WO2008140598A2 - Nouvelles microbactéries immunothérapeutiques, formulations pharmaceutiques et utilisation de celle-ci - Google Patents

Nouvelles microbactéries immunothérapeutiques, formulations pharmaceutiques et utilisation de celle-ci Download PDF

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WO2008140598A2
WO2008140598A2 PCT/US2007/086335 US2007086335W WO2008140598A2 WO 2008140598 A2 WO2008140598 A2 WO 2008140598A2 US 2007086335 W US2007086335 W US 2007086335W WO 2008140598 A2 WO2008140598 A2 WO 2008140598A2
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bcg
composition
mycobacterium
mycobacteria
dna
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WO2008140598A3 (fr
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David M. Hone
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Bacilligen, Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55544—Bacterial toxins

Definitions

  • BCG tuberculosis
  • BCG immunotherapy The apparent effectiveness of BCG in Europe is exemplified by the results of a randomized- controlled clinical trial initiated in 1950 in the United Kingdom where BCG was shown to afford 84% protection over the first 5 years and 77% protection over the 20-year passive surveillance period (2, 5). BCG immunotherapy
  • BCG has been utilized to prevent recurrences of superficial bladder cancer.
  • Local immunotherapy with BCG is clinically established and efficacious against recurrences after transurethral resection of superficial bladder cancer (82, 83, 95).
  • the immunologic mechanism of BCG immunotherapy is still not fully resolved (84, 85); however, when BCG is intravesically
  • 112275/F/l applied to treat bladder cancer, it induces local inflammation and an influx of various immune cells including CD4 + and CD8 + T cells, granulocytes and NK cells (86), which accumulate and form cellular infiltrates in the bladder wall.
  • BCG therapy also induces local secretion of cytokines, which can be detected in the urine of patients (87-92).
  • ThI type 1 T helper
  • Th2 type 2 T helper
  • the second strategy that is believed to enhance the immunopotency of BCG involves genetic manipulations to BCG that result in rBCG strains that promote greater levels of apoptosis (99, 100). Increased apoptosis has been accomplished by reducing expression of anti-apoptotic factors in BCG (100) or by promoting degradation of the endosome (99). However, the precise basis through which apoptosis improves BCG potency has not been delineated. The link between apoptosis and the presentation of antigens by dendritic cells (DCs), termed cross-priming, is discussed elsewhere (32-41).
  • DCs dendritic cells
  • BCG-induced apoptosis may provide a conduit through which BCG antigens are transferred to DCs leading to the induction of effector T cells (40, 41).
  • the prior art also documents the genetic manipulation of nonpathogenic mycobacteria, such as M. vaccae and M. smegmatis, and the use of genetically modified nonpathogenic mycobacteria as TB vaccines (49, 101), as a vaccine vector carrying HIV antigens (50) and cancer immunotherapeutics (51).
  • a genetically modified derivative of M. smegmatis that expressed tumor necrosis factor herein referred to as "TNF" was effective as a cancer immunotherapeutic in the mouse bladder cancer model (51).
  • the invention relates, in part, to novel Mycobacteria with enhanced biological activities, such as, immunogenicity.
  • a Mycobacteria of interest can serve as an improved adjuvant, resulting from modifications providing the bacteria with enhanced tissue attachment and adherence.
  • FIGURE 1 depicts a map of pB ACIL-101.
  • FIGURE 2 depicts a cloning scheme.
  • FIGURE 3 depicts a cloning scheme.
  • FIGURE 4 depicts a cloning scheme.
  • Mycobacterium is defined herein as an acid-fast bacterial genus that includes M. tuberculosis, M. bovis, M. smegmatis, M. microti, M. avium, M. vaccae and other species. This genus is divided into pathogenic organisms, such as M. tuberculosis and M. bovis, and nonpathogenic organisms, such as M. smegmatis and M. avium.
  • Mycobacteria is a vernacular term that refers to organisms in the Mycobacterium genus, wherein mycobacterial is the adjectival form, thereof.
  • Immunogen and "antigen” are used interchangeably herein as a molecule that elicits a specific immune response containing an antibody that binds to that molecule. That molecule can contain one or more sites to which a specific antibody binds. As known in the art, such sites are known as epitopes.
  • a vaccine is a form of immunogen or antigen.
  • An antigen can be polypeptide, polynucleotide, polysaccharide, a lipid and so on, as well as a combination thereof.
  • An immunogenic compound or product, and an antigenic compound or product is one which elicits a specific immune response, which can be a humoral, cellular or both.
  • a vaccine is an immunogen or antigen used to generate an immunoprotective response, that is, the antibody reduces the negative impact of the immunogen or antigen, or entity expressing same, in a host.
  • the dosage is derived, extrapolated and/or determined
  • the successful endpoint of the utility of a vaccine for the purpose of this invention is the resulting presence of an induced serum antibody, or antibody made by the host in any tissue or organ, that binds the antigen or immunogen of interest.
  • the induced antibody in some way, neutralizes and/or eliminates a pathogen, compound, molecule and the like carrying the cognate antigen or immunogen.
  • Immunoprotection for the purposes of the instant invention is the presence of such circulating antibody. That can be determined using any known immunoassay, such as an ELISA.
  • observing immunoprotection of at least thirty days is evidence of efficacy of a vaccine of interest.
  • the time of immunoprotection can be at least 45 days, at least 60 days, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 1 year, at least 2 years or longer.
  • the immunoprotection is observed in outbred populations, and to different forms, strains, variants, alleles and the like of a pathogen.
  • an "immunogenic factor” is one that supplements the immunogenicity of, antigenicity of or immune system reaction inducing ability of the host organism carrying or expressing same; or to an antigen associated or administered therewith.
  • a factor includes a TAP, a GGDEP containing peptide, a molecule with an adjuvant activity, RDl expression, a PAP and so on.
  • Another factor is one which reduces expression of, for example, a mannosylated mannan, such as mannosylated lipoarabinomannan.
  • 112275/F/l fragment or analog of a bacterium such as a cell wall/cell membrane preparation carrying an immunogenic and/or antigenic molecule, or of a foreign antigen is one which stimulates an immune response as does the native bacterium or portion thereof, or foreign antigen.
  • subcellular parts of a Mycobacterium of interest such as a cell wall or cell membrane preparation can be obtained practicing methods known in the art.
  • Recombinant expression of an adjuvant molecule or of a foreign antigen can be realized practicing methods known in the art.
  • a foreign antigen is a molecule that elicits an immune response in a host.
  • the molecule is not of the Mycobacterium host species used as an adjuvant or expressing the foreign antigen.
  • 112275/F/l of the mutation per se need not be predetermined. Similar substitutions can be attempted with other amino acids, depending on the desired property of the scanned residues.
  • a more systematic method for identifying amino acid residues to modify comprises identifying residues involved in immune system stimulation and those residues with little or no involvement with immune system stimulation.
  • An alanine scan of the involved residues is performed, with each ala mutant tested for enhancing immune system stimulation.
