein, the terms "treat," "treatment," and "treating" in the context of administration of a therapy to a subject for a disease refers to the eradication, reduction or amelioration of symptoms of said disease/disorder (e.g., bacterial disorder). [0040] For all the amino acid and nucleic acid sequences disclosed herein, it is understood that equivalent amino acids and nucleotides can be substituted into the sequences without affecting the function of the sequences. Such substitution is within the ability of a person of ordinary skill in the art. Biologically functional equivalent nucleotide sequences of the present invention also include nucleotide sequences that encode conservative amino acid changes within the amino acid sequences of the present polypeptides, producing silent changes therein. Such nucleotide sequences thus contain corresponding base substitutions based upon the genetic code compared to the nucleotide sequences encoding the present polypeptides. Substitutes for an amino acid within the fundamental polypeptide amino acid sequences discussed herein can be selected from other members of the class to which the naturally occurring amino acid belongs. Amino acids can be divided into the following four groups: (1) acidic amino acids; (2) basic amino acids; (3) neutral polar amino acids; and (4) neutral non-polar amino acids. Representative amino acids within these various groups include,, but are not limited to: (1) acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; (2) basic (positively charged) amino acids such as arginine, histidine, and lysine; (3) neutral polar amino acids such as glycine, serine, threonine, cysteine, cystine, tyrosine, asparagine, and glutamine; and (4) neutral nonpolar (hydrophobic) amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Conservative amino acid changes within the present polypeptide sequences can be made by substituting one amino acid within one of these groups with another amino acid within the same group. The encoding nucleotide sequences (gene, plasmid DNA, cDNA; synthetic DNA, or mRNA) will thus have corresponding base substitutions, permitting them to code for the expression of the biologically functional equivalent forms of the present polypeptides.

1011497 vl 15 It should be noted that the present invention encompasses not only the specific DNA sequences disclosed herein and the polypeptides encoded thereby, but also biologically functional equivalent nucleotide and amino acid sequences. The phrase "biologically functional equivalent nucleotide sequences" denotes DNAs and RNAs, including chromosomal DNA, plasmid DNA, cDNA, synthetic DNA, and mRNA nucleotide sequences, that encode polypeptides exhibiting the same or similar activity as that of the polypeptides encoded by the sequences disclosed herein when assayed by standard methods, or by complementation. Such biologically functional equivalent nucleotide sequences can encode polypeptides that contain a region or moiety exhibiting sequence similarity to the corresponding region or moiety of the presently disclosed polypeptides. The phrase "isolated" in the context of a nucleic acid molecule or amino acid sequence, e.g., polypeptide or protein, refers to any such sequence, however constructed or synthesized, which is locationally distinct from the natural location.

[0041] Various embodiments of the present invention rely on altering biological material using molecular techniques. Molecular techniques refers to procedures in which DNA is manipulated in a test tube during at least one stage of the process, such as the direct manipulation of DNA or the use of shuttle host such as bacterium. Additional examples of molecular techniques include, for example, methods of using PCR to multiply a nucleic acid of interest for introduction and expression in a mammal or mammal cell via expression vectors or direct introduction of the nucleic acid; methods of using nucleic acid libraries to determine, isolate, introduce, and express a nucleic acid of interest into a mammal or mammalian cell via expression vectors or direct introduction of the nucleic acid; isolation of nucleic acid segments, concatemerization of said nucleic acid segments into a larger nucleic acid, introduction, and expression of the same in a mammal or mammalian cell via expression vectors or direct introduction of the nucleic acid; and isolation of mRNA from a gene, creation of cDNA from the mRNA by reverse transcription, and introduction and expression of the same in a mammal or a cell via expression vectors or direct introduction of the nucleic acid. Such methods are well known in the art and are described in, for example, Sambrook et al. (1989), "Molecular Cloning, A Laboratory Manual", second ed., Cold Spring Harbor Laboratory Press (hereby incorporated by reference in its entirety).

4. BRIEF DESCRIPTION OF THE DRAWINGS

1011497 vl 16 [0042] FIG. IA-C. Restriction analysis of expression plasmids; digestion of rPA/pET9a and rPA/pETpET24a with varying endonucleases. (A) lanes 1-2, rPA/pET9a digested with Nde I and BamH I; lanes 3-4, rPA/pET24a with Nde I and Xho I; lane 5, molecular weight marker; lanes 6-7, rPA/pET9a with BgI II. (B) lanes 1-2, rPA/pET24a with BgI II; lane 3, marker; lanes 4-5, rPA/pET9a with Nde I and BgI II; lanes 6-7, rPA/pET24a with Nde I and BgI II. (C) lanes 1-2, rPA/pET9a with BamH I and BgI II; lane 3, marker; lanes 4-5, rPA/pET24a with Xho I and BgI II.

[0043] FIG. 2A-B. Schematic of the restriction map for expression plasmids (A) rPA/pET9a and (B) rPA/pET24a. Restriction endonucleases depicted in (A) include Xho I, PaeR7 I, Xma I, Sma I, Tthl 11 I, Bstl 107 I, Sap I, Msc I, Nde I; Xba I, Bsa I, Sal I, PshA I, Eag I, BgI II, SgrA I, BcI I, Mun I, EcoR I, Bspl407 I, Rsr II, Pst I, Nhe I, Bpul 102 I, BamH I, and Dra I. Restriction endonucleases depicted in (B) include Dra III, Xma I, Sma I, BgI I, BgI II, Fsp I, PpuM I, Tthl 11 I, Bstl 107 I, Ace I, Sap I, BstE II, Bspl20 I, MIu I, Apa I, BssH II, Hpa I, PshA I, Nde I, Xba I, SgrA I, Bspl407 I, Rsr II, Mun I, EcoR I, Pst I, Sty I, Bpul 102 I, Xho I, PaeR7 I, and Dra I.

[0044] FIG. 3A-B. Images of SDS-PAGE of cellular proteins from bacterial cultures of comprising rPA/pET9a or rPA/pET24a vectors; precast NO VEX®, 8%, gels. Separated proteins were visualized with Coomassie stain. For both A and B, lanes 2 and 3 are from samples of non-induced cultures, and lanes 5-9 are from cultures 3 h post-induction with ImM IPTG (see Section 6.2, Example2). (A) lanel, blank; lane 2, rPA/pET9a (non-induced); lane 3, rPA/pET24a (non-induced); lane 4, high-molecular weight marker; lane 5, rPA/pET9a; lane 6, rPA/pET9a in the presence of 0.9 μg/ml rifampicin; lane 7, rPA/pET24a; lane 8, rPA/pET24a in the presence of 0.9 μg/ml rifampicin. (B) lanel, See Blue marker (INVITROGEN®); lane 2, rPA/pET9a (non-induced); lane 3, rPA/pET24a (non-induced); lane 4, BSA marker; lane 5, blank; lane 6, rPA/pET9a; lane 7, rPA/pET9a in the presence of 0.9 μg/ml rifampicin; lane 8, rPA/pET24a; lane 9, rPA/pET24a in the presence of 0.9 μg/ml rifampicin.

[0045] FIG. 4. Image of SDS-PAGE of cellular proteins from bacterial cultures comprising rPA/pET24a vectors, cultured at varying temperatures in the presence of varying concentrations of rifampicin (see Section 6.2, Example 2): lanel, blank; lane 2, non-induced; lane 3, See Blue marker (INVITROGEN®); lane 4, 3 h post-induction in the presence of 0.9 μg/ml rifampicin, 37 ⁰C; lane 5, 3 h post induction in the presence of 1.5 μg/ml rifampicin, 37 ⁰C; lane 6, 3 h post induction in the presence of 0.9 μg/ml rifampicin, 38.5 ⁰C; lane 7, 4 h

1011497 vl 17 post-induction in the presence of 0.9 μg/ml rifampicin, 37 ⁰C; lane 8, 4 h post induction in the presence of 1.5 μg/ml rifampicin, 37 ⁰C; lane 9, 4 h post induction in the presence of 0.9 μg/ml rifampicin, 38.5 ⁰C.

[0046] FIG. 5. Image of SDS-PAGE of cellular proteins from bacterial cultures comprising rPA/pET24a vectors cultured at varying temperatures in the presence of 0.9 μg/ml rifampicin (see Section 6.2, Example 2): lane 1, 2 h post induction, 22 ⁰C; lane 2, 2 h post induction, 30 ⁰C; lane 3, 2 h post induction, 42 ⁰C; lane 4, See Blue marker (INVITROGEN®); lane 5, 3 h post induction, 22 ⁰C; lane 6, 3 h post induction, 30 ⁰C; lane 7, 3 h post induction, 42 ⁰C; lane 8, 4 h post induction, 22 ⁰C; lane 9, 4 h post induction, 30 ⁰C; lane 10, 4 h post induction, 42 ⁰C.

[0047] FIG. 6. Image of SDS-PAGE of cellular proteins from bacterial cultures comprising rPA/pET24a vectors, cultured in the presence of 0.9 μg/ml rifampicin (except lanes 5 and 9) at varying temperatures and in varying media: lane 1, no induction; lane 2, See Blue marker (INVITROGEN®); lane 3, 3 h post induction, APS medium, 30 ⁰C; lane 4, 3 h post induction, APS medium, 37 ⁰C; lane 5, 3 h post induction, LB medium, no rifampicin, 37 ⁰C; lane 6, 3 h post induction, LB medium, 37 ⁰C; lane 7, 4 h post induction, APS medium, 30 ⁰C; lane 8, 4 h post induction, APS medium, 37 ⁰C; lane 9, 4 h post induction, LB medium, no rifampicin, 37 ⁰C; lane 10, 4 h post induction, LB medium, 37 ⁰C. [0048] FIG. 7. Image of Western blot of protein samples from gel of FIG. 3 A.

Primary antibody, murine monoclonal anti-PA; secondary antibody, AP-conjugated goat anti- murine IgG, F(ab')₂ fragment.

[0049] FIG. 8. Schematic of Asymmetric Overlap Extension ("AOE") PCR.

[0050] FIG. 9. Image of SDS-PAGE of cellular proteins from bacterial cultures comprising rPA/pET24a, rPA/ElD/M266L/pET24a, or rPA/ElG/M266L/pET24a vectors cultured in APS medium containing 0.9 μg/ml rifampicin at 37 ⁰C: lane 1, rPA/ElG/M266L/pET24a, no induction; lane 2, rPA/ElD/M266L/pET24a, no induction; lane 3, See Blue marker (INVITROGEN®); lane 4, rPA/pET24a, 3 h post induction; lane 5, rPA/pET24a, 3 h post induction; lane 6, rPA/ElG/M266L/pET24a, 3 h post induction; lane 7, rPA/ElD/M266L/pET24a, 3 h post induction; lane 8, rPA/ElG/M266L/pET24a, 3.5 h post induction; lane 9, rPA/ElD/M266L/pET24a, 3.5 h post induction.

[0051] FIG. 10. Image of SDS-PAGE of cellular proteins from bacterial cultures comprising rPA/pET24a or rPA/ElD/ pET24a vectors cultured in APS medium containing 0.9 μg/ml rifampicin at 37 ⁰C: lane 1, rPA/ElD/ pET24a (clone 1), 3 h post induction; lane 2,

1011497 vl 18 rPA/ElD/pET24a (clone 2), 3 h post induction; lane 3, rPA/pET24a, 3 h post induction; lane 4, See Blue marker (INVITROGEN®); lane 5, rPA/ElD/pET24a (clone 1), not induced; lane 6, rPA/ElD/pET24a (clone 2), not induced; lane 7, rPA/ElD/pET24a (clone 1), not induced; lane 8, rPA/ElD/pET24a (clone 1), not induced; lane 9, rPA/ElD/pET24a (clone 2), not induced; lane 10, rPA/pET24a, not induced.

[0052] FIGS. 1 IA-B. Images of SDS-Page (A) and Western Blot (B)of cellular proteins from bacterial cultures comprising rPA/266trunc cultured in APS medium at 37 ⁰C in the presence of 0.9μg/ml rifampicin (unless other wise indicated). For all analyses, samples were collected 3.5 h after induction with ITPG. For the Western blot, the primary antibody was a murine monoclonal anti-PA and the secondary antibody was a AP-conjugated goat anti-murine IgG, F(ab')₂ fragment (see Section 6.4, Example 4). For both A and B: lanes 1 and 10, See Blue marker (INVITROGEN®); lanes 2 and 3, rPA/M266L/pET24a (lane 2, no rifampicin); lanes 4 and 5, rPA/266trunc/pET24a (lane 4, no rifampicin); lanes 6 and 7, rPA/266trunc/pET9a (lane 6, no rifampicin); lanes 8 and 9, rPA/pET24a (for B, lanes 9 and 10 were left blank).

[0053] FIGS. 12A-B. Images of SDS-Page (A) and Western Blot (B)of cellular proteins from bacterial cultures comprising rPA/348trunc/pET24a or rPA/348trunc/pET9a vectors cultured in APS medium at 37 ⁰C in the presence of 0.9μg/ml rifampicin (unless otherwise indicated). For all analyses, samples were collected 3.5 h after induction with ITPG. For the Western blot, the primary antibody was a murine monoclonal anti-PA and the secondary antibody was a AP-conjugated goat anti-murine IgG, F(ab')₂ fragment (see Section 6.4, Example 4). For A: lane 1, rPA/348trunc/pET24a (not induced); lane 2, rPA/348trunc/pET9a (not induced); lane 3, See Blue marker (INVITROGEN®); lanes 4-5, rPA/348trunc/pET24a; lanes 6-7, rPA/348trunc/pET9a; lane 8, rPA/348trunc/pET24a; lane 9, rPA/348trunc/pET9a. For B: lane 1, rPA/348trunc/pET24a (not induced); lane 2, rPA/348trunc/pET9a (not induced); lane 3, See Blue marker (INVITROGEN®); lane 4, rPA/348trunc/pET24a; lane 5, rPA/348trunc/pET9a; lane 6, rPA/348trun/pET24a, no rifampicin; lane 7, rPA/348trunc/pET24a; lane 8, rPA/348trunc/pET9a, no rifampicin; lane 9, rPA/348trun/pET9a; lane 10, rPA/pET24a (not induced).

[0054] FIG. 13. Schematic of column design for protein purification and isolation.

[0055] FIG. 14. Image of SDS-PAGE of flow through and eluate from the recycled tandem column system, Heparin column in tandem with a Blue Sepharose Colum; lanes 1-6, flow through; lanes 7-9, eluate from Blue Sepharose column.

1011497 vl 19 [0056] FIG. 15. Image of SDS-PAGE of flow through and eluate from the recycled tandem column system, Hightrap Q column in tandem with a Blue Sepharose Colum; lane 1,

See Blue molecular weight marker; lanes 2-3, eluate Hightrap Q column; lane 4, eluate

Sepharose Blue column; lane 5, flow through.

[0057] FIG. 16. Image of SDS-PAGE of eluate from the recycled tandem column system, Hightrap Q column in tandem with a Blue Sepharose Colum; lane 1, See Blue molecular weight marker; lanes 2-4, pooled, protein-containing fractions from the eluate of the Hightrap Q column: the three lanes represent three separate procedures.

[0058] FIG. 17. HPLC chromatographs of samples from the eluate of the recycled, tandem column system for protein purification and refolding. (A) sample of eluate from

Hightrap Q column, and (B) sample from Blue Sepharose column.

[0059] FIG. 18. GS-MS spectra of samples from two separate productions of rPA

(PA-6-PK1 and PA-6PK2) as compared to those of control rPA (Acambis) and control E. coli endotoxin (BL21 LOS).

[0060] FIG. 19. MALDI-TOF spectrophrometry analysis of protein from pooled fractions of eluent from the recycled, tandem column system.

[0061] FIG. 20. Capture ELISA: Quantitation of detection signal from varying concentrations of rPA captured by murine monoclonal anti-PA antibody and detected by PA immunized rabbit serum.

[0062] FIG. 21. Inhibition ELISA: Percent inhibition of binding by PA immunized rabbit serum to commercial PA (Acambis) by preincubation with varying concentrations of rPA of the invention, or a fragment thereof.

[0063] FIG. 22. Binding of rPA to RAW 264.7 ANTXRl human macrophage cells.

The dose dependent increase in binding is suggestive of a specific interaction with a cell surface receptor.

[0064] FIG. 23. Antibody cytotoxicity inhibition in RAW 264.7 ANTXRl human macrophage cells. Serum from rabbits immunized with the rPA of the invention or control peptides was assayed for inhibition of rPA/LF cytotoxicity. Immunization with the rPA peptides of the invention resulted in an antibody response which was demonstrated to have an inhibition profile similar to that resulting from both commercially available rPA (Acambis) and wild-type PA (i.e., purified from cultures of B. anthracis).