  • those residues with little or no involvement in immune system stimulation are selected to be modified. Modification can involve deletion of a residue or insertion of one or more residues adjacent to a residue of interest. However, normally the modification involves substitution of the residue by another amino acid.
  • a conservative substitution can be a first substitution. If such a substitution results in a change in immune system stimulation, then another conservative substitution can be made to determine if more substantial changes are obtained.
  • Even more substantial modification in the ability to stimulate the immune system can be accomplished by selecting an amino acid that differs more substantially in properties from that normally resident at a site.
  • a substitution can be made while maintaining: (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • the naturally occurring amino acids can be divided into groups based on common side chain properties:
  • hydrophobic methionine (M or met), alanine (A or ala), valine (V or val), leucine (L or leu) and isoleucine (I or ile);
  • cysteine C or cys
  • serine S or ser
  • threonine T or thr
  • asparagine N or asn
  • glutamine Q or gin
  • H or his histidine
  • K or lys lysine
  • R or arg arginine
  • Non-conservative substitutions can entail exchanging an amino acid with an amino acid from another group.
  • Conservative substitutions can entail exchange of one amino acid for another within a group.
  • Preferred amino acid substitutions can include those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter immune system stimulating activity and/or (4) confer or modify other physico-chemical or functional properties of such analogs.
  • Analogs can include various muteins of a sequence other than the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the naturally- occurring sequence (for example, in the portion of the polypeptide outside the functional domain(s)).
  • a conservative amino acid substitution generally should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence) unless of a change in the bulk or conformation of the R group or side chain (Proteins, Structures and Molecular Principles (Creighton, ed., W. H. Freeman and Company, New York (1984); Introduction to Protein Structure, Branden & Tooze, eds., Garland Publishing, New York, NY (1991)); and Thornton et al. Nature 354: 105 (1991)).
  • the adjuvant or immunogen mutant with improved biological properties will have an amino acid sequence having at least 75% amino acid sequence identity or similarity with the amino acid sequence of the parent molecule, at least 80%, at least 85%, at least 90% and often at least 95% identity.
  • Identity or similarity with respect to parent antibody sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical (i.e., same residue) or similar (i.e., amino acid residue from the same group based on common side-chain properties, supra) with the parent molecule residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.
  • Covalent modifications of the molecules of interest are included within the scope of the invention. Such may be made by chemical synthesis or by enzymatic or chemical cleavage of the molecule, if applicable. Other types of covalent modifications of the molecule can be introduced into the molecule by reacting targeted amino acid residues of the molecule with an organic derivatizing agent that is capable of reacting with selected side chains or with the N-terminal or C-terminal residue.
  • Cysteinyl residues can be reacted with ⁇ -haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to yield carboxylmethyl or carboxyamidomethyl derivatives. Cysteinyl residues also can be derivatized by reaction with bromotrifluoroacetone, ⁇ -bromo- ⁇ -(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercura-4-nitrophenol or chloro-7-nitrobenzo-2-oxa-l,3- diazole, for example.
  • ⁇ -haloacetates such as chloroacetic acid or chloroacetamide
  • Histidyl residues can be derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0.
  • p-bromophenacyl bromide also can be used, the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.
  • Lysinyl and ⁇ terminal residues can be reacted with succinic or other carboxylic acid anhydrides to reverse the charge of the residues.
  • suitable reagents for derivatizing ⁇ -amino-containing residues include imidoesters, such as, methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea and 2,4-pentanedione, and the amino acid can be transaminase-catalyzed with glyoxylate.
  • Arginyl residues can be modified by reaction with one or several conventional reagents, such as, phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione and ninhydrin. Derivatization of arginine residues often requires alkaline reaction conditions. Furthermore, the reagents may react with lysine as well as the arginine ⁇ -amino group.
  • tyrosyl residues can be made with aromatic diazonium compounds or tetranitromethane.
  • N-acetylimidizole and tetranitromethane can be used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.
  • Tyrosyl residues can be iodinated using 125 I or 131 I to prepare labeled proteins for use in a radioimmunoassay or with other radionuclides to serve as an imaging means.
  • aspartyl and glutamyl residues can be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
  • Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues, respectively, under neutral or basic conditions.
  • the deamidated form of those residues falls within the scope of this invention.
  • Another type of covalent modification involves chemically or enzymatically coupling glycosides to the molecules of interest.
  • the sugar(s) may be attached to: (a) arginine and histidine; (b) free carboxyl groups; (c) free sulfhydryl groups, such as those of cysteine; (d) free hydroxyl groups, such as those of serine, threonine or hydroxyproline; (e) aromatic residues such as those of phenylalanine, tyrosine or tryptophan; or (f) the amide group of glutamine.
  • Such methods are described in WO 87/05330 and in Aplin & Wriston, CRC Crit Rev Biochem, pp. 259-306 (1981).
  • Removal of any carbohydrate moieties present on the molecule of interest may be accomplished chemically or enzymatically.
  • Chemical deglycosylation for example, can require exposure of the molecule to the compound, trifluoromethanesulfonic acid, or an equivalent compound, resulting in cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the remainder of the molecule intact.
  • Chemical deglycosylation is described, for example, in Hakimuddin et al. Arch Biochem Biophys 259:52 (1987) and in Edge et al., Anal Biochem 118: 131 (1981).
  • Enzymatic cleavage of carbohydrate moieties on molecules can be achieved by any of a variety of endoglycosidases and exoglycosidases as described, for example, in Thotakura et al., Meth Enzymol 138:350(1987).
  • Another type of covalent modification of the molecule comprises linking the molecule to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol or polyoxylalkylenes, in the manner set forth in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
  • nonproteinaceous polymers e.g., polyethylene glycol, polypropylene glycol or polyoxylalkylenes
  • DNA encoding the adjuvant, immunogen, antigen and the like of the invention is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to the relevant genes, Innis et al. in PCR Protocols. A Guide to Methods and Applications, Academic (1990), and Sanger et al., Proc Natl Acad Sci 74:5463 (1977)). Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E.
  • the DNA also may be modified, for example, by substituting bases to optimize for codon usage in a particular host or by covalently joining to the coding sequence of a heterologous polypeptide.
  • Adherence to target tissue by means of the fibronectin attachment protein is a factor that can determine the success of cancer immunotherapy with Mycobacterium strains, such as BCG (52).
  • Mycobacterium strains such as BCG
  • Mycobacterium strains with modified tissue adherence were produced, and, surprisingly, although Mycobacterium strains such as BCG are capable of binding mammalian tissue, Mycobacterium strains with modified tissue adherence are substantially more effective cancer immunotherapeutics than are unmodified Mycobacterium strains.
  • the precise mechanism through which Mycobacterium strains with modified tissue adherence induce an enhanced cancer immunotherapeutic effect has not been resolved.
  • Mycobacterium strains with modified tissue adherence are retained in the target tissue in greater numbers, therefore invoking a stronger inflammatory response to the target locale in which the tumor resides.
  • An embodiment of the present invention provides mycobacterial strains engineered to express at least one recombinant DNA sequence (herein referred to as "RDS”) comprised of DNA encoding a tissue attachment factor (herein referred to as "TAF”), which may be either derived from a Mycobacterium species, e.g. M. tuberculosis and M. bovis, or a TAF derived from an animal, plant, or fungal, viral, bacterial, protozoan or metazoan organism.
  • RDS recombinant DNA sequence
  • TAF tissue attachment factor
  • the TAFs may be the full-length native protein, chimeric fusions between a TAF and an endogenous protein, heterologous protein or mimetic, or a fragment or fragments of a TAF or TAFs that originate from an animal, plant, or fungal, viral, bacterial, protozoan or metazoan organism.
  • the RDS encoding the TAF is introduced into the chromosome or as part of an extrachromosomal element (i.e. plasmids) using compositions and methods well known in the art (102-108).
  • TAFs include, but are not restricted to, the fibronectin attachment protein of M. tuberculosis strain CDC 1551 (GenBank accession no. AAK46179), the fibronectin attachment protein of M. leprae (GenBank accession no. AAB34676), the fibronectin attachment protein of M. bovis (GenBank accession no. AAB71842), and fibronectin attachment protein of M. avium (GenBank accession no. AAG22111), for example.
  • the fibronectin-binding activity of the aforementioned TAFs to yield a variant, derivative and the like can be enhanced by site-directed mutagenesis to either augment ligand-binding affinity, change the specificity of the ligand-binding activity to include additional targets on fibronectin or through a combination of both these approaches as discussed herein.
  • Methods to make such modification are well-known in the art (e.g. Rauceo et al., Eukaryot Cell, 5(10): 1664-1673, (2006);Roche et al., J. Biol. Chem., 279(37):38433- 38440, (2004); and Terao et al., J. Biol. Chem., 277(49):47428-47435, (2002)).
  • the recombinant mycobacteria of the present invention can express other TAFs derived from an animal, plant, or fungal, viral, bacterial, protozoan or metazoan organism.
  • heterologous TAFs include, but are not limited to, E-selectin (GenBank Accession no. AY367062), L-selectin (GenBank Accession no. AY367061), E. coli fimbrial adhesin subunit F 1845 antigen (GenBank Accession no. M27725), Escherichia coli adhesin (F17b-G) (GenBank Accession no. L14319), Klebsiella pneumoniae type 3 fimbrial adhesin (mrkD) (GenBank Accession no. M24536), B. parapertussis fimbrial adhesin FimD (GenBank Accession no.
  • Burkholderia pseudomallei aidA autotransporter diffuse adhesion protein GenBank Accession no. NC 009075
  • lectins including, but not limited to, hemagglutinins, phytoagglutinins (Sharron and Lis, Glycobiol., 14(11):53R-62R, (2004)), S-type lectins (i.e.
  • galectins a group consisting of prolactins, prolactins, and others.
  • endogenous glycan-binding proteins such as, but not limited to, C-type lectins (collectins or selectins), mannose receptor, I-type lectins (siglecs and others), P-type lectins (phosphomannosyl receptors), pentraxins, tachylectins, etc.
  • the Mycobacterium strains can be engineered to express at least one RDS comprised of DNA encoding an endogenous immunogen, such as, but not limited to, an autoimmune antigen or a tumor antigen.
  • an endogenous immunogen such as, but not limited to, an autoimmune antigen or a tumor antigen.
  • tumor specific antigens include prostate specific antigen (109); TAG-72 and CEA (110); MAGE-I; and tyrosinase (111).
  • tumor specific antigens include prostate specific antigen (109); TAG-72 and CEA (110); MAGE-I; and tyrosinase (111).
  • transplant antigens include the CD3 molecule on T cells (113). Treatment with an antibody to CD3 receptor has been shown to rapidly clear circulating T cells and to reverse cell-mediated transplant rejection (113).
  • An example of an autoimmune antigen includes IAS ⁇ chain (114). Vaccination of mice with an 18 amino acid peptide from IAS ⁇ chain has been demonstrated to provide protection and treatment in mice with experimental autoimmune encephalomyelitis (114).
  • This invention draws a novel distinction between the pro-inflammatory properties of BCG and the immunogenicity of BCG as a vaccine.
  • the pro-inflammatory property of BCG results in an influx of host phagocytes, natural killer cells and lymphocytes to the site of BCG inoculation. Initially, these cells comprise the innate host response, which develops with time into an adaptive host response. The latter is central to the success of BCG as a vaccine and this invention shows that the former is central to the success of BCG as an anticancer therapy.
  • This invention provides novel mycobacterial strains that undergo deregulated expression of immunostimulatory factors.
  • Expression deregulation of an immunostimulatory factor in mycobacteria can be achieved by overexpressing a biosynthetic pathway that produces an immunostimulatory factor.
  • mycobacteria can be engineered to overexpress a protein containing a GGDEF domain from a heterologous bacterium, such as AdrA from Salmonella enteriditis (GenBank Accession no. NP 806207), PIeD from unicellular cyanobacterium Synechocystis sp. strain PCC6803 (GenBank Accession no.
  • mycobacteria are engineered to overexpress a protein containing a GGDEF domain, such as, but not limited to, the GGDEF domain contained within amino acids 1-360 in Mb 1389c (GenBank Accession no. NP 855043; SEQ ID NO: 1), the GGDEF domain contained within amino acids 1-360 in Rv 1354c of M tuberculosis (GenBank Accession no. NP_215870), etc.
  • c-di- GMP cyclic-di(3'- ⁇ 5')-guanylic acid
  • GGDEF (SEQ ID NO: 1) domains are included as a guide; however, given the ubiquitous presence of the GGDEF/cyclase superfamily, which forms a large diversified cluster of orthologous proteins present in bacteria, archaea and eukaryotes (67, 68), those skilled in the art will recognize that other proteins or fragments containing at least one GGDEF (SEQ ID NO: 1) domain exist or can be constructed, or possessing di-GMP cyclase activity, thereof, which are also suitable for use in the present invention.
  • the present invention also provides novel mycobacterial strains that undergo deregulated expression of immunostimulatory factors, which is achieved by diminishing the expression of a biosynthetic pathway that produces an immunosuppressive factor.
  • mycobacteria can be engineered to underexpress or are rendered incapable of expressing mannosylated lipoarabinomannan (herein referred to as "ManLAM").
  • ManLAM mannosylated lipoarabinomannan
  • a preferred embodiment of the present invention provides a novel BCG carrying a defective embC gene (GenBank Accession no. CAB02472), which is incapable of expressing ManLAM.
  • this molecule promotes suboptimal immune responses and blocks apoptosis (59, 61); however, despite there being other immunoregulatory factors in mycobacteria, the absence of ManLAM surprisingly enhances the cancer immunotherapeutic potential of mycobacteria.
  • the present invention also provides novel Mycobacterium strains that carry an RDS encoding an adjuvant, which is useful in eliciting augmented host immune responses, thereby improving the cancer immunotherapeutic efficacy of said mycobacteria.