5. DETAILED DESCRIPTION

1011497 vl 20 [0065] The present invention is generally directed to (i) novel polynucleotides, e.g. , nucleic acid sequences, encoding polypeptides comprising the one or more of the Lethal Factor ("LF"), the Edema Factor ("EF"), or the Protective Antigen ("PA") protein of B. anthracis, or antigenic fragments thereof, (ii) polypeptides comprising novel sequences derived from one or more of a LF, EF or PA protein, or an antigenic fragment thereof, (iii) immunogenic compositions comprising the polypeptides of the invention, and (iv) methods of making and/or use of molecules of the invention. In specific embodiments, the invention encompasses the use of the immunogenic compositions of the invention as vaccines. As disclosed herein, the invention provides methods that facilitate the efficient production of B. anthracis proteins, e.g., PA, from recombinant systems for use in a variety of research and/or therapeutic applications. The invention also provides for novel nucleic acid sequences encoding B. anthracis proteins, which sequences have been modified to improve recombinant processing. The invention also provides for novel amino acid sequences derived from B. anthracis proteins, which sequences are engineered to exhibit improved production and/or improved in vivo properties relative to wild-type counterparts. For example, the modification encompassed by the invention may remove common protease recognition sites, leading to increased protein yield from recombinant systems and also to increased half- life in vivo. The novel amino acid sequences {i.e., polypeptides) of the invention are designated herein by an "r" prefix, e.g., "rLF", "rEF", and "rPA," referencing the recombinant origins of said polypeptides. rLF, rEF and rPA molecules may comprise their full length protein counterparts or may comprise only fragments thereof, preferably antigenic fragments thereof. Unless otherwise indicated, the rLF, rEF and rPA polypeptides of the invention comprise one or more amino acid modifications relative to the wild-type proteins. [0066] In specific embodiments, the methods of the invention also provide immunogenic compositions comprising the polypeptides of the invention, e.g., vaccines, which compositions may exhibit improved properties {e.g. , immunogenicity) relative to similar vaccines known in the art. The methods of the invention are applicable to any antigenic B. anthracis protein, and, in particular, are directed to the making and use of acellular Anthrax vaccines. In specific, nonlimiting embodiments of the invention, the immunogenic compositions comprise rPA and/or an antigenic fragment thereof. The vaccines of the invention are expected to be useful in prevention and/or treatment of Anthrax, i.e., to prevent or treat infection with B. anthracis and/or the symptoms thereof. Unlike vaccines derived from alum precipitated materials of active cultures, the immunogenic

1011497 vl 21 compositions of the instant invention offer the advantage of fully defined and selectable components. The polypeptides of the invention may comprise a full length derivative of a B. anthracis protein (and thus potentially multiple epitopes of said protein), or may comprise an antigenic fragment thereof (and thus potentially only one epitope of said protein). In certain embodiments, it may be desirable to include more that one species of polypeptide in the pharmaceutical compositions of the invention to optimize the immune response to the composition. Such an approach may be particularly advantageous in the prevention or treatment of infections by unknown strains of B. anthracis. Accordingly, in certain embodiments, the immunogenic composition may comprise a plurality of polypeptides of the invention. In other embodiments, the immunogenic composition may comprise a single species of polypeptide of the invention. Moreover, in certain embodiments, the polypeptides of the invention may be fusion proteins of one or more antigenic domains or epitopes from differing proteins. In a specific example in accordance with this embodiment, a polypeptide of the invention may comprise two or more epitopes from at least two differing proteins of one strain of B. anthracis. In another example in accordance with this embodiment, a polypeptide of the invention may comprise two or more epitopes from proteins of at least two differing strains of B. anthracis. Techniques for creating fusion proteins from known nucleic acid sequences are well known in the art.

[0067] The invention encompasses methods generally directed to increasing the economy and efficiency of recombinant production of polypeptides comprising B. anthracis proteins and/or derivatives or fragments thereof , e.g., PA and/or rPA. To this end, the invention encompasses nucleic acid sequences encoding a B. anthracis protein, e.g. , PA, which sequences have been engineered to improve transcription of the polynucleotide and/or translation and processing of the encoded polypeptide in a recombinant system. In certain embodiments, the invention encompasses the codon optimization of a nucleic acid sequence encoding a B. anthracis protein to improve efficiency of transcription and/or translation in a specific expression system, e.g., E. coli (see, e.g., Section 6.2 and 6.3). In other embodiments, the nucleic acid sequence is modified to effect a modification in the encoded amino acid sequence {e.g., by substitution, insertion, or deletion). In a specific example in accordance with this embodiment, the novel amino acid sequence of the protein, e.g., rPA, is altered such that the processing {e.g., post-translational processing) of the protein is altered relative to that of the wild-type amino-acid sequence (see, e.g., Section 6.3). The invention encompasses any nucleic acid or amino acid sequence modification known in the art and/or

1011497 vl 22 described herein to improve the processing and/or expression of a recombinant molecule of the invention.

5.1 POLYNUCLEOTIDES ENCODING THE POLYPEPTIDES OF THE INVENTION

[0068] The present invention encompasses novel polynucleotides that encode B. anthracis proteins and/or their derivatives, or immunogenic fragments thereof, and methods for producing the same. In preferred embodiments the polynucleotides have been optimized for expression in a recombinant system. Modifications to nucleic acid sequences to improve their expression in recombinant systems is well known in the art, and the invention encompasses the use of such modifications and/or any other modifications described herein. Commonly used techniques to improve expression of a recombinant nucleotide sequence that are encompassed by the invention include, but are not limited to, codon optimization, use of special recombinant systems {e.g., the use of commercial host cells specifically designed for the expression of foreign proteins), and elimination of protease recognition sites within the encoded protein. In preferred embodiments, the invention is directed to novel nucleic acid sequences encoding PA, its derivatives and/or antigenic fragments thereof. [0069] A polynucleotide encoding a polypeptide of the invention, e.g., rPA, or fragment thereof, may be obtained, and the nucleotide sequence thereof determined, by any method and from any suitable source described herein and/or known in the art. Non-limiting examples of sources of nucleic acids encoding B. anthracis proteins include, nucleic acids isolated from any cell culture, tissue sample, or cell sample comprising B. anthracis {e.g. , B. Anthracis cell culture, tissue sample comprising a B. anthracis infection) and cDNA libraries of B. anthracis. Nucleic acids can be isolated from sources by methods well known in the art, e.g., hybridization and selection with sequence specific, tagged probes; or PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence encoding the protein. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors for further amplification and sequencing using any method well known in the art.

[0070] Once the nucleotide sequence of a protein of interest, e.g., PA, is determined it may be manipulated using methods well known in the art and/or described herein for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc., to generate molecules of the invention having a different amino acid

1011497 vl 23 sequence, e.g., to create amino acid substitutions, deletions, and/or insertions, (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are both incorporated by reference herein in their entireties and/or Section 6.1), [0071] Alternatively, nucleic acid sequences of the invention may be generated de novo by oligonucleotide synthesis. For example, synthetic oligonucleotides may be prepared according to the manufacturer's directions in an automated synthesizer, e.g., Applied Biosystems 38A DNA Synthesizer. Synthesized constructs may be purified according to any methods known in the art, e.g., high performance liquid chromatography (HPLC). Long, double-stranded polynucleotides, such as a DNA molecule of the present invention, may also be synthesized in stages due to the size limitations inherent in synthetic methods. Thus, for example, a large double-stranded DNA molecule may be synthesized as several smaller segments possessing appropriate cohesive termini for attachment to adjacent segments and then annealed. Adjacent segments may be ligated, for example, by annealing cohesive termini in the presence of DNA ligase to construct the entire protein encoding sequence. A synthetic DNA molecule so constructed may then be cloned and amplified in an appropriate vector.

[0072] The methods of the invention encompass the use of nucleic acid sequences derived from any type or strain of B. anthracis. Worldwide, there is little diversity among B. anthracis isolates, but extensive genetic analysis from disparate samples has revealed the existence of at least six major clones split into at least two major groups (A and B) (Keim P et al, 2000, J Bacteriol. 182:2928-2936; Ryu et al. 2005, Appl Environ Microbiol. 71 : 4664- 4671, each of which is hereby incorporated by reference in its entirety). Type A strains are found around the world and are responsible for most epidemics and outbreaks. In contrast, type B strains are almost exclusively restricted to southern Africa. In a specific embodiment, the invention encompasses the use of an isolated nucleotide comprising SEQ ID NO:2, which encodes a type A protective antigen (PA)..

[0073] The isolated nucleic acids of the invention can be used to produce large quantities of one or more substantially pure B. anthracis proteins, or selected portions thereof. The full-length proteins or selected domains thereof can be used for research, diagnostic and therapeutic purposes, e.g., vaccines. Nucleic acids encoding the polypeptides of the invention, e.g., PA or rPA, may also be used for a variety of other purposes in

1011497 vl 24 accordance with the present invention. For example, isolated DNA or RNA molecules of the invention may be used as probes to detect the presence of and/or expression of genes encoding LF, EF, PA and/or derivatives or fragments thereof. Methods in which nucleic acid molecules of the invention may be utilized as probes include, but are not limited to: (1) in situ hybridization; (2) Southern hybridization (3) northern hybridization; and (4) assorted amplification reactions such as polymerase chain reactions (PCR). [0074] The polynucleotides of the invention include cDNA, genomic DNA, and

RNA, and may be single-or double-stranded. The methods of the invention also encompass polynucleotides that hybridize under various stringency, e.g., high stringency, intermediate or lower stringency conditions, to polynucleotides (e.g., double-stranded or single-stranded) that encode polypeptides of the invention, e.g. PA or rPA. The hybridization can be performed under various conditions of stringency. By way of example and not limitation, procedures using conditions of low stringency are as follows (see also Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci. U.S.A. 78, 6789-6792). Filters containing DNA are pretreated for 6 h at 40° C. in a solution containing 35% formamide, 5 X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500μg/ml denatured salmon sperm DNA. Hybridizations are carried out in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20 X lO⁶ cpm ³²P-labeled probe is used. Filters are incubated in hybridization mixture for 18-20 h at 40°. C, and then washed for 1.5 h at 55° C. in a solution containing 2 X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an additional 1.5 h at 60° C. Filters are blotted dry and exposed for autoradiography. If necessary, filters are washed for a third time at 65-68° C. and re- exposed to film. Other conditions of low stringency which may be used are well known in the art (e.g., as employed for cross-species hybridizations). By way of example and not limitation, procedures using conditions of high stringency are as follows. Prehybridization of filters containing DNA is carried out for 8 h to overnight at 65° C. in buffer composed of 6 X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters are hybridized for 48 h at 65° C. in prehybridization mixture containing 100 μg/ml denatured salmon sperm DNA and 5-20 X lO⁶ cpm of ³²P-labeled probe. Washing of filters is done at 37°. C. for 1 h in a solution containing 2 X SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1 X SSC at 50° C for 45 min before autoradiography. Other conditions of high

1011497 vl 25 stringency which may be used are well known in the art. Selection of appropriate conditions for such stringencies is well known in the art (see e.g., Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.; see also, Ausubel et al., eds., in the Current Protocols in Molecular Biology series of laboratory technique manuals, .COPYRGT. 1987-1997, Current Protocols, .COPYRGT. 1994-1997 John Wiley and Sons, Inc.; see especially, Dyson, 1991, "Immobilization of nucleic acids and hybridization analysis," In: Essential Molecular Biology: A Practical Approach, Vol. 2, T. A. Brown, ed., pp. 111-156, IRL Press at Oxford University Press, Oxford, UK) (each of which is hereby incorporated by reference herein in its entirety).

5.1.1 OPTIMIZATION OF WILD TYPE B. anthracis

POLYNUCLEOTIDES FOR RECOMBINANT PRODUCTION [0075] Low yield or inefficient production of foreign protein in recombinant expression systems may, in part, be attributable to differences in codon usage between the organism from which the nucleotide sequence encoding the protein was derived and the recombinant host. During translation, the rate limiting step is the recognition of each codon by its specific cognate tRNA. However, not all organisms maintain similar relative tRNA populations. Normally, the relative abundance of any particular tRNA in an organism is reflected in the relative frequency of appearance of its cognate codon in its genome. Therefore, when the codon usage of a target protein differs significantly from the average codon usage of the expression host, there is a significant probability that translation will be slowed or stalled due to the scarcity of one or more tRNAs (the condition of "rare" codons/tRNAs). The slowing or stalling of the translation machinery can lead to, e.g., decreased mRNA stability; premature termination of transcription and/or translation (generating nonuniform truncated protein products); frameshifts; deletions; misincorporations; and general decrease in translation rate or inhibition of protein synthesis. Rare codons/tRNAs can also completely abrogate recombinant expression in cases where the rare codons are present at the 5 '-end of the mRNA or where they are present in clusters. [0076] Codon optimization is a method to resolve codon usage issues by replacing codons rarely found in the recombinant organism with more favorable codons throughout the coding nucleotide sequence. Commonly, the codon preference of a particular organism is determined by analyzing the nucleotide sequences of highly or constitutively expressed genes in the organism and calculating the frequency of codon appearance. For example, the codon

1011497 vl 26 frequency of the common recombinant system E. coli is widely known in the art and available from a number of sources (see, e.g., Table 1; adapted from Henaut and Danchin, 1996, Analysis and Predictions from Escherichia coli sequences, Escherichia coli and Salmonella Vol. 2 (F.C. Neidhardt ed., ASM press, Washington D.C.) pages, 2047-2066; hereby incorporated by reference in its entirety).

Table 1: Codons encoding Arg, GIy, He, Leu, and Pro in the native PA nucleotide sequence (SEQ ID NO:1) and their usage in E. coli.

Amino Acid Codon Fraction in All Genes Fraction in Class II*

Arg R AGG 0.022 0.003

Arg R AGA 0.039 0.006

Arg R CGG 0.098 0.008

Arg R CGA 0.065 0.011

Arg R CGU 0.378 0.643

Arg R CGC 0.398 0.330

GIy G GGG 0.151 0.044

GIy G GGA 0.109 0.020

GIy G GGU 0.337 0.508

GIy G GGC 0.403 0.428

He I AUA 0.073 0.006

He I AUU 0.507 0.335

He I AUC 0.420 0.659

Leu L UUG 0.129 0.034

Leu L UUA 0.131 0.055

Leu L CUG 0.496 0.767

Leu L CUA 0.037 0.008

Leu L CUU 0.104 0.056

Leu L cue 0.104 0.080

Pro P CCG 0.525 0.719

Pro P CCA 0.191 0.153

Pro P ecu 0.159 0.112

Pro P CCC 0.124 0.016

* Genes are clustered by using factorial correspondence analysis into three classes; class II genes correspond to genes highly and continuously expressed during exponential growth of the target sequence

1011497 vl 27 [0077] Considering Table 1, non-limiting examples of codons of the native PA nucleotide sequence that are expected to be associated with translation problems in E. coli include, e.g., AGG (arginine); AGA (arginine); CGG (arginine); CGA (arginine); GGA (glycine); AUA (isoleucine); CUA (leucine); and CCC (proline). Codon incompatibility can be resolved by any method known in the art or described herein for modification of nucleic acid sequences (see, e.g., Section 6.1 for Asymmetric Overlap Extension (AOE) PCR). For example, nucleic acid sequences can be modified by constructing a synthetic gene comprising altered codons by using a template, e.g., SEQ ID NO:1, inexpensive oligonucleotides and PCR. This can be accomplished, for example, using a one-step reaction for small genes (see e.g., Casimiro et al, 1997, Structure 5:407-1412, hereby incorporated by reference in its entirety). Briefly, staggered internal oligos may be used at intermediate {e.g., 20 nM) concentrations to serve as both template and internal primers. This intermediate concentration of the internal primer/template should be between that of a typical template and that of the standard primers {e.g., about 200 nM). The reaction is driven by outside primers at the concentration of standard primers. A full-length template is made within the first few cycles.

[0078] The invention thus encompasses isolated nucleic acid sequences encoding B. anthracis proteins, their derivatives, and/or fragments thereof, that have been codon optimized for expression in a particular recombinant system. In certain embodiments, the recombinant expression system is E. coli. In specific embodiments, the invention encompasses an isolated nucleic acid sequence encoding PA that has been codon optimized for recombinant expression in E. coli, wherein said sequence is SEQ ID NO: 19. In another embodiment, the invention encompasses an isolated nucleic acid sequence encoding an antigenic fragment of PA that has been codon optimized for recombinant expression in E. coli, wherein said sequence is SEQ ID NO: 17 or SEQ ID NO: 15. In still another specific embodiment, the invention encompasses an isolated nucleic acid sequence encoding a derivative of PA, i.e., rPA, that has been codon optimized for expression in E. coli, wherein said sequence is SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14. In specific embodiments, the nucleotide sequences encoding the polypeptides of the invention can be commercially synthesized, e.g., by Integrated DNA Technologies Inc. (Coralville, IA).

[0079] Other methods of optimizing expression of the polynucleotides of the invention, which may be used independently or in conjunction with codon optimization,

1011497 vl 28 include co-expressing the polynucleotide of the invention with genes encoding one or more rare codon tRNAs. Several commercial bacterial strains are available comprising genes that code for the, relatively, rare tRNAs. For example, such commercial strains include, but are not limited to, the following E. coli strains: BL21 (DE3) CodonPlus-RIL comprising genes encoding AGG and AGA (arginine), AUA (isoleucine), and CUA (leucine) tRNAs; BL21 (DE3) CodonPlus-RP comprising genes encoding AGG and AGA (arginine) and CCC (proline) tRNAs; and Rosetta or Rosetta (DE3) comprising genes encoding AGG , AGA and CGG (arginine), AUA (isoleucine), CUA (leucine), CCC (proline), and GGA (glycine) tRNAs. In a specific embodiment, the polynucleotides of the invention are expressed in BL21(DE3) E. coli.