  • the specific adjuvant encoded by the RDS expressed by Mycobacterium is not critical to the present invention and may be, for example, the A subunit of cholera toxin (i.e. CtxA; GenBank accession no. X00171, AF175708, D30053, or D30052,) or parts, and/or mutant derivatives thereof (e.g. the Al domain of the A subunit of Ctx (i.e. CtxAl; GenBank accession no.
  • K02679 from classical Vibrio cholerae (e.g. V. cholerae strain 395, ATCC # 39541) or from El Tor V. cholerae (e.g. V. cholerae strain 2125, ATCC # 39050) strain.
  • El Tor V. cholerae e.g. V. cholerae strain 2125, ATCC # 39050
  • the A subunit of heat-labile toxin (referred to herein as "EItA"; GenBank accession no. M35581) from enterotoxigenic Escherichia coli (ATCC# 35401) may be used in place of CtxA.
  • Secretion of CtxA and EItA by recombinant mycobacteria of the present invention is accomplished by generating a genetic fusion between DNA encoding a molecule which facilitates secretion, such as a leader sequence, a signal peptide, a targeting signal and
  • the Ag85A leader peptide SEQ ID NO:2
  • LPA g 85A DNA encoding the mature CtxA protein
  • EItA protein amino acids 18-258.
  • the sequences encoding QxA 18-258 and EltA 18-2 58 can be optimized for expression in mycobacteria by using the preferred codon bias of this genus (69, 70).
  • LP Ag85A ::CtxA 18-258 and LP Ag85A ::EltA 18-258 are accomplished by functionally linking synthetic DNA encoding said genetic fusions to, for example, the antigen 85A promoter (herein referred to as "P Ag8 5A"; SEQ ID NO:3).
  • synthetic DNA encoding recombinant genes P Ag8SA -LP Ag8SA -QxA 18-258 and PA g8 5A::LPA g8 5A::EltA 18-258 can be purchased from commercial sources (i.e. Picoscript, Houston, Texas) and are introduced into mycobacterial strains as described here (see, for example, Example 1).
  • interference can play an important role in limiting the effectiveness of mycobacterial vaccine BCG. Therefore, interference may limit the usefulness of BCG as a cancer immunotherapeutic, since the target population is adults who had been exposed to environmental mycobacteria over their lifetimes. To date, there is no guidance in the art as to whether a modified BCG that overcomes such interference displays improved rates of success when treating superficial bladder cancer or has therapeutic applications in other cancers. But that can be overcome, for example, by having a Mycobacterium of interest express RDl.
  • BCG-RDl + strains in which the RDl region has been functionally restored, display improved vaccinal properties in animals that have been pre-exposed to environmental mycobacteria (71).
  • BCG-RDl + strains also invoke a stronger influx of CD4 + and CD8 + T cells to the site of inoculation (27).
  • BCG-RDl + strains are only marginally more
  • BCG-RDl + strains induce immunity to ESAT-6 (Rv3875; GenBank Accession no. AAL 16895) and CFPlO (Rv3874; GenBank Accession no. CAAl 7966), which are important diagnostic antigens, since these two antigens distinguish between TB and BCG exposures (30). For this reason, there has been little impetus to move BCG-RDl + strains forward as TB vaccines.
  • BCG-RDl + strains are more effective as cancer immunotherapeutics than are the parental strains.
  • Strategies and methods for introducing RDl into target mycobacteria are well known (23, 72, 73). This region can be introduced in its entirety or as smaller components that are sufficient to complement deletions in the RDl region of the target strain (23, 72, 73).
  • BCG strains are made RDl + by introducing SEQ ID NO:4 or functional portions thereof.
  • BCG strains are made RDl + by introducing SEQ ID NO:4, which carries a Q4L mutation in esxA (substituting Leu for GIn at position 4 of EsxA, Brodin et al., J Biol Chem 280(4):33953-33959, 2005) resulting in production of ESAT-6 Q4L that partially reduces the toxicity of the resulting recombinant strain BCG-RD I + -ES AT-6 Q4L .
  • the mutation in esxA can be introduced by site-directed mutagenesis procedures using the QuikChange ® Site-Directed Mutagenesis Kit (Stratagene, La Jolla CA; Cat. No. 200518) according to the manufacturer's directions.
  • nonpathogenic mycobacteria can be made RDl + by introducing SEQ ID NO:4.
  • nonpathogenic mycobacteria strains can be made RDl + by introducing SEQ ID NO:4, which carries a Q4L mutation in ESAT-6 that partially reduces the toxicity of the resulting BCG-RDl -ES AT-6 Q4L + strains.
  • the RDl sequences capable of complementing the RDl deletion in BCG can be obtained by PCR amplification of M. tuberculosis genomic DNA using SEQ ID NO: 5 as a
  • An RDl sequence capable of complementing the RDl deletion in M. microti can be obtained by PCR amplification of M. tuberculosis genomic DNA using SEQ ID NO: 7 as a forward primer and SEQ ID NO: 6 as a reverse primer.
  • Mutant RDl derivatives, such as RD1-ESAT-6 Q4L + can be made by site- directed mutagenesis of full-length or truncated RDl subclones using the QuikChange ® Site- Directed Mutagenesis Kit (Stratagene, La Jolla CA; Cat. No. 200518) according to the manufacturer's directions.
  • the RDS expressed by said mycobacteria strains for cancer immunotherapy can also encode any combination of TAFs, immunostimulatory factors, immunoregulatory factors and adjuvants described herein.
  • the RDS expressed by said mycobacteria strain for cancer immunotherapy can encode at least a TAF and can overexpress c-di-GMP.
  • the RDS expressed by said mycobacteria strain for cancer immunotherapy can encode a TAF and the RDl region.
  • the RDS expressed by said mycobacteria strain for cancer immunotherapy can encode a TAF, the RDl region and overexpress c-di-GMP.
  • this invention provides mycobacteria that encode a molecule that induces, stimulates, precipitates, causes etc. apoptosis, identified herein as a pro-apoptosis protein (herein referred to as "PAP"), and direct tumor antigens to cross-prime antigen presentation pathways and to elicit effector CD4 + and CD8 + T-cell responses.
  • PAP pro-apoptosis protein
  • BCG-induced apoptosis provides a mechanism for the delivery of tumor-specific antigens to DCs, thereby leading to the induction of said T cells. It has also been observed that macrophages undergoing apoptosis are more effective at killing BCG than are macrophages undergoing necrosis (42), suggesting that apoptosis may improve the safety of cancer immunotherapeutics comprised of live attenuated or nonpathogenic mycobacteria, while facilitating the distribution of tumor antigens to DCs. To date, however, there is no guidance in the literature as to how mycobacterial strains can be engineered to promote apoptosis.
  • the present invention provides mycobacteria capable of expressing a PAP, such as, but not limited to, the mature activated form of caspase-8 + (GenBank Accession no. NP033942; i.e., amino acids 99-480).
  • a preferred embodiment provides derivatives of mycobacteria capable of expressing a PAP from a microbial source, such as, but not limited to, the proteolytic domain of NS3 (spans amino acids 1-190; SEQ ID NO:8; herein designated "NS3 Pr ") encoded by base pairs 6469-7039 of West Nile virus isolate Mex03 (GenBank Accession no. AY660002), the hepatitis C virus core protein (GenBank Accession no.
  • AAXl 1912 the cytomegalovirus-encoded chemokine receptor (GenBank Accession no. AAQ24855), the human herpes virus chemokine receptor US28 (GenBank Accession no. AAN37944), the lyssavirus matrix protein (GenBank Accession no. AY540348), the IpaB protein of Shigella flexneri (GenBank Accession no. AAM89543), and the SipB protein of Salmonella enterica (GenBank Accession no. 2123407B).
  • sequences encoding the PAP can be generated synthetically by a commercial source (e.g. Picoscript, Houston Texas) and can be optimized for expression in mycobacteria by using the preferred codon bias of this genus (69, 70).
  • Secretion of the PAP by recombinant mycobacteria of the present invention can be accomplished by generating a genetic fusion between DNA encoding, for example, the Ag85A leader peptide (SEQ ID
  • LBP intercellular trafficking protein
  • VP22 human herpes virus tegument protein
  • Tat protein human immunodeficiency virus Tat protein
  • a spacer between the ITP and the PAP such as a flexible spacer (e.g. Serine-Glycine-Glycine-Glycine-Glycine-Serine; SEQ ID NO:9), an inflexible linker (e.g. Serine-Proline-Proline-Proline-Proline-Proline-Proline-Serine; SEQ ID NO: 10) or flexible linker with a furin degradation motif (e.g. Serine-Glycine-Glycine- Glycine-Glycine- Arginine-Threonine-Lysine-Arginine-Glycine-Glycine-Glycine-Glycine-Glycine-Gly cine- Serine; SEQ ID NO: 11), for example.
  • a flexible spacer e.g. Serine-Glycine-Glycine-Glycine-Glycine-Glycine-Glycine-Serine; SEQ ID NO:9
  • an inflexible linker e.g. Serine-Pro
  • the RDS expressed by said mycobacteria strain for cancer immunotherapy can encode a TAF, the RDl region, overexpress c-di-GMP and a pro- apoptosis factor. 4.
  • Useful attenuated and non-pathogenic Mycobacteria can encode a TAF, the RDl region, overexpress c-di-GMP and a pro- apoptosis factor.
  • the Mycobacterium strain that is genetically modified as set forth hereinabove is attenuated, as exemplified by BCG.
  • Attenuated Mycobacterium strains can be derived from M. tuberculosis strain H37Rv
  • Examples of attenuated Mycobacterium strains include, but are not restricted to, M. tuberculosis pantothenate auxotroph strain (119), lysine and pantothenate auxotrophic strain M. tuberculosis AlysA, ApanCD (120), leucine auxotrophic strain M. tuberculosis AleuD (118), BCG Danish strain (ATCC # 35733), leucine and pantothenate auxotrophic strain M. tuberculosis AleuD, ApanCD (121), M.
  • tuberculosis fadD26 mutant with impaired synthesis of phthiocerol dimycocerosates (122), Mycobacterium mce mutants with impaired synthesis of mammalian cell entry ⁇ mce) proteins (123), Mycobacterium sigC mutant strains (124), Mycobacterium leuD mutant strains (125), BCG Japanese strain (ATCC # 35737), BCG Copenhagen strain (ATCC #: 27290), BCG Pasteur strain (ATCC #: 35734), BCG Glaxo strain (ATCC #: 35741), and BCG Connaught strain (ATCC # 35745), for example.
  • non-pathogenic mycobacteria useful to the present invention include, but are not limited to, M. fortuitum (ATCC#: 15073), M. smegmatis (ATCC#: 12051 or 12549), M. intracellular e (ATCC#:35772 or 13209), M. kansasii (ATCC#:21982 or 35775), M avium (ATCC#: 19421 or 25291), and M microtti (ATCC#: 11152).
  • Methods for genetic manipulation of nonpathogenic mycobacterial strains are extensively documented (21, 50, 79-81, 118). 5. Strategies to produce rBCG strains that meet regulatory standards
  • biological pharmaceutics must meet purity, safety and potency standards defined by the pertinent regulatory agency.
  • the recombinant organisms should be maintained in culture media that is, for example, certified free of transmissible spongiform encephalopathies (herein referred to as "TSE").
  • TSE transmissible spongiform encephalopathies
  • Plasmids harboring an RDS of interest are introduced into mycobacteria by electroporation and selection of mycobacterial strains carrying such plasmids is achieved, for example, by antibiotic selection, such as hyg, encoding hygromycin resistance (GenBank accession no. AF025746; AF025747) and aph from Tn903, which confers kanamycin resistance (herein referred to as "Kan R "; GenBank accession no. U75323).
  • antibiotic selection such as hyg, encoding hygromycin resistance (GenBank accession no. AF025746; AF025747) and aph from Tn903, which confers kanamycin resistance (herein referred to as "Kan R "; GenBank accession no. U75323).
  • plasmids harboring the RDS carry a non-antibiotic selection marker, since it is not always ideal to use antibiotic resistance markers for selection and maintenance of plasmids in mycobacteria that are designed for use in humans and veterinary pharmaceutics.
  • the present invention provides a novel selection strategy in which, for example, a catabolic enzyme is utilized as a selection marker
  • catabolic enzyme includes, but is not restricted to, lacYZ encoding lactose uptake and ⁇ -galactosidase (Genbank accession no. J01636, J01637, K01483, or K01793).
  • Other selection markers that provide a metabolic advantage in defined media include, but are not restricted to, galTK (GenBank Accession no. X02306) for galactose utilization, sacPA (GenBank Accession no. J03006) for sucrose utilization, trePAR (GenBank Accession no.
  • the selection can involve the use of antisense mRNA to inhibit a toxic allele, such as the sacB allele (GenBank Accession no. NP 391325), which renders Mycobacterium strains sensitive to sucrose.
  • a suicide plasmid harboring the RDS of interest can be introduced into mycobacteria by electroporation and selection of mycobacterial strains carrying such plasmids can be achieved by antibiotic selection, such as hyg encoding hygromycin resistance (GenBank accession no. AF025746; or AF025747) and Kan R (GenBank accession no. U75323).
  • the suicide plasmid can carry a sequence that is identical to a genomic homolog. The sequence allows recombination between the suicide plasmid and the genome resulting in integration of the suicide plasmid into the mycobacterial genome. Methods for allelic exchange are described in detail elsewhere (21, 79, 81).
  • Selective medium containing the metabolite as a carbon source can be a modified Sauton's medium (herein defined as "MSM") containing 0.5 g KH 2 PO 4 (Sigma Cat. No. P9666), 0.5 g MgSO 4 7H2O (Sigma Cat. No. M5921-500G), 0.1 ml of 1% (w/v) ZnSO 4 (Sigma Cat. No. 35392-1L) solution, 5 ml of a 5% (v/v) Triton WR1339 (Sigma Cat. No. T8761) solution, 2.0 g citric acid (Sigma Cat. No. 251275), 0.05 g ferric ammonium citrate (Sigma Cat. No. F5879), 4.0 g asparagine (Sigma Cat. No. A4159), and 0.6 ml oleic acid
  • the recombinant mycobacteria of the present invention can be used to vaccinate against TB.