[0080] Still other methods to optimize expression of the polynucleotides of the invention, which may be used independently or in combination with any other optimization scheme known in the art or described herein, include the modification of key codons to improve transcription or translational processing. For example, Looman et al. (1987, EMBO J. 6:2489-2492, hereby incorporated by reference in its entirety) has shown that the expression efficiency of a recombinant IacZ gene in E. coli varied at least 15 fold depending on the particular codon immediately following that encoding the N-terminal methionine {e.g. , the codon encoding the residue at position 1 of PA (SEQ ID NO:2), see also, e.g., Hirel et al., 1989, Proc. Natl Acad. Sci. USA 86:8247-8251; Lathrop et al., 1992, Prot. Exp. Purif. 3:512- 517, each of which is hereby incorporated by reference in its entirety). Endogenous, i.e., native and not recombinant, E. coli proteins exhibit a distinct preference in this penultimate amino acid residue. The most frequently appearing residue at this position is lysine, encoded by the codon, AAA, while a number of other codons are not used at all. Thus, modifying the codon encoding the residue at this position to a positionally preferred codon can improve expression levels. Because, in most instances, modification of the positionally preferred codons alters the amino acid sequence of the encoded protein, the expressed protein will be a derivative of the originally encoded polypeptide. Accordingly, in certain embodiments, the invention encompasses a novel isolated nucleic acid sequence encoding a derivative of a B. anthracis protein that has been optimized by modification of one or more positionally preferred codons. In a specific embodiment, the invention encompasses a nucleic acid sequence encoding a derivative of PA, e.g., an rPA, comprising a modification of a positionally preferred codon relative to the wild type sequence for optimized expression in E. coli. In a specific example in accordance with this embodiment, the positionally preferred

1011497 vl 29 codon is the codon encoding the residue at position 1 of the encoded rPA. In specific embodiments of this example, the nucleotide sequence of the codon encoding the residue at position 1 is GGT (glycine) or GAC (aspartic acid). In another specific example in accordance with this embodiment, the nucleotide sequence of the codon encoding the residue at position 1 of the encoded rPA is GGT (glycine) and the optimized sequence encoding rPA is SEQ ID NO: 12. In another specific example in accordance with this embodiment, the nucleotide sequence of the codon encoding the residue at position 1 of the encoded rPA is GAC (aspartic acid) and the optimized sequence encoding rPA is SEQ ID NO: 10, SEQ ID or SEQ ID:14.

[0081] Yet other methods to optimize polynucleotides for recombinant production include modifying nucleic acid sequence to reduce formation of secondary structures in or near the translation initiation region. The consideration of secondary structure is particularly important in embodiments where the nucleic acid sequence of the invention contains a high GC content in the 5' end of the sequence. The high GC content can lead to formation of secondary structures in the transcribed mRNA leading to interruption of translation and/or lower than expected expression levels of encoded protein. Accordingly, in certain embodiments, the invention encompasses optimizing the expression of a recombinant polynucleotide by abrogation of formation of secondary structure in said polynucleotide. In specific examples of this embodiment, the abrogation of secondary structure is effected by substitution of G or C residues with A or T residues. Where possible the substitution of specific nucleotide residues should not alter the encoded amino acid residue and/or sequence. [0082] The nucleic acid sequence of the invention may also be manipulated by standard techniques in the art to modify the encoded amino acid sequence (e.g., by insertion, deletion or substitution) to improve polypeptide processing and/or polypeptide stability. Improvements to the in vivo processing or stability of the polypeptide in the recombinant system can improve protein yield. In certain embodiments, addition of a transcription terminator (or an additional one if one is already present)will increase levels of expression. For example, fusion of the N-terminus of a heterologous protein to the C-terminus of a highly-expressed fusion partner can result in high level expression of the fusion protein. Also, the use of cell strains carrying mutations that eliminate the production of proteases as the recombinant host can enhance accumulation of polypeptides of the invention by reducing proteolytic degradation. For example, BL21 E. coli is deficient in two proteases encoded by the Ion (cytoplasmic) and ompT (periplasmic) genes. Alternately, the amino acid sequence

1011497 vl 30 of the encoded protein can be altered by standard techniques to eliminate common sites of protease recognition. In certain embodiments, the invention encompasses B. anthracis proteins comprising one or more amino acid modifications that eliminate one or more protease cleavage sites in the amino acid sequence. Is specific embodiments, the invention encompasses a modified PA polypeptide comprising one or more amino acid modifications, wherein said modification alters a chymotrypsin recognition sequence. In a specific embodiment, the invention encompasses a modified PA polypeptide, or antigenic fragment thereof, comprising at least one amino acid modification that alters at least one chemotrypsin recognition sequence, which at least one modification is at position 285. In a specific example in accordance with this embodiment, the modification at position 285 is a substitution at position 285. In a preferred embodiment, the substitution at position 285 is a substitution with glutamic acid. In another specific embodiment, the invention encompasses a modified PA polypeptide, or antigenic fragment thereof, comprising one or more amino acid modifications to alter a chemotrypsin recognition sequence, which one or more modifications is at position 308. In a specific example in accordance with this embodiment, the modification at position 308 is a substitution at position 308. In a preferred embodiment, the substitution at position 308 is a substitution with aspartic acid. In still another specific embodiment, the invention encompasses a modified PA polypeptide, or antigenic fragment thereof, comprising one or more amino acid modification to alter one or more chemotrypsin recognition sequences, which one or more modification is at position 313. In a specific example in accordance with this embodiment, the modification at position 313 is a deletion of residue 313. In yet another specific embodiment, the invention encompasses a modified PA polypeptide, or antigenic fragment thereof, that has been modified, e.g. , at one or more amino acid residues, to alter at least one chemotrypsin recognition sequence, which one or more modifications is at position 314. In a specific example in accordance with this embodiment, the modification at position 314 is a deletion of residue 314. In another embodiment, the invention encompasses a modified PA polypeptide, or antigenic fragment thereof, comprising one or more amino acid modifications to alter one or more furin recognition sequences. In a specific embodiment, the invention encompasses a modified PA polypeptide, or antigenic fragment thereof, comprising one or more amino acid modifications to alter one or more furin recognition sequences, wherein the one or more furin recognition sequence is the sequence spanning residues corresponding to residues 164-167 of SEQ ID NO:2, i.e., SEQ ID NO:3. In other embodiments, the invention encompasses a modified PA polypeptide, or antigenic

1011497 vl 31 fragment thereof, comprising one or more amino acid modifications to alter one or more furin recognition sequences, wherein the one or more furin recognition sequence does not span residues corresponding to residues 164-167 of SEQ ID NO:2. The furin recognition sequence, SEQ ID NO:3, may be modified by modification at one or more of residues 1, 2, 3, or 4, e.g., corresponding to residues 164, 165, 166, or 167 of SEQ ID NO:2, respectively. In a more preferred embodiment, the furin recognition sequence SEQ ID NO: 3 is modified at one or more of residues 1, 2, or 4. In a still more preferred embodiment, the furin recognition sequence SEQ ID NO:3 is modified at each of residues 1, 2 and 4 by substitution at 1, 2, and 4. In a specific example in accordance with this embodiment, SEQ ID NO: 3 is modified by substitution at positions 1, 2, and 4 to produce SEQ ID NO:4.

[0083] The amino acid sequence of the polypeptide of the invention may also be designed to reduce or eliminate formation of secondary transcription products. For example, in some cases, a nucleic acid sequence may, once transcribed to RNA, comprise favorable locations for the initiation translation other than at the 5' end of the gene (e.g., at an internal ribosome recognition/entry site; at a codon encoding methionine (i.e., an internal methionine) etc.). Such sites are known as internal entry sites and lead to parallel translation from a single mRNA. Parallel translation can produce non- functional truncations of a polypeptide of interest and/or directs the use of cellular resources that would otherwise be used for the full- length polypeptide of interest. Accordingly, identifying and eliminating the internal translation ignition sites can lead to improved recombinant production. In certain embodiments, the invention encompasses a modified B. anthracis polypeptide comprising a modification to alter an internal ribosyme entry site or to modify an internal methionine residue. In a specific embodiment, the invention encompasses a modified PA comprising a modification to an internal methionine residue, which methionine residue corresponds to M266 of SEQ ID NO: 2. In a specific example, the modification to M266 is a substitution. In a preferred embodiment, the substitution at M226 is M266L. In another specific embodiment, the invention encompasses a modified PA comprising a modification to an internal methionine residue, which methionine residue corresponds to M350 of SEQ ID NO: 2. In a specific example, the modification to M350 is a substitution. In a preferred embodiment, the substitution at M350 is M266L or M350I. The levels of expression of the target protein can be optimized by varying the time and/or temperature of induction and the concentration of the inducer. Such optimization of temperature and concentrations of the inducer, e.g., IPTG, are within the skill of one with ordinary knowledge in the art.

1011497 vl 32 5.2 RECOMBINANT EXPRESSION OF POLYPEPTIDES OF THE INVENTION

[0084] One aspect of the invention encompasses an efficient and economical method for producing and purifying B. anthracis proteins. In preferred embodiments, the invention is directed to the production and use of recombinant PA, i.e., rPA. The rPA so produced is a recombinant PA polypeptide (rPA) able to elicit an immune response protective against B. anthracis infection and or against one or more components of Anthrax toxin.. [0085] Once a nucleic acid sequence encoding a polypeptide of the invention has been obtained, the vector for the production of the polypeptide, e.g., rPA, may be produced by recombinant DNA technology using techniques well known in the art. Methods which are well known to those skilled in the art can be used to construct expression vectors containing the coding sequences of the polypeptides of the invention and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al. eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY). [0086] One aspect of the invention is a vector comprising the nucleic acids of the invention. In one embodiment, a vector of the invention comprises any of SEQ IS NOS:5, 8, 10, 12, 14, 15, 17, and 19.

[0087] The polynucleotide sequences of the invention can be part of an expression cassette or vector that comprises, operably linked in the 5' to 3' direction, a promoter, a polynucleotide of the present invention, and a transcriptional termination signal sequence functional in a host cell. The promoter can be of any of the types discussed herein, for example, a tissue specific promoter, a developmental regulated promoter, an organelle specific promoter, etc. The expression cassette or vector can further comprise an operably linked targeting, transit or secretion peptide coding region capable of directing transport of the protein produced. The expression cassette or vector can also further comprise a nucleotide sequence encoding a selectable marker and a purification moiety. In addition, the expression cassette or vector can further comprise an additional sequence encoding an enzyme capable of cleaving the polypeptide of the present invention between the tandem repeats in order to produce non-repeating peptide units. The enzyme encoding sequence can

1011497 vl 33 be under the control of a separate promoter, for example an inducible or developmentally regulated promoter so that production of the enzyme is triggered only after substantial amounts of the repeating polypeptide of the present invention has been produced. [0088] Accordingly, in some embodiments, the expression vector comprises a nucleic acid sequence encoding one or more polypeptides of the invention, e.g., rPA and/or fragments thereof. In other embodiments, the expression vector comprises an antisense nucleic acid sequence encoding a nucleic acid complementary to the polypeptide encoding nucleic acid sequence. A nucleic acid sequence that encodes a B.anthracis protein of the invention, e.g., rPA, can be operably linked to expression control sequences. An expression control sequence operably linked to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences. As used herein, the term "expression control sequence" refers to a nucleic acid sequence that regulates the expression of a nucleic acid sequence to which it is operably linked. Expression control sequences are operably linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and., as appropriate, translation of the nucleic acid sequence. Thus expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon in front of a protein-encoding gene, a splicing signal for introns or maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The term "control sequences" is intended to comprise components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Expression control sequences can include a promoter. The expression control elements used to regulate the expression of the protein or antisense coding region can either be the expression control element that is normally found associated with the coding sequence (homologous expression element) or can be a heterologous expression control element. A variety of homologous and heterologous expression control elements are known in the art and can readily be used to make expression units for use in the present invention.

[0089] Included within the meaning of promoter is a sequence sufficient to direct transcription. Also included are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue-specific, environmentally- or developmentally-regulated, or expression that is inducible by external signals or agents. Such elements may be located in the 5 ' or 3' regions of the gene. Both

1011497 vl 34 constitutive and inducible promoters, are included in the invention (see e.g. Bitter et al., Methods in Enzymology, 1987, -153, 516-544, hereby incorporated by reference in its entirety).

[0090] A non-limiting example of a useful vector for cloning and expression of B. anthracis proteins of the invention, e.g., rPA, in E. coli includes the pET vector (Novagen). The pET system is based upon the T7-promoter-driven system. The target gene is cloned into pET plasmids under control of strong bacteriophage T7 transcription and translation signals. Expression is induced by providing a source of T7 RNA polymerase in the host cell. Generally, the desired product can comprise more than 50% of the total cell protein a few hours after induction. In one embodiment, expression of the target protein via the pET system constitutes at least about 80% of the total cell protein (see e.g. Section 6.4, Example 4). The various pET expression systems are well known and extensively characterized in the art.

[0091] Either a constitutive promoter or an inducible promoter is typically ligated to the protein or antisense encoding region using standard techniques known in the art. The promoter may be operably linked to the protein, nucleic acid, or antisense coding region in any manner known to one of skill in the art. The expression unit may be further optimized by employing supplemental elements such as transcription terminators and/or enhancer elements. In the construction of heterologous promoter/structural gene or antisense combinations, the promoter is preferably positioned about the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.

[0092] In addition to a promoter sequence, the expression cassette can also contain a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes. If the mRNA encoded by the structural gene is to be efficiently processed, DNA sequences which direct polyadenylation of the RNA are also commonly added to the vector construct.

[0093] Thus, for expression in E. coli, the expression units will typically contain, in addition to the nucleic acid sequence or antisense sequence coding for one or more B. anthracis proteins of the invention, e.g., rPA, a promoter region, a transcription initiation site, and a transcription termination sequence. Alternatively, the expression unit may contain, in

1011497 vl 35 addition to a nucleic acid sequence or antisense coding sequence, a promoter, one or more enhancers or enhancer elements, a transcription initiation site, and a transcription termination sequence. Unique restriction enzyme sites at the 5' and 3' ends of the expression unit are typically included to allow for easy insertion into a preexisting vector. [0094] The resulting expression unit is ligated into or otherwise constructed to be included in an appropriate vector. Transformation vectors capable of introducing nucleic acid sequences encoding a B. anthracis protein of the invention, e.g., rPA, are easily designed, and generally contain one or more nucleic acid sequences of interest under the transcriptional control of 5' and 3' regulatory sequences. Such vectors generally comprise, operably linked in sequence in the 5' to 3' direction, a promoter sequence that directs the transcription of a downstream heterologous structural DNA; optionally, a 5' non-translated leader sequence; a nucleic acid sequence that encodes a protein of interest; and a 3' non- translated region that encodes a polyadenylation signal which functions to cause the termination of transcription and the addition of polyadenylate nucleotides to the 3' end of the mRNA encoding said protein. Typical 5 '-3' regulatory sequences include a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.

[0095] The vector will also typically contain a selectable marker gene by which transformed cells can be identified in culture. The marker may generally be associated with the heterologous nucleic acid sequence, i.e., the structural, gene operably linked to a promoter. As used herein, the term "marker" refers to a gene encoding a trait or a phenotype that permits the selection of, or the screening for, an organism or cell containing the marker. The marker gene may encode antibiotic resistance. This allows for selection of transformed cells from among cells that are not transformed. Other suitable markers will be known to those of skill in the art.

[0096] Replication sequences, of bacterial or viral origin, are generally also included to allow the vector to be cloned in a bacterial or phage host, preferably a broad host range prokaryotic origin of replication is included. A selectable marker for bacteria should also be included to allow selection of bacterial cells bearing the desired construct. [0097] Generally, E. coil express proteins larger than about 70 kDa poorly. Choosing a smaller fragment of the target protein can improve expression levels and solubility. In several embodiments, custom genes encoding a truncated B. anthracis protein, e.g. , a fragment of rPA, are utilized. The increased expression of the truncated forms of the proteins

1011497 vl 36 described herein may also be due in part to elimination of secondary products (see Examples). The solubility of a poorly soluble (or insoluble) protein can also be improved by selecting only a soluble domain for expression.

[0098] A wide variety of expression vectors are available that can be modified to express the novel DNA sequences of this invention. The specific vectors exemplified below are merely illustrative, and are not intended to limit the scope of the invention. Expression methods are described by Maniatis, et al. Molecular Cloning: A Laboratory Manual or Current Protocols in Molecular Biology 16.3-17.44 (1989).