  • mycobacteria that overexpress a fibronectin attachment protein such as FapB encoded by the fapB gene (herein referred to as FapB, GenBank accession no. AAB71842) can be used as a TB vaccine using procedures described elsewhere (Horwitz and Harm, US Pat. No. 6,471,967; Bloom et al., US Pat. No. 5,504,005).
  • mycobacteria that overexpress FapB and express a factor that enhances immunostimulating properties of said mycobacteria are used a TB vaccines.
  • an rBCG strain that overexpresses FapB and expresses a fusion protein comprised of the Ag85A leader peptide, VP22 and NS3 Pr has improved vaccinal properties due to the enhanced ability of this strain to adhere to tissue and to form a depot, and to promote apoptosis through delivery of NS3 Pr to the cytoplasm of host antigen-presenting cells triggering activation of caspase-8 and apoptosis.
  • Recombinant mycobacteria are useful as vaccine vectors, wherein the recombinant strains are engineered to express at least one passenger or foreign antigen, for example, from a second pathogen.
  • the second pathogen can be a bacterium, virus, metazoa or a protozoa. Methods to produce vaccine vectors and antigen that are useful thereof are described in detail elsewhere (Bloom et al., US Pat. No. 5,504,005; and Sun et al., US Patent Publ. No. 20060121054).
  • Attenuated mycobacteria such as BCG
  • mycobacteria have long been used as adjuvants, compounds or microbes (either live or inactivated) that quantitatively and/or qualitatively improve an immune response to an immunogen that is co-administered with the adjuvant.
  • mycobacteria are the immunostimulating component in Freund's Complete Adjuvant (e.g. Difco, Detroit, MI, Cat. No. 231131). More recently, mycobacteria have been used to increase the immunogenicity of sub unit vaccines and nucleic acid vaccines (129).
  • the recombinant mycobacteria of the present invention can be used as adjuvants.
  • the particular recombinant mycobacterium strain of the present invention that is utilized as an adjuvant is not important and can be selected from, but not restricted to, recombinant mycobacteria that overexpress FapB.
  • the adjuvant is selected from recombinant mycobacteria that overexpress FapB and a factor that enhances the immunostimulatory properties of said mycobacteria (e.g. an rBCG strain that overexpresses FapB and expresses a fusion protein comprised of the Ag85A leader peptide, VP22 and NS3p r ).
  • kits e.g., comprising a bacterium or functional portion thereof of interest, homolog, derivative thereof and so on, for use, such as a vaccine or an adjuvant, and instructions for the use of same and so on.
  • the instructions may include directions for using the bacterium, derivative and so on.
  • the bacterium can be in liquid form or presented as a solid form, generally, desiccated or lyophilized.
  • the kit can contain suitable other reagents, such as a buffer, a reconstituting solution and other necessary ingredients for the intended use. A packaged combination of reagents in predetermined
  • 112275/F/l amounts with instructions for use thereof, such as for a therapeutic use is contemplated.
  • other additives may be included, such as, stabilizers, buffers and the like.
  • the relative amounts of the various reagents may be varied to provide for concentrates of a solution of a reagent, which provides user flexibility, economy of space, economy of reagents and so on.
  • the bacterium of the present invention may be used to treat a mammal.
  • the bacterium of interest is administered to a nonhuman mammal for the purpose of obtaining preclinical data, for example.
  • exemplary nonhuman mammals include nonhuman primates, dogs, cats, rodents and other mammals.
  • Such mammals may be established animal models for a disease to be treated with the formulation, or may be used to study toxicity of the bacterium of interest.
  • dose escalation studies may be performed in the mammal.
  • the specific method used to formulate the novel rdsRP vaccines and formulations described herein is not critical to the present invention and can be selected from a physiological buffer (Feigner et al., U.S. Pat. No. 5,589,466 (1996)); aluminum phosphate or aluminum hydroxyphosphate (e.g. Ulmer et al., Vaccine, 18: 18 (2000)), monophosphoryl- lipid A (also referred to as MPL or MPLA; Schneerson et al. J. Immunol., 147:2136-2140 (1991); e.g. Sasaki et al. Inf. Immunol., 65:3520-3528 (1997); Lodmell et al.
  • a physiological buffer Feigner et al., U.S. Pat. No. 5,589,466 (1996)
  • aluminum phosphate or aluminum hydroxyphosphate e.g. Ulmer et al., Vaccine, 18: 18 (2000)
  • the formulation herein also may contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary
  • 112275/F/l activities that do not adversely impact each other.
  • Such molecules suitably are present in combination in amounts that are effective for the purpose intended.
  • the bacterium can be used with a second component, such as a foreign antigen or a therapeutic moiety conjugated to or mixed with same, administered as a conjugate, separately in combination, mixed prior to use and so on as a therapeutic.
  • a second component such as a foreign antigen or a therapeutic moiety conjugated to or mixed with same
  • the recombinant mycobacteria of the present invention are produced as live, inactivated or cell wall preparations as described above and can be admixed with an antigen, inactivated bacteria or live bacteria using methods well known in the art (e.g. Levine et al., Eds., New Generation Vaccines. 2 nd edition. Marcel Dekker, Inc., New York, N.Y. (1997)).
  • the amount of antigen, inactive bacteria or live bacteria is not critical to the present invention but is typically an amount sufficient to induce the desired humoral and cell mediated immune response in the target host.
  • the Mycobacteria of interest also can be configured to express a foreign antigen, or the adjuvant of interest can be administered sequentially, before of after antigen administration.
  • the adjuvant of interest can be used in any known manner where an enhancement of the immune response is desired or needed.
  • an adjuvant of interest can be administered with a foreign antigen, a therapeutic agent and so on.
  • the therapeutic agent can be any drug, vaccine and the like used for an intended purpose.
  • the therapeutic agent can be a biological, a small molecule and so on.
  • small molecule as well as the “foreign antigen” and analogous terms include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogues, polynucleotides, polynucleotide analogues, carbohydrates, lipids, nucleotides, nucleotide analogues, organic or inorganic compounds (i.e., including heterorganic and/organometallic compounds) having a molecular weight less than about 10,000 grams per
  • organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, combinations thereof and other pharmaceutically acceptable forms of such compounds which stimulate an immune response or are immunogenic, or have a desired pharmacologic activity.
  • the bacterium of the invention may be administered alone or in combination with other types of cancer treatments, including conventional chemotherapeutic agents (paclitaxel, carboplatin, cisplatin, methotrexate and doxorubicin), anti-EGFR agents (gefitinib, erlotinib and cetuximab), anti-angiogenesis agents (bevacizumab and sunitinib), as well as immunomodulating agents, such as interferon- ⁇ and thalidomide.
  • the bacterium of the invention can be administered with a cancer antigen, such as, CEA or TAG-72, or other isolated cancer-specific cell surface molecule.
  • the bacterium or product thereof of the instant invention may be conjugated to various effector molecules such as heterologous polypeptides, drugs, radionucleotides or toxins, see, e.g., WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EPO 396,387.
  • a bacterium or product thereof may be conjugated to a therapeutic moiety such as a cytotoxin (e.g., a cytostatic or cytocidal agent), a therapeutic agent or a radioactive metal ion (e.g., ⁇ emitters such as, for example, 213 Bi).
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin and analogs or homologues thereof.
  • Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-
  • alkylating agents e.g., mechlorethamine, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU)
  • alkylating agents e.g., mechlorethamine, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU)
  • cyclothosphamide busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin
  • anthracyclines e.g., daunorubicin, daunomycin and doxorubicin
  • antibiotics e.g., dactinomycin, actinomycin, bleomycin, mithramycin and anthramycin (AMC)
  • anti-mitotic agents e.g., vincristine and vinblastine.
  • the present invention also is directed to Mycobacterium-based therapies which involve administering a bacterium or derivative of the invention to an animal, a mammal, or a human, for treating, for example, TB, HIV, an infectious disease, such as, malaria, a cancer, such as, bladder cancer, ocular squamous cell carcinoma, vulval papilloma and so on, or other disorder when used as an adjuvant.
  • a bacterium or derivative of the invention to an animal, a mammal, or a human, for treating, for example, TB, HIV, an infectious disease, such as, malaria, a cancer, such as, bladder cancer, ocular squamous cell carcinoma, vulval papilloma and so on, or other disorder when used as an adjuvant.
  • the animal or subject may be a mammal in need of a particular treatment, such as a mammal having been diagnosed with a particular disorder, e.g., TB or bladder cancer.
  • disease symptoms may be ameliorated or prevented in the treated mammal, particularly humans.
  • Therapeutic compounds of the invention alleviate at least one symptom associated with Mycobacterium or any other disease, disorder, or condition amenable for treatment with an adjuvant of interest.
  • the products of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
  • physiologically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S.
  • the products of interest can be administered to a mammal in any acceptable manner.
  • Methods of introduction include, but are not limited to, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intranasal, epidural, inhalation and oral routes, and if desired for immunosuppressive treatment, intralesional administration.
  • Parenteral infusions include intramuscular, intradermal, intravenous, intraarterial or intraperitoneal administration.
  • the products or 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 or local.
  • the product can be suitably administered by pulse infusion, particularly with declining doses of the products of interest.
  • the dosing is given by injection, preferably intravenous or subcutaneous injections, depending, in part, on whether the administration is brief or chronic.
  • Various other delivery systems are known and can be used to administer a product of the present invention, including, e.g., encapsulation in liposomes, microparticles or microcapsules (see Langer, Science 249: 1527 (1990); Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein et al., eds., (1989)).
  • the active ingredients may be entrapped in a microcapsule prepared, for example, by coascervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule,
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules
  • macroemulsions for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules
  • Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
  • the composition of interest may also be administered into the lungs of a patient in the form of a dry powder composition, see e.g., U.S. Pat. No. 6,514,496.
  • the therapeutic products or compositions of the invention may be administered locally to the area in need of treatment; that may be achieved by, for example, and not by way of limitation, local infusion, topical application, by injection, by means of a catheter, by means of a suppository or by means of an implant, said implant being of a porous, non-porous or gelatinous material, including hydrogels or membranes, such as sialastic membranes or fibers.
  • care is taken to use materials to which the protein does not absorb or adsorb.
  • the product can be delivered in a controlled release system.
  • a pump may be used (see Langer, Science 249: 1527 (1990); Sefton, CRC Crit Ref Biomed Eng 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); and Saudek et al., N Engl J Med 321 :574 (1989)).
  • polymeric materials can be used (see Medical Applications of Controlled Release, Langer et al., eds., CRC Press (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen et al., eds., Wiley (1984); Ranger et al., J Macromol Sci Rev Macromol Chem 23:61 (1983); see also Levy et al., Science 228: 190 (1985); During et al., Ann Neurol 25:351 (1989); and Howard et al., J Neurosurg 71 : 105 (1989)).
  • a controlled release system can be placed in proximity of the therapeutic target.
  • Therapeutic formulations of the product may be prepared for storage as lyophilized formulations or aqueous solutions by mixing the product having the desired degree of purity with optional pharmaceutically acceptable carriers, diluents, excipients or stabilizers typically employed in the art, i.e., buffering agents, stabilizing agents, preservatives, isotonifiers, non-ionic detergents, antioxidants and other miscellaneous additives, see Remington's Pharmaceutical Sciences, 16th ed., Osol, ed. (1980). Such additives are generally nontoxic to the recipients at the dosages and concentrations employed, hence, the excipients, diluents, carriers and so on are pharmaceutically acceptable.
  • An "isolated” or “purified” bacterium is substantially free of contaminating proteins from the medium from which the cell is obtained, or substantially free of chemical precursors or other chemicals in the medium used which contains components that are chemically synthesized.
  • the language “substantially free of subcellular material” includes preparations of a cell in which the cell is separated from subcellular components of the cells, such as dead cells, and portions of cells, such as cell membranes, ghosts and the like, from which same is isolated or recombinantly produced.
  • a bacterium that is substantially free of subcellular material includes preparations of the cell having less than about 30%, 20%, 25%, 20%, 10%, 5%, 2.5% or 1%, (by dry weight) of non-bacterial, subcellular contaminants.
  • the terms "stability" and “stable” in the context of a liquid formulation comprising a bacterium or product thereof refer to the resistance of the bacterium of product thereof in the formulation to thermal and chemical aggregation, degradation or fragmentation under given manufacture, preparation, transportation and storage conditions, such as, for one month, for two months, for three months, for four months, for five months, for six months or more.
  • the “stable” formulations of the invention retain biological activity equal to or more than 80%, 85%, 90%, 95%, 98%, 99% or 99.5%
  • the stability of said bacterium preparation can be assessed by degrees of aggregation, degradation or fragmentation by methods known to those skilled in the art, including, but not limited to, physical observation, such as, with a microscope, particle size and count determination and so on, compared to a reference.
  • carrier refers to a diluent, adjuvant, excipient or vehicle with which the therapeutic is administered.
  • physiological carriers can be 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. Water is a suitable carrier when the pharmaceutical composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions also can be employed as liquid carriers, particularly for injectable solutions.
  • 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.
  • compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations, depots and the like.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate etc. Examples of suitable carriers are described in "Remington's Pharmaceutical Sciences,” Martin.
  • Such compositions will contain an effective amount of the bacterium or functional portion of variant thereof, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration
  • the formulation will be constructed to suit the mode of administration.
  • Buffering agents help to maintain the pH in the range which approximates physiological conditions. Buffers are preferably present at a concentration ranging from about 2 mM to about 50 mM.