[0099] Also encompassed by the invention are chimeric or fusion proteins, in particular for increasing production, solubility, and/or purification. A non-5, anthracis polypeptide can be fused to the N-terminus or C-terminus of polypeptide of the invention. Generally, vector-encoded peptide tags enable convenient detection and/or purification of target proteins, or potentiate their localization within a cell. Non-limiting examples of such fusion tags include T7 Tag, S-Tag, His-Tag, HSV-Tag, pet B/ompT, KSI, Trx-Tag, and CBD (Novagen). For example, in one embodiment, the fusion protein is a GST-rPA fusion protein in which the rPA sequence is fused to the C-terminus of the GST sequence. In another embodiment, the fusion protein is a His-rPA fusion protein in which the His-tag is fused to the C-terminus of the rPA sequence. In certain host cells, expression and/or secretion of B. anthracis proteins of the invention can be increased through use of a heterologous signal sequence. In a further embodiment, the fusion protein is an polypeptide of the invention containing a heterologous signal sequence at its N-terminus. A non-limiting example of such a heterologous sequence is the 11 amino acid T7-Tag sequence (Novagen). [00100] Preferably, the chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see e.g. Current Protocols in Molecular

1011497 vl 37 Biology, Eds. Ausubel et al. John Wiley & Sons, 1992, hereby incorporated by reference in its entirety). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide, a metal ion affinity tag, a c-myc epitope tag, etc.). An polypeptide encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in- frame to the one or more proteins of the invention. [00101] Other DNA sequences encoding additional functions may also be present in the vector, as is known in the art.

[00102] An expression vector comprising the nucleotide sequence of a polypeptide of the invention can be transferred to a host cell by conventional techniques (e.g., electroporation, liposomal transfection, heat shock, and calcium phosphate precipitation) and the transfected cells are then cultured by conventional techniques to produce the polypeptide of the invention. Generally, methods have been developed that are well known in the art for optimal transformation of a given host system. In certain embodiments, the transformation method is not electroporation. In specific embodiments, the expression of the polypeptide is regulated by a constitutive, an inducible or a tissue, specific promoter.

[00103] Generally, transformation involves splicing the gene of interest into a plasmid.

The host cell, usually a bacteria such as E. coli, is then "infected" with the plasmid and incorporates the new gene into its DNA. To separate the infected from the uninfected, the plasmid may also contain a gene giving the bacteria immunity to a certain antibiotic. By treating the sample with the antibiotic, all host cells not taking up the plasmid are killed. This process results in a new strain of E. coli bacteria expressing the inserted gene that can be cultured in bulk to create the polypeptide of interest. Transformation methodology is well known in the art. See e.g. Sambrook and Russel, Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press (2001), hereby incorporated by reference in its entirety.

[00104] Transformation is the process of incorporating the recombinant vector from a reaction mixture or vector solution into host cells (e.g., E. coli host cells). As is commonly known in the art, so-called chemically competent cells are best able to take up circular vector DNA. The method for the preparation of competent cells depends on the transformation method used and transformation efficiency required. As a non-limiting example, heat shock transformation can be used. Other methods of transformation include electroporation and electroporation-competent cells. Each of these techniques are within the understanding of one skilled in the arts. Several companies, including Bio-Rad, CLONTECH, Invitrogen, Life

1011497 vl 38 Technologies, Novagen, Promega, Qbiogene, Sigma-Aldrich, and Stratagene, offer competent cell lines and individualized protocols useful in the present invention. [00105] The choice of the E. coli host strain will depend on the goal of the transformation. The transformation of a ligation mix can be done in a cloning strain, such as DH5a, NovaBlue, or XLl-Blue. Depending on the background of non-recombinants (from a ligation mix containing only digested vector), a number of transformants (as a non- limiting example, 3-12 transformants) can be picked and checked for the presence of the right insert by restriction analysis or colony PCR. The transformation of a vector for multiplication can also be done in a recA- strain, such as DH5a, NovaBlue, or XLl-Blue. In one embodiment, transformation of ligation mixture occurs in DH5a cells. In a further embodiment, transformation of vector for multiplication occurs in -DH5a cells. In one embodiment, the cloning vector is pSP72. In another embodiment, the cloning vector is pUC18. [00106] The host cells used to express the recombinant polypeptides of the invention may be either bacterial cells such as Escherichia coli, or, eukaryotic cells. Non-limiting examples of expression vectors and appropriate E. coli hosts are as follows: pBAD vectors with Top 10 or LMG 194 hosts; pET vectors with BL21(DE3) host; pGEX vectors with BL21 host; pMal vectors with BL21 or TBl hosts; pProEx vectors with BL21 or DHlOB hosts; pQE vectors with M 15 or M 15 [pREP4] hosts; pRSET vectors with BL21 (DE3) or pLysS hosts; and pTrcHis vectors with BL21 host. In several embodiments, transformation for protein expression is done with pET vectors in BL21(DE3) hosts. In preferred embodiments a pET vector is used, which vector is pET9a or pET24a.

[00107] As known to the art, a popular protein expression system is based on T7 RNA polymerase because of the high level of expression attainable and ease of culture of E. coli. As a non- limiting example of a T7 expression system, Invitrogen, Stratagene, Novagen, and Promega each carry BL21 strains optimized for protein expression. The BL21 strain is naturally deficient in the OmpT and Lon proteases, resulting in a higher yield of intact recombinant proteins. The suffix "DE3" indicates that the host is a lysogen of *DE3, carrying a chromosomal copy of the T7 RNA polymerase gene under the control of the IPTG- inducible IacUVS promoter. Such strains can be used to induce high-level protein expression in T7 promoter-based systems.

[00108] A variety of other host-expression vector systems may be utilized to express the polypeptides of the invention. Such host-expression systems represent alternatives by which the coding sequences of the polypeptides may be produced and subsequently purified,

1011497 vl 39 but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express the polypeptides of the invention in situ. The choice of a particular expression system over another is within the skill of one with ordinary knowledge in the art. Examples include, but are not limited to, microorganisms such as bacteria {e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing polypeptide coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing polypeptide coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the polypeptide coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing polypeptide coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 293T, 3T3 cells, lymphotic cells (see U.S. Pat. No. 5,807,715), Per C.6 cells (rat retinal cells developed by Crucell)) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).

[00109] In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the polypeptide being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of a polypeptide of the invention, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the recombinant, optimized polypeptide coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free gluta-thione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

1011497 vl 40 [00110] In an insect system, Autographa californica nuclear polyhedrosis virus

(AcNPV) can be used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The recombinant, optimized polypeptide coding sequence may be cloned individually into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter). As is known in the art, insect expression systems may offer some advantages over those of bacterial cells, e.g., glycosylation of glycoprotein.

[00111] In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the recombinant, optimized polypeptide coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g. , the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the recombinant polypeptide molecule in infected hosts, (e.g., see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81 :355-359). Specific initiation signals may also be required for efficient translation of inserted polypeptide coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al, 1987, Methods in Enzymol. 153:51-544).

[00112] In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, HeIa, COS,

1011497 vl 41 MDCK, 293, 293T, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030 and Hs578Bst.

[00113] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express a polypeptide of the invention may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the recombinant polypeptide of the invention. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the recombinant polypeptide of the invention.

[00114] A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11 :223), hypoxanthine- guanine phosphoribosyltransferase (Szybalska & Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and Wu, 1991, 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIB TECH 11(5): 155-215). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY.; Colberre-

1011497 vl 42 Garapin et al., 1981, J. MoI. Biol. 150: 1; and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147) (each of the above references hereby incorporated in their entireties).

[00115] The expression levels of a polypeptide of the invention can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)). When a marker in the vector system expressing an recombinant polypeptide is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the nucleotide sequence of a polypeptide of the invention, production of the polypeptide will also increase (Crouse et al., 1983, MoI. Cell. Biol. 3:257) (each of the above references hereby incorporated in their entireties). [00116] Once the recombinant polypeptide of the invention has been expressed, it may be purified by any method known in the art for purification of a protein for pharmaceutical purposes, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. [00117]

5.2.1 RECOMBINANT PRODUCTION IN E. COLI HOST SYSTEMS [00118] In a specific embodiment, an E. coli host transformed with recombinant B. anthracis protective antigen (rPA) is cultured to produce rPA by: inoculating a host E. coli cell in growth media (pH of about 5.0 to about 8.0) containing a suitable antibiotic; incubating the inoculated media at a predetermined temperature, all the while agitating, until the optical density at 600 nm (OD600) is within a predetermined range; adding an operon inducer to induce expression of rPA; further incubating the host cell culture, which now contains host cells, media, and operon inducer, for about 2 to about 4 hours at a predetermined temperature; and, finally, cooling the sample and harvesting cells. [00119] In another embodiment, an E. coli host transformed with recombinant B. anthracis protective antigen (rPA) is cultured to produce rPA by: inoculating a host E. coli cell in growth media (pH of about 5.0 to about 8.0) containing a suitable antibiotic; incubating the inoculated media at a predetermined temperature, all the while agitating, until the OD600 is within a predetermined range; adding operon inducer to induce expression of

1011497 vl 43 rPA; incubating the culture for a pre-determined period of time; adding Rifampicin at a concentration of about 30 to about 250 ug per milliliter of host cell culture; continuing incubation of the host cell culture, which now contains host cells, media, operon inducer, and Rifampicin for a predetermined period of time and at a predetermined temperature; and, finally, cooling the sample and harvesting cells.

[00120] In a further embodiment, an E. coli host transformed with a gene encoding recombinant B. anthracis protective antigen (rPA), the rPA capable of eliciting an immune response protective against B. anthracis infection, is cultured by: inoculating a host E. coli cell in growth media (pH of about 5.0 to about 8.0) containing a suitable antibiotic; incubating the inoculated media at a predetermined temperature, all the while agitating, until the OD600 is within a predetermined range; adding Rifampicin at a concentration of about 30 to about 250 μg per milliliter of host cell culture; incubating the culture for a pre-determined period of time; adding enough IPTG to the incubated, inoculated media to bring the host cell culture to a concentration of between about 0.05 mM to about 2.0 mM IPTG; continuing incubation of the host cell culture, which now contains host cells, media, operon inducer, and Rifampicin for a predetermined period of time and at a predetermined temperature; and, finally, cooling the sample and harvesting cells.

[00121] The recombinant host may be cultured by any method known in the art suitable for maintenance of the host and enabling optimal production of the recombinant protein. In most embodiments, the recombinant host is cultured according to manufacturer's directions. In several of the above described embodiments, the growth media may be APS growth media. In specific embodiments, the APS growth media is at a pH of about 7.0. [00122] Generally, expression of transgenic polypeptide through culturing a transformed host involves isolation of a single transformed colony, growing a starter culture, inoculation and incubation of the main culture, and induction of protein expression. [00123] To isolate a transformed colony, a single colony can be picked from a freshly streaked plate of the expression host containing the recombinant vector. When the heterologous protein is toxic for the cells, higher expression levels can be obtained by using the so-called "plating" method. A starter culture can be grown by inoculating the picked colony in to rich medium containing the appropriate antibiotic. Appropriate antibiotic can be determined by referencing which antibiotic gene is present in the vector constructs described above and below. For E. coli cultures, starter cultures may, for example, be grown overnight at 30⁰C or lower; or, the culture can be incubated at 37°C until the OD600 is approximately 1

1011497 vl 44 and then stored overnight at 4°C. The cells can then be collected by centrifugation, resuspended in fresh medium, and used to inoculate the main culture.

[00124] In one embodiment, both a starter culture and a main culture are generated. In another embodiment, the main culture is generated directly from a single isolated cell or colony.

[00125] The main culture can then be inoculated until reaching some reference OD600 value. In one embodiment, the main culture is incubated until OD600 reaches from about 0.4 to about 0.6. In another embodiment, the main culture is incubated until OD600 reaches approximately 0.5. In a further embodiment, the main culture is incubated until OD600 reaches approximately 0.6. The optimal OD value may depend on the culture method and the medium, as commonly understood in the art. For good aeration, the medium will usually not exceed about 20% of the total flask volume. Usually, agitation will accompany incubation. Temperature of incubation can range from below 30° C to around 42° C. In one embodiment, the cultures are held at about 37° C until the OD for induction is reached. The 37° C incubation temperature is thought to facilitate increased growth rate. In another embodiment, the cultures are held at about 30° C until the OD for induction is reached. The 30° C incubation temperature is thought to minimize the expression of secondary rPA products. After incubation, the main cultures are induced to express the recombinant gene. In several embodiments, the operon inducer is isopropyl-beta-D-thiogalactopyranoside (IPTG). In one embodiment, the IPTG concentration in the cell culture is about 0.01-0.05 mM, e.g., for the "plain" T& promoter, e.g., of the pET9a system. In other embodiments, the IPTG concentration in the cell culture may be about 1 mM or higher, e.g., for a regulated TlI lac promoter, e.g., of the pET 24a system. The determination of IPTG concentrations suitable for the expression of the recombinant polypeptide is well within the knowledge of one of skill in the art. In some embodiments, the main culture is cooled, for example on ice, to an optimal induction temperature. Protein expression can be induced in the host cells by the addition of the proper inducer or by changing the growth conditions. [00126] Non-limiting examples of induction conditions for various promoters within recombinant bacterial systems are as follows: for trc (hybrid) promoter, add IPTG at 0.05 - 2.0 mM, typically 0.2 mM; for araBAD promoter, add I-arabinose at 0.002% - 0.4%, typically 0.2%;for PL promoter, shift the temperature from 37 to 42°C; and for T7-lac operator promoter, add IPTG at 0.05 -2.0 mM, typically 0.2 mM.

1011497 vl 45 [00127] The induced cell culture is then incubated for a predetermined interval of time.

In one embodiment, the induced cell culture is incubated for about 2 to about 6 hours. In another embodiment, the induced cell culture is incubated for about 3 to about 4 hours. In a further embodiment, the induced cell culture is incubated for about 3.5 hours. After induction, the cells will usually use most of their resources for the production of the target protein and further growth is limited. Incubation time will in part depend upon induction temperature. Non- limiting examples of incubation time and temperatures are as follows: 15°C, overnight; 20⁰C, overnight; 25°C, overnight; 30⁰C, 5-6 hours; and 37°C, '3-4 hours. In one embodiment of the invention, after induction, the culture is incubated at about 37° C for about 3.5 hours.

[00128] In some embodiments, Rifampicin is added to the main culture. Rifampicin is known as rifampin in the United States, and is manufactured, for example, by Merrell under the tradename Rifadin and by Ciba Geigy under the tradename Rimactane. In one embodiment, Rifampicin is added at about 30 to about 250 micrograms per milliliter of host cell culture. In another embodiment,. Rifampicin is added at about 150 micrograms per milliliter of host cell culture. In several embodiments, addition of Rifampicin is performed after induction with IPTG. In one embodiment, Rifampicin is added 30 minutes after IPTG induction commences. In non- limiting exemplary studies described below, adding Rifampicin was observed to significantly increase expression of the target rPA in E. coli. 5.3 PURIFICATION, ISOLATION AND PREPARATION OF

RECOMBINANT PROTEIN

[00129] One aspect of the invention entails recovering recombinant protein from transformed E, coli cells. In one embodiment, rPA is recovered from a transformed E. coli host by: disrupting the cells of the E. coli host; isolating the rPA-containing inclusion bodies from the disrupted cells; suspending these inclusion bodies in a solubilizing solution containing about 8 M Urea; adding this mixture to buffer so that the urea concentration of the sample decreases to within the range of about 6 to about 2 M; recycling this buffered sample through a Heparin column in tandem with a Blue Sepharose column for a predetermined period of time; washing the Blue Sepharose column containing bound rPA with buffer; eluting the rPA from the washed Blue Sepharose column; and finally, collecting the column void fractions that contain rPA.

[00130] In specific embodiments, the recovered rPA is encoded by any of SEQ ID

NOS:5, 8, 10, 12, 14, 15, and 17.

1011497 vl 46 [00131] Recovery of recombinant protein entails disrupting the cultured E. coli host cells and isolating the protein therefrom. E. coli cells can be disrupted by high pressure homogenization. Optional lysozyme treatment can follow. It is beneficial that cell lysis is complete, because intact cells can sediment together with the inclusion bodies, thus potentially contaminating the preparation. In one embodiment, disruption of cells occurs via cell bomb, where the cells are first polytroned in buffer (as a non-limiting example, 2 mM EDTA / 50 mM TRIS, Ph 8.5) and then bombed for 20 minutes. In a further embodiment, the cells are bombed for 30 minutes.

[00132] In several embodiments of the invention, the target polypeptide is expressed in inclusion bodies of the cultured E. coli host cells. Generally, when target protein is expressed in inclusion bodies, the protein is recovered, denatured, and refolded in vitro. This procedure can be carried out in three phases:, isolation of the inclusion bodies; solubilization and denaturation of the target protein; and refolding by removal of denaturant. [00133] In the cell there is competition between folding and aggregations. In many cases and in several host systems, recombinant proteins accumulate intracellularly in insoluble aggregates, especially under conditions of high level expression. The proteins in these so-called inclusion bodies are mostly inactive and denatured. In addition, dimers and multimers may be present. However, the expression of recombinant proteins in inclusion bodies can also be advantageous. Non- limiting examples of the advantages of inclusion body expression include: the recombinant protein deposited in inclusion bodies can be 50% or more of the total cellular protein; the inclusion bodies often contain almost exclusively the over-expressed protein; the protein in inclusion bodies can be protected from proteolytic degradation; and expression in inclusion bodies can protect the cell against the toxicity of the recombinant protein. It is recognized in the art that a major problem is to recover biologically active and/or soluble protein in high yield from inclusion bodies. In order to accomplish this, the protein in the inclusion bodies must by solubilized and refolded in vitro (see generally Lilie et aL, Advances in refolding of proteins produced in E. colt, 1998, Current Opinion Biotechnol. 9, 497-501, incorporated herein by reference in its entirety). [00134] Inclusion bodies can be isolated from the disrupted cells, e.g., E. coli cells.

Inclusion bodies have a relatively high density and, therefore, can be pelleted by centrifugation (as a non-limiting example, 30 minutes at 12,000 x g). After centrifugation of disrupted E. coli cells, the pellet can be washed with buffer (as non-limiting examples, TEN or PET) that may contain low concentrations of chaotropic agents (as non- limiting examples,

1011497 vl 47 0.5-1 M guanidine-HCI or urea) or detergents (as non- limiting examples, 1 % Triton X-IOO or 1 mg/ml sodium deoxycholate). This wash step aids in removal of contaminants, especially proteins (as a non-limiting example, proteases), that may have absorbed onto the hydrophobic inclusion bodies during processing.

[00135] In some embodiments, the pellet is washed twice. In other embodiments, the pellet is washed three times. In certain embodiments, the pellet is further washed in ethanol to facilitate the removal of Rifampicin that may be present. In some embodiments, the pellet can be incubated with Benzonase (as a non-limiting example, for 30 minutes at 37 C) to hydrolyze DNA and RNA. The washed pellet is usually re-centrifuged. [00136] The inclusion bodies can then be resuspended and incubated in buffer that may contain a strong denaturant (see generally Protein purification. Handbook, Amersham Pharmacia biotech, p.71 (1999), hereby incorporated by reference in its entirety). Non- limiting examples of strong denaturants and the typical concentrations used for the solubilization of protein from inclusion bodies include: urea at 2-8 M, typically 8 M; guanidine hydrochloride at 3-8 M, typically 6 M; sarkosyl (N-lauroylsarcosine) at 2%; Triton X-100 at 2% + sarkosyl at 1.5%; N-cetyl trimethylammonium chloride at 5%; N- octylglucoside at 2%;sodium dodecyl sulphate at 0.1-0.5%,typically 0.1 %;and alkaline pH above pH 9 (NaOH). The addition of a reducing agent (as non- limiting examples, 20 mM DTT or b-mercaptoethanol) can keep all cysteines in the reduced state and cleaves disulfide bonds formed during the preparation.

[00137] Incubation temperatures above 30⁰C can be used to facilitate the solubilization process. Optimal conditions for solubilization are protein specific and can be determined for each protein, as is within the ordinary skill in the art. A non- limiting example of re-folding conditions is as follows: buffer composition (pH, ionic strength) 50 mM Tris-HCI, pH 7.5; incubation temperature 30⁰C; incubation time 60 min; concentration of solubilizing agent 6 M guanidine-HCI or 8 M urea; and total protein concentration 1-2 mg/ml. After solubilization, the solution can optionally be centrifuged (as a non-limiting example, ultracentrifugation for 30 min at >100,000 xg) to remove remaining aggregates which could act as nuclei to trigger aggregation during refolding.

[00138] The invention encompasses the use of any solubilizing agent known in the art to be useful for the solubilization of pharmaceutical compounds from inclusion bodies. In several embodiments of the invention, the solubilizing solution is 8 M urea and the refolding buffer is 0.025% PEG in 50 mM Tris (PEG buffer).

1011497 vl 48 [00139] Refolding of the solubilized proteins is usually initiated by the removal of the denaturant. The efficiency of refolding may depend on the competition between correct folding and aggregation. To slow down the aggregation process, refolding is usually carried out at low protein concentrations (as a non- limiting example, 10-100 mg/ml). Furthermore, refolding conditions can be optimized for each individual protein. Non-limiting examples of refolding variables include: buffer composition, e.g., pH or ionic strength; temperature; and additives (often in combination). Refolding additives may include chaotropic agents, salts, sugars, detergents and surfactants, and short chain alcohols (see e.g. De Bernardez Clark, 1998, Current Opinion Biotechnol. 9, 157-163). Non-limiting examples of useful in vitro folding aids include guanidine-HCI at 1 M; urea at 4 M; L-arginine at 0.5 M; ammonium sulphate at 1 M; sucrose; glucose at 1 M; N-acetylglucosamine at 1 M; Glycerol at 10 - 50%; Sarcosine at 1 - 4 M; Chaps at 31 mM; Tween; SDS; N-lauroylsarcosine (Sarkosyl) at 0.4%; dodecylmaltoside at 2 mM; polyethylene glycol at 10-100 mM; octaethylene glycol monolauryl at 10-100 mM; phospholipids; Sulphobetaines; Non-detergent sulphobetaines (e.g. NDSB 195) at 1-4 M; n-pentanol at 1-10 mM;~ n-hexanol at 10-100 mM; and cyclohexanol at 1-10 mM. Commercial protein folding screens are also available (as a non- limiting example, Foldlt by Hampton Research).

[00140] Non-limiting examples of refolding methods include slow dilution, rapid dilution, dialysis, pulse renaturation, and chromatography.

[00141] In slow dilution refolding, the concentration of the solubilizing agent is decreased by dilution allowing the protein to refold. Usually the dilution is carried out slowly by step-wise addition of buffer or by continuous addition using a pump. The refolding protein is exposed for some period of time to an intermediate concentration of the solubilizing agent (as non-limiting examples, 2-4 M urea or guanidine-HCI).

[00142] In one embodiment, inclusion bodies solubilized in about 8 M urea are added dropwise at a rate of 5 ml/min to buffer, until the final concentration of urea after dilution is about 3-5 M, all the while continuing to stir the buffer. In some embodiments, the buffer is a Tris buffer containing PEG. In other embodiments, the final concentration of urea after dilution is about 4 M. In certain embodiments, the buffer contains MgC12. In some embodiments, the buffer contains CaC12. In several embodiments, the buffer contains benzamidine. As should be apparent, various combination of these conditions are possible.

1011497 vl 49 [00143] In rapid dilution refolding, solubilized protein solution is rapidly diluted into the refolding buffer. Aggregation during this process can be limited by adding mild solubilizing agents to the refolding buffer, such as non-detergent sulfobetaines. [00144] In dialysis refolding, the concentration of the solubilizing agent decreases slowly which allows the protein to refold optimally. The ratio of the volumes of the sample and the dialysis buffer can be such that at the equilibrium concentration of the solubilizing agent, the protein has completely refolded.

[00145] In pulse renaturation refolding, in order to keep the concentration of the unfolded protein low, thus limiting aggregation, aliquots of denatured protein are added at defined time points to the refolding buffer. The time intervals between two pulses can be optimized for each individual protein. The process is stopped when the concentration of denaturant reaches a critical level with respect to refolding of the specific protein. [00146] In chromatography refolding, the solubilizing agent is removed using a chromatographic step. Non- limiting examples of different chromatography methods useful in refolding include: size exclusion chromatography (e.g. gel filtration on a Superdex 75 column); ion exchange chromatography; and affinity chromatography (e.g. IMAC using Chelating Sepharose or Ni-NTA agarose). The denaturant is removed while the protein is slowly migrating through the column or bound to the matrix. This usually gives a high yield of active protein even at protein concentrations in the mg/ml range. [00147] Alternatively, it is possible to carry out chromatography under denaturing conditions before refolding the protein. Most modern chromatography resins are stable under the commonly used conditions for solubilization.

[00148] In various embodiments of the invention, the urea solubilized inclusion bodies are recycled through a Heparin column in tandem with a Blue Sepharose column as the urea concentration is continually decreased from about 8 M to about 6 M-2 M. In other embodiments, the urea concentration is continually decreased from about 8 M to about 5 M-3 M. In further embodiments, the urea concentration is continually decreased from about 8 M to about 4 M. It is thought that while refolding rPA does not bind to the heparin column, low molecular weight proteins do, thus facilitating purification. The refolding rPA binds to the matrix of the Blue Sepharose column as the urea denaturant is decreased in concentration. In one embodiment, the urea solubilized inclusion bodies are recycled for two hours. In another embodiment, the urea solubilized inclusion bodies are recycled for three hours. In certain embodiments, the void from the Heparin and Blue Sepharose columns are passed through a Q

1011497 vl 50 column. In several embodiments, the void from columns is salted out (as a non-limiting example, a 60% ammonium sulfate cut), as commonly understood in the art (see generally Arakawa and Timasheff, 1985, Methods in Enzymology, 113, 49-77, hereby incorporated by reference in its entirety), and the cut fraction reintroduced to the recycling sample. [00149] The Blue Sepharose column containing bound rPA can be eluted with a salt gradient. In one embodiment, the gradient is a NaCI gradient from about 0 to about 5 M. Eluted fractions containing successfully refolded rPA can then be collected. In certain embodiments, these fractions may be pooled and/or further purified. [00150] As an alternative to the refolding procedure, the expression system can be designed with the intent to maintain the protein in solubilized form. For example, reducing the rate of protein synthesis can improve the solubility of the expressed protein. Lowering the growth temperature can decrease the rate of protein synthesis and usually more soluble protein is obtained. Using a weaker promoter (e.g. trc instead of T7) can reduce the rate of protein synthesis and improve the solubility of the expressed protein. Using a lower copy number plasmid can reduce the rate of protein synthesis and improve the solubility of the expressed protein. Lowering the inducer concentration can reduce the rate of protein synthesis and improve the solubility of the expressed protein.

[00151] Changing the growth medium can improve the solubility of the expressed protein. Non- limiting examples include: addition of prosthetic groups or co-factors which are essential for proper folding or for protein stability; addition of buffer to control pH fluctuation in the medium during growth; addition of 1 glucose to repress induction of the lac promoter by lactose, which is present in most rich media; addition of polyols (e.g. sorbitol) and sucrose which increase osmotic pressure leading to accumulation of osmoprotectants in the cell, which stabilize the native protein structure; and addition of ethanol, low molecular weight thiols and disulfides, and NaCI (see, e.g., Georgiou and Valax, 1996, Current Opinion Biotechnol. 7, 190-197, hereby incorporated by reference in its entirety). [00152] Molecular chaperones can be used to promote the proper isomerization and cellular targeting by transiently interacting with folding intermediates. Non- limiting examples in E. coli systems include: GroES-GroEL; DnaK-DnaJ-GrpE; and CIpB. [00153] Foldases can accelerate rate-limiting steps along the folding pathway. Non- limiting examples of foldases that can play an important role include: peptidyl prolyl cis/trans isomerases (PPI's); disulfide oxidoreductase (DsbA); disulfide isomerase (DsbC); and protein disulfide isomerase (PDI).

1011497 vl 51 [00154] Co-expression of one or more of the above described proteins with the target protein can lead to higher levels of soluble protein. The levels of co-expression of the different chaperones/foldases can be optimized for each individual case. DsbA and DsbC can also show positive effects on expression levels when used as a fusion partner. [00155] Addition of a fusion partner can improve the solubility of the expressed protein. Fusion of the N-terminus of a heterologous protein to the C-terminus of a soluble fusion partner can improve the solubility of the fusion protein.

5.4 COMPOSITIONS AND METHODS OF ADMINISTERING

[00156] The invention provides methods and pharmaceutical compositions comprising the derivative B. anthracis polypeptides of the invention, e.g., rPA and/or fragments thereof. The invention also provides methods of treatment, prophylaxis, and amelioration of one or more symptoms associated with a disease, disorder or infection, e.g. , Anthrax, by administering to a subject an effective amount of a protein or a conjugated molecule of the invention, or a pharmaceutical composition comprising a protein or conjugated molecules of the invention. In a preferred aspect, an polypeptide or conjugated molecule, is substantially purified (i.e., substantially free from substances that limit its effect or produce undesired side- effects). In a specific embodiment, the subject is an animal, preferably a mammal such as non-primate (e.g., cows, pigs, horses, cats, dogs, rats etc.) and a primate (e.g., monkey such as, a cynomolgous monkey and a human). In a preferred embodiment, the subject is a human.

[00157] Various delivery systems are known and can be used to administer a composition comprising the polypeptides of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the polypeptide or fusion protein, receptor-mediated endocytosis (See, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as part of a retroviral or other vector, etc. [00158] Methods of administering a polypeptide of the invention include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural, and mucosal (e.g., intranasal and oral routes). In a specific embodiment, the polypeptides of the invention are administered intramuscularly, intravenously, or subcutaneously. The compositions may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic

1011497 vl 52 or local. In addition, pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968; 5,985, 20; 5,985,309; 5,934,272; 5,874,064; 5,855,913; 5,290,540; and 4,880,078; and PCT Publication Nos. WO 92/19244; WO 97/32572; WO 97/44013; WO 98/31346; and WO 99/66903, each of which is incorporated herein by reference in its entirety. [00159] The amount of the composition of the invention which will be effective in the treatment, prevention or amelioration of one or more symptoms associated with a disorder can be determined by standard clinical techniques. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the condition, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[00160] Treatment of a subject with a therapeutically or prophylactically effective amount of the pharmaceutical compositions, i.e., comprising one or more of the polypeptides of the invention, can include a single treatment or, preferably, can include a series of treatments.

5.5 PHARMACEUTICAL COMPOSITIONS

[00161] Preferably, a composition (e.g., pharmaceutical composition) includes, in admixture, a pharmaceutically acceptable excipient, carrier, or diluent, and one or more of a bioactive agent (e.g., rPA protein or antigenic fragment thereof), as described herein, as an active ingredient. The preparation of pharmaceutical compositions that contain bioactive agents as active ingredients is well understood in the art. Typically, such compositions are prepared as liquid solutions or suspensions, however, solid forms suitable for solution in, or suspension in, liquid prior to administration can also be prepared. The preparation can also be emulsified. The active therapeutic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient, e.g., a permeation enhancer. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. Preferred carriers, excipients, and diluents of the invention comprise physiological saline (i.e., 0.9% NaCl). In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH- buffering agents, which enhance the effectiveness of the active ingredient. [00162] The compositions of the invention include bulk drug compositions useful in the manufacture of pharmaceutical compositions (e.g., impure or non-sterile compositions)

1011497 vl 53 and pharmaceutical compositions (i.e., compositions that are suitable for administration to a subject or patient) which can be used in the preparation of unit dosage forms. Such compositions comprise a prophylactically or therapeutically effective amount of a prophylactic and/or therapeutic agent disclosed herein or a combination of those agents and a pharmaceutically acceptable carrier. In certain embodiments, the compositions of the invention comprise an immunogenic amount of an rPA as disclosed herein and/or an antigenic fragment thereof and, optionally, a pharmaceutically acceptable carrier. In other embodiments, the compositions of the invention comprise a prophylactically or therapeutically effective amount of an rPA as disclosed herein and/or an antigenic fragment thereof and, optionally, a pharmaceutically acceptable carrier.

[00163] In a specific embodiment, the term "pharmaceutically acceptable" means physiologically compatible. Preferably, pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, excipient, permeation enhancer (in the art as described above), or vehicle with which the therapeutic is administered. Such pharmaceutical carriers include, but are not limited to, sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Common suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.

[00164] The pharmaceutical compositions of the invention comprising an immunogenic rPA and/or an antigenic fragment thereof as set forth above are referred to herein as "vaccines." The term vaccine is used to indicate that the compositions of the invention may be used to induce a prophylactic or therapeutic immune response. A vaccine of the invention may comprise a fragment of an rPA as described herein comprising a single antigenic domain or epitope, or an rPA polypeptide comprising a plurality of antigenic domains or epitopes. Further, a vaccine may comprise an admixture of rPAs of the invention and/or fragments thereof or any combination of the foregoing. Pharmaceutical compositions

1011497 vl 54 comprising vaccines of the invention can offer various advantages over conventional vaccines, including reduced costs, enhanced immunogenicity, potential reduction in the amount of antigen used, and less frequent booster immunizations.

[00165] A vaccine composition comprising one or more rPA proteins and/or fragments thereof in accordance with the invention may be administered cutaneously, subcutaneously, intradermally, intravenously, intramuscularly, parenterally, intrapulmonarily, intravaginally, intrarectally, nasally, orally or topically. The vaccine composition may be delivered by injection, particle bombardment, orally or by aerosol.

[00166] Vaccine compositions in accordance with the invention may further include various additional materials, such as a pharmaceutically acceptable carrier. Suitable carriers include any of the standard pharmaceutically accepted carriers, such as phosphate buffered saline solution, water, emulsions such as an oil/water emulsion or a triglyceride emulsion, various types of wetting agents, tablets, coated tablets and capsules. An example of an acceptable triglyceride emulsion useful in intravenous and intraperitoneal administration of the compounds is the triglyceride emulsion commercially known as Intralipid.RTM.. Typically such carriers contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid, talc, vegetable fats or oils, gums, glycols, or other known excipients. Such carriers may also include flavor and color additives or other ingredients. [00167] The vaccine composition of the invention may also include suitable diluents, preservatives, solubilizers, emulsifϊers, adjuvants (e.g., aluminum phosphate, hydroxide, or sulphate) and/or carriers. Such compositions may be in the form of liquid or lyophilized or otherwise dried formulations and may include diluents of various buffer content (e.g., Tris- HCl, acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), solubilizing agents (e.g. glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfϊte), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), bulking substances or tonicity modifiers (e.g., lactose, mannitol, sorbitol), covalent attachment of polymers such as polyethylene glycol to the protein, complexing with metal ions, or incorporation of the material into or onto particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, hydrogels, etc. or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance. The choice of compositions will depend on the

1011497 vl 55 physical and chemical properties of the vaccine. For example, a product derived from a membrane-bound form of a polysaccharide and/or carrier protein may require a formulation containing detergent. Controlled or sustained release compositions include formulation in lipophilic depots (e.g. fatty acids, waxes, oils). Other embodiments of the compositions of the invention incorporate particulate forms protective coatings, protease inhibitors or permeation enhancers for various routes of administration, including intramuscular, parenteral, pulmonary, nasal and oral.

[00168] The compositions of the invention can be formulated as neutral or salt forms.

Pharmaceutically acceptable salts include, but are not limited to those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

[00169] The compositions of the invention, e.g., vaccines, may further comprise one or more adjuvants to enhance immunogenic effectiveness of the composition. The adjuvant used can be any adjuvant known in the art to be suitable for use with antigenic protein or polypeptide -based vaccines. Suitable adjuvants include, but are not limited to oil-in- water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides or bacterial cell wall components), such as for example (a) MF59.TM. (WO 90/14837; Chapter 10 in Vaccine design: the subunit and adjuvant approach, eds. Powell & Newman, Plenum Press 1995), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing MTP-PE) formulated into submicron particles using a microfluidizer, (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP either micro fluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) RIBI. TM. adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components such as monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox.TM.). Other adjuvants include saponin adjuvants (such as QS21 or Stimulon.TM. (Cambridge Bioscience, Worcester, Mass.) or particles generated therefrom such as ISCOMs (immunostimulating complexes), which ISCOMS may be devoid of additional detergent e.g WO 00/07621); Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); cytokines (such as interleukins (e.g. IL-I, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 (WO99/44636), etc.),

1011497 vl 56 interferons (e.g. gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.); monophosphoryl lipid A (MPL) or 3-0-deacylated MPL (3dMPL) (e.g. GB-2220221, EP-A-0689454, optionally in the substantial absence of alum when used with pneumococcal saccharides e.g. WO 00/56358); combinations of 3dMPL with, e.g., QS21 and/or oil-in-water emulsions (e.g. EP-A-0835318, EP-A-0735898, EP-A- 0761231); oligonucleotides comprising CpG motifs (Krieg Vaccine 2000, 19, 618-622; Krieg Curr opin MoI Ther2001 3:15-24; Roman et al, Nat. Med, 1997, 3, 849-854; Weiner et al, PNAS USA, 1997, 94, 10833-10837; Davis et al, J. Immunol, 1998, 160, 810-876; Chu et al., J. Exp. Med, 1997, 186, 1623-1631; Lipford et al, Ear. J. Immunol, 1997, 27, 2340-2344; Moldoveami e/ al., Vaccine, 1988, 16, 1216-1224, Krieg et al., Nature, 1995, 374, 546-549; Klinman et al., PNAS USA, 1996, 93, 2879-2883; Ballas et al, J. Immunol, 1996, 157, 1840- 1845; Cowdery et al, J. Immunol, 1996, 156, 4570-4575; Halpern et al, Cell Immunol, 1996, 167, 72-78; Yamamoto et al, Jpn. J. Cancer Res., 1988, 79, 866-873; Stacey et al, J. Immunol, 1996, 157,2116-2122; Messina et al, J. Immunol, 1991, 147, 1759-1764; Yi et al, J. Immunol, 1996, 157,4918-4925; Yi et al, J. Immunol, 1996, 157, 5394-5402; Yi et al, J. Immunol, 1998, 160, 4755-4761; and Yi et al, J. Immunol, 1998, 160, 5898-5906; International patent applications WO 96/02555, WO 98/16247, WO 98/18810, WO 98/40100, WO 98/55495, WO 98/37919 and WO 98/52581] i.e. containing at least one CG dinucleotide, where the cytosine is unmethylated); a polyoxyethylene ether or a polyoxyethylene ester (e.g. WO 99/52549); a polyoxyethylene sorbitan ester surfactant in combination with an octoxynol (WO 01/21207) or a polyoxyethylene alkyl ether or ester surfactant in combination with at least one additional non-ionic surfactant such as an octoxynol (WO 01/21152); a saponin and an immunostimulatory oligonucleotide (e.g. a CpG oligonucleotide) (WO 00/62800); an immunostimulant and a particle of metal salt (e.g. WO 00/23105); a saponin and an oil-in-water emulsion e.g. WO 99/11241; a saponin (e.g QS21)+3dMPL+IM2 (optionally+a sterol) e.g WO 98/57659; and/or other substances that act as immunostimulating agents to enhance the efficacy of the composition. [00170] The pharmaceutical compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of antigen in these formulations can vary widely (e.g. , from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight), and will

1011497 vl 57 be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs. The resulting compositions may be in the form of a solution, suspension, tablet, pill, capsule, powder, gel, cream, lotion, ointment, or aerosol.

[00171] Conjugates prepared according to the preferred embodiment are administered to a subject in an immunologically effective dose in a suitable form to treat and/or prevent infectious diseases. The term "subject" as used herein, refers to animals, such as mammals. For example, mammals contemplated include humans, primates, dogs, cats, sheep, cattle, goats, pigs, horses, mice, rats, rabbits, guinea pigs, and the like. The terms "subject", "patient", and "host" are used interchangeably. As used herein, an "immunologically effective" dose of the conjugate vaccine is a dose which is suitable to elicit an immune response. The particular dosage depends upon the age, weight and medical condition of the subject to be treated, as well as on the method of administration. Suitable doses can be readily determined by those of skill in the art.

[00172] In practicing immunization protocols for treatment and/or prevention of specified diseases, a therapeutically effective amount of an rPA protein and/or fragment thereof is administered to a subject. As used herein, the term "effective amount" means the total amount of therapeutic agent (e.g., rPA protein or fragment thereof) or other active component that is sufficient to show a meaningful benefit to the subject, such as, enhanced immune response, treatment, healing, prevention or amelioration of the relevant medical condition (disease, infection, or the like), or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When "effective amount" is applied to an individual therapeutic agent administered alone, the term refers to that therapeutic agent alone. When applied to a combination, the term refers to combined amounts of the ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously. As used herein, the phrase "administering an effective amount" of a therapeutic agent means that the subject is treated with said therapeutic agent(s) in an amount and for a time sufficient to induce an improvement, and preferably a sustained improvement, in at least one indicator that reflects the severity of the disease, infection, or disorder. [00173] The anthrax vaccines of the invention can be administered as a single dose or in a series including one or more boosters. For example, an infant or child can receive a single dose early in life, then be administered a booster dose up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years later. The booster dose generates antibodies from primed B-cells, i.e., an

1011497 vl 58 anamnestic response. The vaccine of the invention may elicit a high primary functional antibody response in infants or children, and may be capable of eliciting an anamnestic response following a booster administration, demonstrating that the protective immune response elicited by the vaccine is long-lived.

[00174] Vaccines of the invention can be formulated into liquid preparations for, e.g., oral, nasal, anal, rectal, buccal, vaginal, peroral, intragastric, mucosal, perlinqual, alveolar, gingival, olfactory, or respiratory mucosa administration. Suitable forms for such administration include suspensions, syrups, and elixirs. The conjugate vaccines can also be formulated for parenteral, subcutaneous, intradermal, intramuscular, intraperitoneal or intravenous administration, injectable administration, sustained release from implants, or administration by eye drops. Suitable forms for such administration include sterile suspensions and emulsions. Such conjugate vaccines can be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, and the like. The vaccines of the invention can also be lyophilized. The conjugate vaccines can contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as "Remington: The Science and Practice of Pharmacy", Lippincott Williams & Wilkins; 20th edition (Jun. 1, 2003) and "Remington's Pharmaceutical Sciences", Mack Pub. Co.; 18.sup.th and 19.sup.th editions (December 1985, and June 1990, respectively), incorporated herein by reference in their entirety, can be consulted to prepare suitable preparations, without undue experimentation. Such preparations can include complexing agents, metal ions, polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, dextran, and the like, liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. The presence of such additional components can influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance, and are thus chosen according to the intended application, such that the characteristics of the carrier are tailored to the selected route of administration.

[00175] The pharmaceutical compositions of the invention are preferably isotonic with the blood or other body fluid of the recipient. The isotonicity of the compositions can be attained using sodium tartrate, propylene glycol or other inorganic or organic solutes.

1011497 vl 59 Sodium chloride is particularly preferred. Buffering agents can be employed, such as acetic acid and salts, citric acid and salts, boric acid and salts, and phosphoric acid and salts. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. [00176] The pharmaceutical compositions and/or vaccines of the invention can be administered to subject that is at risk for acquiring a disease or disorder (e.g., bacterial infection) to prevent or at least partially arrest the development of disease an/or a symptom or complication associated therewith. Amounts effective for therapeutic use will depend on, e.g., the antigen composition, the manner of administration, the weight and general state of health of the patient, and the judgment of the prescribing physician. Single or multiple doses of the antigen compositions may be administered depending on the dosage and frequency required and tolerated by the patient, and route of administration.

5.5.1 IMMUNIZATION REGIMEN

[00177] The recombinant proteins produced according to the methods of the invention retain the immunogenic properties of their wild-type counterparts, and thus may be used in immunogenic compositions (e.g., vaccines) for the treatment or prevention of B. anthracis infection and/or symptoms thereof. A protective immune response against B. anthracis may be characterized by vaccinated subjects responding with significant IgG anti-PA levels. A protective immune response exists when, for example, sera from vaccinated subjects have toxin neutralizing titers that correlate with their antibody levels as measured, for example, by ELISA. Western blot may also be utilized to check for protective immune response. The pharmaceutical compositions and/or vaccines of the invention are administered to a host in a manner that provides for production of selective anti-B. anthracis, e.g., anti- rPA, antibodies, preferably, with little or no detectable host autoantibody production. [00178] In particular embodiments, the vaccine compositions described herein are administered serially. First, an immunogenically effective dose of a vaccine of the invention is administered to a subject. The first dose is generally administered in an amount effective to elicit an immune response (e.g., activation T cells). Amounts for the initial immunization generally range from about 0.001 mg to about 1.0 mg per 70 kilogram patient, more commonly from about 0.001 mg to about 0.2 mg per 70 kilogram patient, usually about 0.005 mg to about 0.015 mg per 70 kilogram patient. Dosages from 0.001 up to about 10 mg per patient per day may be used, particularly when the antigen is not administered into the blood

1011497 vl 60 stream, such as into a body cavity or into a lumen of an organ. Substantially higher dosages (e.g. 10 to 100 mg or more) are possible in oral, nasal, or topical administration. [00179] After administration of the first vaccine dosage, a therapeutically effective second dose of the vaccine of the invention is administered to the subject after the subject has been immunologically primed by exposure to the first dose. The booster may be administered days, weeks or months after the initial immunization, depending upon the patient's response and condition.

[00180] The existence of an immune response to the first vaccine administration may be determined by known methods (e.g. by obtaining serum from the individual before and after the initial immunization, and demonstrating a change in the individual's immune status, for example an immunoprecipitation assay, or an ELISA, or a bactericidal assay, or a Western blot, or flow cytometric assay, or the like) and/or demonstrating that the magnitude of the immune response to the second injection is higher than that of control animals immunized for the first time with the composition of matter used for the second injection (e.g. immunological priming). Immunologic priming and/or the existence of an immune response to the first vaccine administration may also be assumed by waiting for a period of time after the first immunization that, based on previous experience, is a sufficient time for an immune response and/or priming to have taken place— e.g. 2, 4, 6, 10 or 14 weeks. Boosting dosages of the second immunization are typically from about 0.001 mg to about 1.0 mg of antigen, depending on the nature of the immunogen and route of immunization. [00181] In certain embodiments, a therapeutically effective dose of third vaccine composition is administered to the subject after the individual has been primed and/or mounted an immune response to the second vaccine composition. The third booster may be administered days, weeks or months after the second immunization, depending upon the subject's response and condition.

[00182] The present invention further contemplates the use of a fourth, fifth, sixth or greater booster immunization, using either the same or differing vaccine formulations. [00183] In certain embodiments, the antigen compositions are administered to a mammalian subject (e.g., human) that is immunologically naive with respect to B. anthracis. In particular embodiments, the mammal is a human adult, teenager or child. Immunizations with vaccine compositions of the invention may begin at any age, e.g., to human adult 25 years or younger, 20 years or younger or 18 years or younger; to a human child about five years or younger or two years old or younger.

1011497 vl 61 [00184] In preferred embodiments, administration to any mammal is initiated prior to the first sign of disease symptoms, or at the first sign of possible or actual exposure to infection or disease (e.g., due to exposure or infection by B. anthracis, or a product thereof, e.g. Anthrax toxin).

[00185] Pharmaceutical or vaccine compositions can be administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered depends on the subject to be treated, capacity of the subject's immune system to utilize the active ingredient, degree of modulation required the severity and type of disease, the route of administration, as well as age, body weight, response, and the past medical history of the patient. Suitable regimens can be selected by one skilled in the art by considering such factors and by following, for example, dosages reported in the literature and recommended in the Physician 's Desk Reference (56^th ed., 2002). [00186] The pharmaceutical or vaccine compositions of the invention can be administered in combination with various vaccines either currently being used or in development, whether intended for human or non-human subjects. Examples of vaccines for human subjects and directed to infectious diseases include the combined diphtheria and tetanus toxoids vaccine; pertussis whole cell vaccine; the inactivated influenza vaccine; the 23-valent pneumococcal vaccine; the live measles vaccine; the live mumps vaccine; live rubella vaccine; Bacille Calmette-Guerin (BCG) tuberculosis vaccine; hepatitis A vaccine; hepatitis B vaccine; hepatitis C vaccine; rabies vaccine (e.g., human diploid cell vaccine); inactivated polio vaccine; meningococcal polysaccharide vaccine; quadrivalent meningococcal vaccine; yellow fever live virus vaccine; typhoid killed whole cell vaccine; cholera vaccine; Japanese B encephalitis killed virus vaccine; adenovirus vaccine; cytomegalovirus vaccine; rotavirus vaccine; varicella vaccine; anthrax vaccine; small pox vaccine; and other commercially available and experimental vaccines. [00187] Polypeptides of the present invention that function as a prophylactic and or therapeutic agents against a disease, disorder, or infection can be administered to an animal, preferably a mammal, and most preferably a human, to treat, prevent or ameliorate one or more symptoms associated with the disease, disorder, or infection. The polypeptides of the invention can be administered in combination with one or more other prophylactic and/or therapeutic agents useful in the treatment, prevention or management of a disease, disorder, or infection, e.g. Anthrax. In certain embodiments, one or more polypeptides of the invention are administered to a mammal, preferably a human, concurrently with one or more other

1011497 vl 62 therapeutic agents useful for the treatment of a disease, e.g., Anthrax. The term "concurrently" is not limited to the administration of prophylactic or therapeutic agents at exactly the same time, but rather it is meant that polypeptides of the invention and the other agent are administered to a subject in a sequence and within a time interval such that the polypeptides of the invention can act together with the other agent to provide an increased benefit than if they were administered otherwise. For example, each prophylactic or therapeutic agent may be administered at the same time or sequentially in any order at different points in time; however, if not administered at the same time, they should be administered sufficiently close in time so as to provide the desired therapeutic or prophylactic effect. Each therapeutic agent can be administered separately, in any appropriate form and by any suitable route.

[00188] In various embodiments, the prophylactic or therapeutic agents are administered less than 1 hour apart, at about 1 hour apart, at about 1 hour to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 11 hours apart, at about 11 hours to about 12 hours apart, no more than 24 hours apart or no more than 48 hours apart. In preferred embodiments, two or more components are administered within the same patient visit.

5.5.2 CHARACTERIZATION AND DEMONSTRATION OF THERAPEUTIC UTILITY

[00189] Several aspects of the pharmaceutical compositions of the invention are preferably tested in vitro, e.g., in a cell culture system, and then in vivo, e.g., in an animal model organism, such as a rodent animal model system, for the desired therapeutic activity prior to use in humans. Assays which can be used to assess the likelihood of generating a therapeutic immune response to a particular vaccine composition are well known in the art. [00190] Combinations of prophylactic and/or therapeutic agents can be tested in suitable animal model systems prior to use in humans. Such animal model systems include, but are not limited to, rats, mice, chicken, cows, monkeys, pigs, dogs, rabbits, etc. Any animal system well-known in the art may be used. In a specific embodiment of the invention, combinations of prophylactic and/or therapeutic agents are tested in a mouse model system. Prophylactic and/or therapeutic agents can be administered repeatedly. Several aspects of the

1011497 vl 63 procedure may vary such as the temporal regime of administering the prophylactic and/or therapeutic agents, and whether such agents are administered separately or as an admixture.

5.5.3 TOXICITY STUDIES

[00191] The toxicity and/or efficacy of the compositions of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Therapies that exhibit large therapeutic indices are preferred. While therapies that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[00192] The data obtained from animal studies can be used in formulating a range of dosage of the therapies for use in subjects. The dosage of such agents lies preferably within a range of concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any therapy used in the method of the invention, the therapeutically effective dose can be estimated initially from animal assays. A dose may be formulated in animal models to achieve an administered concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in animal models. Such information can be used to more accurately determine useful doses in subjects (e.g., humans).

5.6 KITS

[00193] The invention also encompasses kits, having a unit dose of the composition of the invention present in a storage-stable form, dissolvable or dilutable to the desired dosage together with appropriate packaging and handling devices for convenience of mixing and to maintain sterility prior to instillation. Such a kit can include, for example, a first container containing active ingredient (e.g. , an rPA protein and/or an antigenic fragment thereof) in a stable storage form, either as a unit dose in a stock solution or a unit dose as lyophilized powder; and a second container containing diluent, or solvent and diluent, either separate or combined, the volume of which will provide a unit dose of therapeutic compound in a volume appropriate for administration; means for combining diluent with the stock solution or

1011497 vl 64 lyophilized powder; and optionally, means for administering the dose to the patient. Means for transferring diluent to the stock solution or lyophilized powder can include, but are not limited to, syringes or multi-chambered containers having a breachable internal seal separating active ingredient from diluent.

[00194] The invention provides a pharmaceutical pack or kit comprising one or more containers filled with the pharmaceutical composition of the invention or a portion thereof. Additionally, one or more other prophylactic or therapeutic agents useful for the treatment of a disease or disorder can also be included in the pharmaceutical pack or kit. The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

[00195] Generally, the ingredients of compositions of the invention are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. In certain embodiments, the compositions of the invention further comprise bulking agents such as sodium chloride, mannitol, polyvinylpyrrolidone and the like, to provide sufficient matter for ease of handling after lyophilization.

[00196] The present invention provides kits that can be used in the above methods. In one embodiment, a kit comprises one or more pharmaceutical compositions of the invention. In another embodiment, a kit further comprises one or more other prophylactic or therapeutic agents useful for the treatment of an infectious disease or a symptom associated therewith, in one or more containers.

6. EXAMPLES

6.1 Example 1: Codon Optimization of Recombinant Protective Antigen ("rPA")

[00197] The nucleotide sequence of native PA from B. anthracis was searched for codons of low E. coli frequency according to published sources (e.g., Table 1) and replaced

1011497 vl 65 with those of highest frequency, thereby optimizing the nucleotide sequence for recombinant expression in E. coli systems. [00198] Methods

[00199] Optimization: Analysis of the native nucleotide sequence encoding a PA

(SEQ ID NO:1) revealed a number of codons inconvenient for protein synthesis in E. coli (see, Table 1). The nucleotide sequence encoding this native PA (SEQ ID NO:1) was redesigned using the preferred codons of E. coli (see Table 2) for optimized expression in the recombinant system. Additionally, SEQ ID NO:1 was modified to alter the encoded amino acid sequence (i.e., that of native PA; SEQ ID NO:2) in order to improve protein stability. In particular, amino acid substitutions were engineered into the protein sequence to eliminate multiple protease cleavage sites: the modifications Q285E , E308D and the deletion of F313 and F314 of SEQ ID NO:2 eliminated sites for potential chymotrypsin digestion; the furin cleavage site spanning amino acid residues 164 to 167 of SEQ ID NO:2, i.e. RKKR (SEQ ID NO:3), was eliminated by modification to SNKE (SEQ ID NO:4). [00200] Results

[00201] The nucleotide sequence of wild-type PA was optimized for recombinant expression in E. coli as SEQ ID NO: 5, encoding recombinant PA ("rPA") having the amino acid sequence of SEQ ID NO:6.

Table 2: Codon Utilization in native PA, SEQ ID NO:1, and rPA, SEQ ID NO:5

Codon Amino Acid native PA rPA

TTT phe F 19 18

TTC phe F 5 4

TTA leu L 38 -

TTG leu L 7 -

CTT leu L 6 -

CTC leu L 2 -

CTA leu L 3 -

CTG leu L 1 57

ATT ile I 28 34

ATC ile I 10 20

ATA ile I 16 -

ATG met M 8 8

GTT val V 11 11

1011497 vl 66 GTC val V 2 2

GTA val V 18 17

GTG val V 10 10

TCT ser S 21 21

TCC ser S 3 3

TCA ser S 14 14

TCG ser S 7 7

CCT pro P 9 -

CCC pro P 3 -

CCA pro P 10 -

CCG pro P 6 28

ACT thr T 20 20

ACC thr T 5 5

ACA thr T 21 21

ACG thr T 10 10

GCT ala A 13 13

GCC ala A 2 2

GCA ala A 16 17

GCG ala A 7 7

TAT tyr Y 22 22

TAC tyr Y 6 6

TAA OCH Z 1 1

TAG AMB Z - -

CAT his H 9 9

CAC his H 1 1

CAA gin Q 27 26

CAG gin Q 4 4

AAT asn N 52 52

AAC asn N 16 17

AAA lys K 44 43

AAG lys K 13 13

GAT asp D 41 41

GAC asp D 6 7

GAA glu E 37 37

GAG glu E 12 13 TGT cys C - -

TGC cys C - -

TGA OPA Z - -

TGG trp W 7 7

CGT arg R 3 14

CGC arg R 3 14

CGA arg R - 12

CGG arg R 2 -

AGT ser R 4 -

AGC ser S 23 23

AGA arg R 16 -

AGG arg R 3 -

GGT giy G 3 -

GGC giy G 2 20

GGA giy G 19 -

GGG giy G 11 -

6.2 Example 2: Cloning Plasmid Generation and Expression of rPA

[00202] The engineered rPA sequence was cloned into commercially available vectors for amplification, sequence verification and expression. DH5a or BL21(DE3) competent cells were transfected with a pET9a or pET24a vector containing verified rPA CDNA (SEQ ID NO:5) and expressed protein analyzed by SDS PAGE electrophoresis and Western blot. The effect of Rifampicin in the culture media was also investigated as a means of increasing recombinant protein production. [00203] Methods

[00204] Cloning and Sequence Analysis: The engineered rPA nucleotide sequence

(SEQ ID NO:5) was commercially synthesized or generated via multiple AOE PCR and cloned into the E. coli pUC18 vector (ampicillin resistant; Integrated DNA Technologies Inc, Coralville, IA) for amplification and sequence analysis. Plasmid containing the optimized sequence (rPA/pUC18) was isolated from stock bacterial cultures using standard procedures, and the presence of the insert initially confirmed using molecular weight analysis on a 1.3% agarose gel. The concentration of stock cultures of DNA was determined by UV spectrophotometry. pUC18 vectors lack promoter regions and thus cannot express the target

1011497 vl 68 gene, preventing plasmid instability due to expression of proteins potentially toxic to the host cell.

[00205] rPA sequences isolated from the pUC18 vectors were recloned into pET9a or pET24a (Novagen) plasmids for expression. Briefly, the rPA/pUC 18 plasmid was digested by restrictases Ndel (NEB) and Xhol for 10 hours at 37 ⁰C and the resulting fragments purified by gel electrophoresis. After extraction from the gel (QIAquick Gel Extraction Kit, Qiagen), the rPA cDNA was ligated into pET9a or pET24a plasmid using T4 DNA ligase (NEB) according to the manufacturer's directions.

[00206] Correct insertion of the rPA cDNA into the expression plasmids was verified by comparing restriction fragments (FIG. IA-C) to the putative restriction map of the expression vectors (FIG. 2A-B). Plasmids identified as having proper inserts by restriction fragment analysis were sequenced using a Thermal cycler GeneAmp PCR System 9600 and a model 310 ABlprism sequinator or ABIprism 310 sequencing machine according to the manufacturer's directions. Sequencing primers were synthesized by a DNA/RNA synthesizer model 392 (Applied Biosystems) and purified using OPC mini-columns (Applied Biosystems). Plasmids containing sequence verified rPA were amplified, isolated and stored at -80 ⁰C according to standard practice.

[00207] Transformation : DH5α or BL21(DE3) competent cells were transfected using a heat shock method according to the procedure in Novagen pET System Manual, 6th Edition (hereby incorporated by reference in its entirety). rPA/pET24a or rPA/pET9a plasmid stocks were diluted to approximately 0.020 ug/ul. DH5a E. coli competent cells (Invitrogen/Gibco) were transformed by adding 1.5 ul of diluted plasmid stock to 100 ul of cell culture. The cultures were then incubated in ice for about 60 minutes and subsequently heat shocked for about 45 seconds at 42 ⁰C. The cultures were incubated on ice for 2 minutes. SOC media was then added (900 ul per 100 ul of transformed cells) and the cultures incubated at 37 ⁰C, 222-250 rpm for one hour. The resulting culture was then plated onto a LB/ APS (Difco) 1.5% agar coated plate containing 40 ug/ml kanamycin and incubated at 37 ⁰C overnight. [00208] BL21(DE3) E. coli competent cells (Novagen) were transformed by adding 1 uL of diluted plasmid stock to 100 μl of cell culture. The cultures were incubated on ice for about 5 minutes and then heat-shocked for about 30 seconds a 42 ⁰C. The cultures were allowed to recover by incubation on ice for about 2 minutes. APS LB media (800 ul) containing kanamycin (30 ug/ml) was added to each tube, followed by incubation at 37 ⁰C

1011497 vl 69 (250 rpm) for 30 minutes. The resulting culture was plated on an LB/ APS (Difco) 1.5% agar plates containing kanamycin (40 ug/ml) and incubated at 37 ⁰C overnight. [00209] Induction and Expression of rPA: Single colonies of DH5a or BL21(DE3) bacteria containing rPA/pET24a or rPA/pET9a prepared as described above, were collected, inoculated into APS LB media containing 40 μg/ml kanamycin and expanded to a volume of about 100 ml. The 100 ml cultures were incubated at 37 ⁰C, 230 rpm until an OD500 of 0.4 to 0.6 was reached. IPTG (Novagen) was added to a final concentration of ImM. For cultures containing rifampicin, 30 minutes after IPTG induction, rifampicin was added to a final concentration of 0.9 μg/ml. All cultures were incubated for another 2.5 to 4.0 hr after IPTG induction at 30 to 42 ⁰C. The OD of the cultures was monitored throughout. [00210] Cells were harvested by centrifugation (6,000 x g for 15 minutes at 4 ⁰C), the supernatant decanted, and the cell pellet immediately prepared for protein isolation and/or analysis, or for storage at -70 ⁰C.

[00211] SDS PAGE Gel Electrophoresis and Western Blot: For SDS PAGE analysis, the cell pellet was resuspended in 1 X SDS Sample buffer (Novex), heated at 70 ⁰C for 5 minutes and loaded on a precast PAA gel (NOVEX) followed by electrophoresis in Xcell II Mini-cell unit at 120 V.

[00212] Western blot analysis was performed according to Towbin et al., 1979, PNAS

USA 76:4350-4354 (hereby incorporated by reference in its entirety). Briefly, after SDS- PAGE electrophoresis as described, the gel was rinsed with transfer buffer and placed on PVDF membrane prepared by a 1 min. soak in methanol, a wash in distilled water, and a final equilibration in transfer buffer. Transb lotting was performed at 30 volts for 2 hr using a XCeIl II Blot Module (Invitrogen). Following the transb lot, the membrane was rinsed with TBS at pH 7.2 and blocked by incubation for 3 hours in PBS at pH 7.4, containing 1% (w/v) nonfat powdered milk (Carnation) and 0.02% sodium azide. The membrane was then washed three times with TBS /0.5% BSA/0.02% sodium azide and once with TBS. [00213] The membrane was incubated with primary antibody (mouse monoclonal antibody to B. anthracis PA) 1 :2000 in PBS containing 1% BSA / 0.02% sodium azide. Following a 3 X wash with TBS / 0.5% BSA / 0.02% sodium azide and a 1 X wash with TBS, the membrane was incubated with an AP-conjugated goat F(ab')₂ specific for murine IgG (ICN) in PBS containing 1% BSA / 0.02% sodium azide. The membrane was then washed 6 X with a total of 500 ml of TBS / 0.5% BSA / 0.02% sodium azide followed by a

1011497 vl 70 final rinse with TBS. The secondary antibody was detected using BCIP-NBT substrate solution (KPL) according to the manufacturer's directions. [00214] Results

[00215] Cultures of BL21 (DE3) E. coli comprising rPA/pET9a or rPA/pET24a were amplified and induced as described, supra. The OD500 of the cultures ranged from 0.3 to 0.6 prior to addition of IPTG and from 1.7 to 2.00 at protein harvest (3 to 4 hr post induction). SDS PAGE analysis revealed an induced protein at the expected molecular weight of ~82 kDa, which accounted for approximately 50% of total protein post-induction in all cultures (FIG. 3). The recombinant expression of the 82 kDa protein from rPA/pET24a was slightly increased relative to that from rPA/pET9a (FIG. 3A, lanes 7/8 versus, lanes 5/6; FIG. 3B, lanes 8/9 versus lanes 6/7), and also showed increased expression in response to the presence of 0.9 μg/ml rifampicin {e.g., FIG. 3B, lane 9). Increasing the concentration of Rifampicin in the media to 1.5 μg/ml failed to further enhance expression of full length rPA (FIG. 4). [00216] Expression dependency on temperature was investigated by inducing clones comprising rPA/pET24a as described above, but, after induction, maintaining the cultures at 22, 30 or 42 ⁰C. FIG. 5 demonstrates that maximal expression from the pET24a vector was achieved by incubation at 30 ⁰C for 4 hr.

[00217] Optimal expression conditions for rPA/pET24a were further investigated by modifying culture media. Cultures were grown and induced in either APS or LB media containing 40 μg/ml kanamycin, as described, at either 30 or 37 ⁰C. FIG. 6 demonstrates that optimal production of rPA occurred in the presence of APS media, while the incubation temperature of 30 or 37 ⁰C resulted in essentially equivalent expression. [00218] In each experiment, a smaller induced protein of ~52 kDa was also observed in post-induction samples {e.g., FIGS. 3 and 4). Western blot analysis revealed that both the protein at the expected molecular weight of rPA (~82 kDa) and the smaller protein (~52 kDa) reacted strongly with the murine anti-PA antibody (FIG. 7). Although the smaller protein could have been a degradation product of full length rPA, the results from sequencing analysis indicated that it was a fragment of the C-terminal portion of rPA beginning at methionine 266 of SEQ ID NO:6. The uniformity of the ~52 kDa fragment suggested that the smaller protein was produced by parallel initiation of translation from the rPA mRNA, both a the initial methionine and at methionine 266 of SEQ ID NO:6.

6.3 Example 3: Mutagenesis of rPA

1011497 vl 71 [00219] To further optimize recombinant expression of rPA, the nucleic acid sequence encoding rPA, i.e., SEQ ID NO:5, was modified such that the encoded protein contained the following modifications: M266L, to eliminate the competing translation of the ~52 kDa rPA fragment; and ElD or ElG, to improve protein processing. Modification of the penultimate N-terminal amino acid failed to further improve protein yield, while M266L improved protein yield slightly. [00220] Methods

[00221] Mutagenesis: Mutagenesis of the nucleotide sequence to effect the desired nucleic acid, or amino acid, modifications was achieved by Asymmetric Overlap Extension ("AOE") PCR (FIG. 8). The protocol used 6 primers, 4 "standard" primers (primers 2, 4, 5 and 6 in FIG. 8) and 2 primers bearing the desired mutations in the sequence of the gene of interest (primers 1 and 3 in FIG. 8). The "standard" outer primers are nested at the ends of the gene of interest, i.e., the forward primer 6 and the reverse primer 5 are nested within the forward and reverse primers 4 and 2, respectively. Forward primer 1 and reverse primer 3 are complements and bear the desired mutations on the sense and antisense stand, respectively. [00222] For Step I of AOE PCR, two separate PCR are performed: tube A containing the template strand and primers 1 and 2, and tube B containing the template strand and primers 3 and 4. In Step I, the mutation bearing primers, i.e., primers 1 and 3, are used in minimal concentrations, e.g., at a concentration of about 5 to 10 times lower than that of the other "standard" primer or about 10 ng per reaction

[00223] Step II of AOE PCR is the overlap extension step. Contents of both tubes from Step I are combined and a second round of PCR is performed to yield a full length sequence {i.e., bounded by primers 2 and 4) containing the mutation originally in primer 1 or 3.

[00224] Step III of AOE PCR comprises a further amplification of the full length sequence using primers nested within the outer primers of Steps I and II. Amplified sequences are digested and cloned into vectors for amplification and sequence analysis. [00225] AOE PCR was used to modify the optimized nucleic acid sequence, SEQ ID

NO:5, to generate the following mutant proteins: rPA comprising M266L (SEQ ID NO:7, encoded by SEQ ID NO:8); rPA comprising ElD and M266L (SEQ ID NO:9, encoded by SEQ ID NO: 10); rPA comprising ElG and M266L (SEQ ID NO: 11, encoded by SEQ ID NO: 12); and rPA comprising ElD (SEQ ID NO: 13, encoded by SEQ ID NO: 14).

1011497 vl 72 [00226] Nucleic acid sequences encoding the mutant rPA proteins were cloned into pET24a vectors as described in Section 6.2. The vector comprising SEQ ID NO:8 was named rPA/L266/pET24a; that comprising SEQ ID NO:10, rPA/5'D/L266/pET24a; that comprising SEQ ID NO: 12, rPA/5'G/L266/pET24a; and that comprising SEQ ID NO: 14, rPA/5'D/pET24a. BL21(DE3) E. coli were transformed with the vectors, the protein expressed, and SDS PAGE and Western blot analyses were performed as described in Section 6.2.

[00227] Results

[00228] AOE PCR allowed reproducible insertions of desired modifications into nucleotide sequences with high fidelity. The deficiency of mutation bearing primers in Step I of the process allowed relatively few amplifications to occur, and those that did occur were transcribed with high fidelity. Using AOE PCR, over 70% of collected mutants were found to have the expected sequence.

[00229] Replacement of methionine at 266 with leucine was designed to reduce additional translation of the ~52 kDa protein from M266 of SEQ ID NO:6, and replacement of El with either aspartic acid or glycine was designed to improve N-terminal processing. Replacement of the methionine at position 266 with leucine slightly improved protein expression relative to clones comprising rPA with 266M {e.g., comprising SEQ ID NO:5). In contrast, FIGS 9 and 10 show that clones expressing rPA variants ElD or ElG with or without M266L failed to improve protein yield and, in fact, slightly lowered protein expression relative to those clones comprising IE, e.g., SEQ ID NO:5 (rPA). The results suggested that the N-terminal processing provided by the glutamic acid residue at position 1 is optimal for rPA expression.

6.4 Example 4: Generation and Expression of Amino-terminal Truncated rPA

[00230] The results presented in Section 6.2, FIG.7, wherein the -52 kDa RPA fragment was found to react with anti-rPA antibody, lead to the investigation of expression of truncated forms of rPA. Truncated forms of rPA were developed based on positions of internal methionines, and were designed in order to increase yield of an antigenic rPA polypeptide isolated from E, coli while minimizing the formation of secondary expression products. The truncated rPAs were found to be expressed at levels equal to or greater than those of full length rPA and to exhibit high levels of reactivity with anti-rPA antibody. The

1011497 vl 73 use of truncated forms of rPA may therefore offer an alternative means to efficiently produce high yields of an antigenic rPA polypeptide for use in vaccine formulations. [00231] Methods

[00232] Site specific PCR was used to modify the optimized nucleic acid sequence to introduce an Ndel restriction at the methionine corresponding to positions 266 or 350 of PA {i.e., residues 266 and 348, respectively of SEQ ID NO:5). The nucleotide sequences encoding the 5' to position 265 deleted rPA (SEQ ID NO: 15, encoding SEQ ID NO: 16 (rP A/266 trunc)) or the 5 'to residue 347 of SEQ ID NO:5 deleted rPA (SEQ ID NO: 17, encoding SEQ ID NO: 18 (rP A/348 trunc)) were first inserted into cloning vector pSP72 and then into expression vectors pET24a and/or pET9a as described in Section 6.2. BL21(DE3) E. coli were transformed with the vectors, the protein expressed, and SDS PAGE and Western blot analyses were performed as described in Section 6.2. [00233] Results

[00234] SDS PAGE analysis showed that the expression levels of expression of rPA/266trunc (SEQ ID NO: 16) from either pET24a or pET9a in the presence of rifampicin was higher than that of the original clone, rPA (SEQ ID NO: 6) or rPA comprising M266L (SEQ ID NO: 7) in the presence of rifampicin (FIG 1 IA). In the absence of rifampicin, the expression of the truncated rPA (SEQ ID NO: 16) was lower than that of the original clone original clone, rPA (SEQ ID NO:6) or rPA comprising M266L (SEQ ID NO:7). [00235] Interestingly, Western blot analysis revealed a conflicting result (FIG. 1 IB).

Although the signal from the truncated rPA expressed in the presence of rifampicin was again greater that that of the full length clones, the Western blot signal from the truncated clones expressed in the absence of rifampicin was now greater than that of the full length clones. The results indicate that the truncated forms of rPA may be, despite the apparent SDS PAGE results, expressed in greater amounts than the full length clones or that the truncated forms exhibit increased affinity for the monoclonal anti-rPA antibody. Such increased affinity could lead to improved vaccine formulations that stimulate an increased or greater immune response relative to those formulations comprising full length proteins. [00236] Expression of rPA/348trunc (SEQ ID NO: 18), revealed similarly increased protein yields, approaching 80% of total protein at 3.5 hr post induction (FIG 12A). This yet smaller truncation also showed a high level of reactivity with the anti-rPA antibody (FIG. 12 B)

1011497 vl 74 6.5 Example 5: Isolation and Purification of rPA

[00237] Cell pellets from induced cultures of bacteria comprising the recombinant vector were prepared as described in Section 6.2. Because of the high level of production, the majority of the recombinant protein was found in inclusion bodies. A continuous, recycled method was developed for solubilization, isolation and refolding of the inclusion body protein. [00238] Methods

[00239] Frozen E. coli cells (3.39 g) prepared as described in Section 6.2 were polytron resuspensed in 30 ml E. T. buffer (2 mM EDTA/50 mM Tris, pH 8.5) and placed in a cell bomb for 30 minutes. The suspension was then centrifuged for 30 minutes at 12,000 X g and the supernatant discarded. The pellet of cellular material was washed 3 X with NET buffer by repeated resuspension and cetrifugation. On the last wash, 5 uL of benzonase was added after resuspension and the mixture incubated 30 minutes at 37 ⁰C. [00240] The washed pellet was polytron resuspended in 50 ml or 8 M Urea ethanolamine/50 mM Tris, pH 8.5, and the protein concentration determined by BCA assay. The sample was diluted to a working volume of 500 ml in 8 M Urea/50 mM Tris/0.0025 M Ca CL₂, pH 8.5, with a protein concentration of about 0.5 to 7.5 mg/ml ("Urea sample"). [00241] A recycled column system was developed for protein refolding. The system comprised Heparin or a Hightrap Q column (15 x 5.0 cm; for binding of low molecular weight proteins) in tandem with a Blue Sepharose column (15 x 5.0 cm; for binding of rPA), wherein the column void was emptied into a stirred reservoir containing 0.025% PEG in 50 mM Tris ("PEG buffer"). The reservoir material was continually recycled through the tandem columns using a MASTERFLEX® peristaltic pump (FIG 13). [00242] For the refolding procedure, the stirred reservoir was filled with 500 ml PEG buffer and recycling pump started. After column equilibration, the Urea sample was added to the reservoir (i.e., PEG buffer) at a rate of 5 ml/min until the entire sample had been loaded. Recycling was maintained throughout the Urea sample addition and, after completion of sample loading, continued for about 2 hours. The final urea concentration of the Urea sample/PEG buffer mixture was slightly less than 4 M.

[00243] For rPA elution, the Blue Sepharose column was first washed with 50 mM

Tris [HCI], pH 8.5. A gradient from 0 - 0.3 M NaCI/50 mM Tris, pH 8.5 was then applied to the washed column at a flow rate of 10 ml/min. rPA appeared to elute at approximately 0.15M NaCI.

1011497 vl 75 [00244] Alternatively, the void from the Blue Sepharose column may be applied to a

Q-Sepharose column XK 50 (Pharmacia) and allowed to recycle for about 2 hours at 180 ml/min. A salt gradient from 0 - 0.8 M NaCI may then be applied to isolate the bound proteins. Fractions coinciding with distinct large peaks are collected, diluted in 400 ml of 0.05 M Tris (HCI), pH 8.5 and reapplied to the Blue Sepharose column. Elution from the Blue Sepharose is performed as described.

[00245] Eluate and flow-through were analyzed for protein content by SDS PAGE analysis as described in Section 6.2. Proteins isolated by SDS PAGE were further subjected to high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) to assess purity and matrix-assisted laser desorption/ionization - time of flight mass spectrometry (MALDI-TOF) for confirmation of molecule identity. [00246] Results

[00247] rPA prepared as described in Section 6.2 was purified and refolded as described using a heparin column in tandem with a Blue Sepharose column. Six fractions of the flow through and three fractions of the eluate indicated to contain protein were compared using SDS-PAGE (FIG. 14). Figure 14 demonstrates that the Blue Sepharose column preferentially bound the higher-molecular weight species of rPA (FIG. 14. lanes 7-9), and that the tandem column effectively separated the ~82 kDa rPA from the ~52 kDa fragment. Analysis of the eluate from the initial column in the tandem system, e.g., a heparin or Hightrap Q column, revealed that a significant fraction of the higher molecular weight species of rPA was also bound by this column (FIG. 15, lanes 2-3), but that the initial column was unable to separate the high- and low-molecular weight species of rPA. FIG. 15 also confirms that the flow through was comprised apparently only of the lower-molecular weight (~52 kDa) species of rPA (FIG. 15, lane 5). Pooled protein fractions from three separate purification/refolding procedures were analyzed for purity by SDS-PAGE. Purity was calculated based on percentage of protein in the ~82 kDa band relative to total protein in the respective lane. The pooled samples had a purity of between 98.2 % (FIG. 16, lane 1) to 86.9 % (FIG. 16, lane 3).

[00248] The results from the SDS-PAGE analysis suggested that the recombinant product collected form the tandem column system of the invention was of high purity. The purity of the product obtainable by the methods of the invention was further confirmed by HPLC (FIGS. 17A-B). Figure 17 A is the chromatogram of the eluate from the Hightrap Q column demonstrating that the two species of rPA were readily identified and separated,

1011497 vl 76 without the Blue Sepharose column. Figure 17 B is the chromatogram of the eluate of the Blue Sepharose column. Again, FIG. 17 B shows that, in the tandem column system, the Blue Sepharose eluate comprised only the higher molecular weight species of rPA. For all peaks in FIGS. 17 A-B, the purity was 97% or greater.

[00249] The purity of the bacterial product was also assessed by GC-MS, in particular, to test for the presence of contaminant endotoxin. Figure 18 presents the GS-MS spectra of two samples of purified, refolded product, i.e., Blue Sepharose eluate, as compared to a commercial control rPA (Acambis) and commercially available E. coli endotoxin. The presence of endotoxin contaminant is classically determined by the presence of one or both of the unique sugars associated with the core polysaccharide of the LPS complex of gram- negative bacteria: 2-keto-3-deoxyoctonoic acid (KDO) and/or heptose. Figure 18 demonstrates that neither test sample contained detectable amounts of KDO or heptose, and thus did not comprise detectable endotoxin.

[00250] The eluent protein was also subjected to MALDI-TOF analysis (FIG. 19). The analysis revealed the presence of three ion peaks, consistent with a highly pure sample of recombinant product.

[00251] Together the SDS PAGE, HPLC, GS-MC, and MALDI-TOF analyses demonstrated that the purification and refolding methods of the invention were able to produce highly pure forms of rPA and that said methods were further able to separate rPA from fragment by-products of the expression reaction.

6.6 Example 6: Characterization of rPA Immunoreactivity and Immunogenicity

[00252] rPA purified and refolded according to the methods of section 6.5 was tested for reactivity in immunochemical assays to evaluate proper folding and/or conformation. The rPA produced by the methods of the invention was recognized by anti-rPA antibodies and exhibited similar affinity for anthrax receptor as PA isolated from B. anthracis. Further, the antibody response generated by rabbits immunized with the rPA according to the invention was compared to that generated by commercially available and/or control peptides in a cytotoxicity inhibition assay. The peptides of the invention were shown to generate an inhibitory antibody responses equivalent to that of control peptides. The results suggest that the rPA of the invention is immunogenically equivalent to wild-type PA. [00253] Results

1011497 vl 77 [00254] An ELISA capture assay performed according to standard methods in the art

(FIG. 20) demonstrated that the purified, refolded rPA produced by the methods of the invention was recognized both by a commercially available murine monoclonal anti-rPA antibody and serum from rabbits immunized with commercially available PA. The results suggest that the rPA of the invention is immunogenically similar to and, at least, shares common epitopes with wild-type PA.

[00255] The antigenic similarity of rPA with that of control rPA (Acambis) was confirmed in an ELISA based inhibition assay (FIG. 21). Briefly, various concentrations of full length rPA of the invention and/or of rPA fragments according to the invention were added to a defined concentration of serum isolated from a rabbit immunized with control PA. The depleted serum was then tested for remaining anti-PA activity and the results compared to a sample prior to deletion. The full length rPA of the invention exhibited an inhibition curve identical to that of control rPA (Acambis), while the inhibition curve determined for the rPA fragment was quite different. The results suggest that all of the epitopes recognized by the rabbit antisera on the control PA were also present in the refolded full length rPA of the invention, but that the rPA fragment lacked one or more epitopes of the control protein. [00256] The rPA of the invention was also found to dose-dependently bind RAW

264.7 ANTXRl human macrophages, suggesting that the protein was specifically interacting with, e.g., binding to, a cell surface protein (FIG. 22). Because these cells overexpress the anthrax receptor, ANTXRl, the results are consistent with and support a specific rPA-anthrax receptor interaction.

[00257] The immunogenic similarity of the rPA of the invention and control peptides was further confirmed by comparing the inhibitory activity of serum isolated from rabbits immunized separately with rPA of the invention or control peptides. As discussed, PA is not cytotoxic to cells alone. The cytotoxic activity of anthrax toxin requires the presence of lethal factor ("LF") and/or edema factor ("EF"), which cytotoxicity is mediated by the specific interaction of PA with ANTXRl cells (see, e.g., Salles et al., 2006, Cell. Microbiol. 8:1272-1281, hereby incorporated by reference in its entirety). Serum was isolated from rabbits immunized separately with rPA of the invention, a commercially available rPA (Acambis) and wild-type PA (isolated from B. anthracis). In each case, serum containing the polyclonal anti-PA antibodies was able to inhibit the PA/LF cytotoxic activity in cultures of RAW 264.7 ANTXRl human macrophages; moreover, the inhibition profile of the antibodies generated with the rPA of the invention was equivalent to that of the wild-type peptides (FIG.

1011497 vl 78 23). The results demonstrate that, despite the amino acid modifications relative to wild-type PA, the rPA of the invention is immunogenically equivalent to the wild-type protein. [00258] It is to be understood that the description, specific examples and data, while indicating exemplary embodiments, are given by way of illustration and are not intended to limit the present invention. Various changes and modifications within the present invention will become apparent to the skilled artisan from the discussion, disclosure and data contained herein, and thus are considered part of the invention.

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Claims

WHAT IS CLAIMED IS:

1. A modified B. anthracis Protective Antigen (PA) polypeptide or antigenic fragment thereof comprising one or more amino acid modifications relative to the wild type PA, wherein said one or more modifications is at one or more residues corresponding to position 1, 164, 165, 167, 266, 285, 308, 313, or 314 of SEQ ID NO:2.

2. The modified PA polypeptide or antigenic fragment thereof according to claim 1, wherein said wild type PA comprises SEQ ID NO:2.

3. The modified PA polypeptide or antigenic fragment thereof according to claim 1, wherein said one or more modification is a substitution at a position corresponding to position 1, 164, 165, 167, 266, 285, or 308 of SEQ ID NO:2; or a deletion a position corresponding to position 313 or 314 of SEQ ID NO:2.

4. The modified PA polypeptide or antigenic fragment thereof according to claim 3, wherein said one or more modification is a substitution, which substitution is a substitution at position 1 with aspartic acid or glycine, a substitution at position 164 with serine, a substitution at position 165 with asparagine, a substitution at position 167 with glutamic acid, a substitution at position at 266 with lysine, a substitution at position 285 with glutamic acid, or a substitution at position 308 with aspartic acid.

5. The modified PA polypeptide according to claim 1 , wherein said polypeptide comprises SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, or SEQ ID NO: 13.

6. The polypeptide according to claim 1, which polypeptide is an antigenic fragment of said modified PA, wherein said fragment comprises SEQ ID NO:16.

7. An isolated nucleic acid sequence encoding the modified PA polypeptide or antigenic fragment thereof of any one of claims 1-6.

8. The isolated nucleic acid sequence according to claim 7, wherein said sequence is optimized for recombinant expression.

9. The isolated nucleic acid sequence according to claim 8, wherein said sequence comprises SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:14, or SEQ ID NO:15.

10. An isolated nucleic acid sequence encoding an antigenic fragment of B. anthracis PA polypeptide, wherein said sequence has been optimized for recombinant expression and wherein said sequence comprises SEQ ID NO: 17.

1011497 vl 80

11. A method for producing a modified B. anthracis PA polypeptide or antigenic fragment thereof, said method comprising:

(i) transforming an E. coli host cell with the nucleotide sequence of claim 7;

(ii) culturing said host cell under suitable conditions for expression of said polypeptide or antigenic fragment thereof; and (iii) isolating said polypeptide or antigenic fragment thereof from said culture.

12. The method according to claim 11, wherein step (ii) further comprises addition of rifampicin at a concentration sufficient to increase expression of said polypeptide or antigenic fragment thereof.

13. The method according to claim 12, wherein said concentration is about 0.5 to about 1.5 μg/ml.

14. The method according to claim 11, wherein step (iii) further comprises:

(a) isolating and solubilizing inclusion bodies from said host cells; and

(b) refolding the solubilized polypeptide obtained from step (a).

15. A pharmaceutical composition comprising the polypeptide or antigenic fragment thereof of claim 1 and a pharmaceutically acceptable carrier.

16. The pharmaceutical composition according to claim 15 further comprising one or more adjuvants.

17. A method of inducing an immune response to B. anthracis comprising administering to a subject in need thereof a thereof a therapeutically effective amount of the pharmaceutical composition of claim 15.

18. A method of treating or preventing Anthrax in a subject in need thereof comprising administering to said subject a therapeutically effective amount of the pharmaceutical composition of claim 15.

19. The method according to claim 17 or 18, wherein said method further comprises administering one or more booster doses of said pharmaceutical composition.

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