  • Suitable buffering agents for use with the instant invention include both organic and inorganic acids, and salts thereof, such as citrate buffers (e.g., monosodium citrate-di sodium citrate mixture, citric acid-tri sodium citrate mixture, citric acid-monosodium citrate mixture etc.), succinate buffers (e.g., succinic acid- monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid- disodium succinate mixture etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture, fumaric acid-d
  • Preservatives may be added to retard microbial growth, and may be added in amounts ranging from 0.2%-l% (w/v).
  • Suitable preservatives for use with the present invention include phenol, benzyl alcohol, m-cresol, octadecyldimethylbenzyl ammonium chloride, benzyaconium halides (e.g., chloride, bromide and iodide),
  • alkyl parabens such as, methyl or propyl paraben
  • catechol resorcinol
  • cyclohexanol and 3-pentanol.
  • Isotonicif ⁇ ers are present to ensure physiological isotonicity of liquid compositions of the instant invention and include polhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.
  • Polyhydric alcohols can be present in an amount of between about 0.1% to about 25%, by weight, preferably 1% to 5% taking into account the relative amounts of the other ingredients.
  • Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the therapeutic agent or helps to prevent denaturation or adherence to the container wall.
  • Typical stabilizers can be polyhydric sugar alcohols; amino acids, such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine etc.; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, arabitol, erythritol, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol and the like, including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur
  • Additional miscellaneous excipients include bulking agents, (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine or vitamin E) and cosolvents.
  • bulking agents e.g., starch
  • chelating agents e.g., EDTA
  • antioxidants e.g., ascorbic acid, methionine or vitamin E
  • cosolvents e.g., ascorbic acid, methionine or vitamin E
  • surfactant refers to organic substances having amphipathic structures, namely, are composed of groups of opposing solubility tendencies, typically an oil-soluble hydrocarbon chain and a water-soluble ionic group. Surfactants can be classified, depending on the charge of the surface-active moiety, into anionic, cationic and nonionic surfactants. Surfactants often are used as wetting, emulsifying, solubilizing and dispersing agents for various pharmaceutical compositions and preparations of biological materials.
  • Non-ionic surfactants or detergents may be added to help solubilize the therapeutic agent, as well as to protect the therapeutic protein against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stresses without causing denaturation of the protein.
  • Suitable non-ionic surfactants include polysorbates (20, 80 etc.), polyoxamers (184, 188 etc.), Pluronic ® polyols and polyoxyethylene sorbitan monoethers (TWEEN-20 ® , TWEEN-80 ® etc.).
  • Non-ionic surfactants may be present in a range of about 0.05 mg/ml to about 1.0 mg/ml, preferably about 0.07 mg/ml to about 0.2 mg/ml.
  • inorganic salt refers to any compound, containing no carbon, that results from replacement of part or all of the acid hydrogen or an acid by a metal or a group acting like a metal, and often is used as a tonicity adjusting compound in pharmaceutical compositions and preparations of biological materials.
  • the most common inorganic salts are NaCl, KCl, NaH 2 PO 4 etc.
  • the present invention provides liquid formulations of a bacterium or product thereof, having a pH ranging from about 5.0 to about 7.0, or about 5.5 to about 6.5, or about 5.8 to about 6.2, or about 6.0, or about 6.0 to about 7.5, or about 6.5 to about 7.0.
  • the instant invention encompasses formulations, such as, liquid formulations having stability at temperatures found in a commercial refrigerator and freezer found in the office of a physician or laboratory, such as from about -20° C to about 5° C, said stability assessed, for example, by microscopic analysis, for storage purposes, such as for about 60 days, for about 120 days, for about 180 days, for about a year, for about 2 years or more.
  • the liquid formulations of the present invention also exhibit stability, as assessed, for example, by particle analysis, at room temperatures, for at least a few hours, such as one hour, two hours or about three hours prior to use.
  • diluents include a phosphate buffered saline, buffer for buffering against gastric acid in the bladder, such as citrate buffer (pH 7.4) containing sucrose, bicarbonate buffer (pH 7.4) alone, or bicarbonate buffer (pH 7.4) containing ascorbic acid, lactose, or aspartame.
  • carriers include proteins, e.g., as found in skim milk, sugars, e.g., sucrose, or polyvinylpyrrolidone. Typically these carriers would be used at a concentration of about 0.1-90% (w/v) but preferably at a range of 1-10% (w/v).
  • formulations to be used for in vivo administration must be sterile.
  • the subcellular formulations of the present invention may be sterilized by filtration.
  • Sustained-release preparations may be prepared for use with the products of interest. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the bacterium, or functional portion or variant thereof, and/or foreign antigen, which matrices are in the form of
  • sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethylmethacrylate), poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (such as injectable microspheres composed of lactic acid-glycolic acid copolymer) and poly-D-(-)-3-hydroxybutyric acid.
  • polyesters for example, poly(2-hydroxyethylmethacrylate), poly(vinylalcohol)
  • polylactides U.S. Pat. No. 3,773,919
  • copolymers of L-glutamic acid and ethyl-L-glutamate non-degradable ethylene-vinyl acetate
  • the bacterium or product thereof composition will be formulated, dosed and administered in a manner consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
  • the "therapeutically effective amount" of the bacterium or product thereof to be administered will be governed by such considerations, and can be the minimum amount necessary to prevent, ameliorate or treat a Mycobacterium based disease, condition or disorder.
  • the amount of the recombinant mycobacteria of the present invention to be administered as live bacteria, inactivated bacteria or as cell wall preparations will vary depending on the species of the subject, as well as the disease or condition that is being treated. Generally, the dosage employed will be about 10 3 to 10 11 viable organisms, preferably about 10 5 to 10 9 viable organisms. Alternatively, when infecting individual cells, the dosage of viable organisms to administered will be at a multiplicity of infection ranging from about 0.1 to 10 6 , preferably about 10 2 to 10 4 . The number of inactivated bacteria may
  • 112275/F/l vary and is adjusted based on a comparison of efficacy with live bacteria.
  • the amount of a subcellular product or component of interest may vary and is adjusted based on a comparison of efficacy with live bacteria.
  • the term "effective amount” refers to the amount of a therapy (e.g., a prophylactic or therapeutic agent), which is sufficient to reduce the severity and/or duration of a Mycobacterium-caused disease, ameliorate one or more symptoms thereof, prevent the advancement of a Mycobacterium-based disease or cause regression of a Mycobacterium-based disease, or which is sufficient to result in the prevention of the development, recurrence, onset, or progression of a Mycobacterium-based disease or one or more symptoms thereof, or enhance or improve the prophylactic and/or therapeutic effect(s) of another therapy (e.g., another therapeutic agent) useful for treating a disease where the bacterium or product thereof is used as an adjuvant.
  • a therapy e.g., a prophylactic or therapeutic agent
  • a treatment of interest can increase survivability of the host, based on baseline or a normal level, by at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, 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 100%.
  • an effective amount of a therapeutic or a prophylactic agent reduces the symptoms of a Mycobacterium-based disease, such as a symptom of TB by at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, 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 100%. Also used herein as an equivalent is the term, "therapeutically effective amount.”
  • the composition may also include a solubilizing agent and a local anesthetic such as lidocaine or other "caine” anesthetic to ease pain at the site of the injection.
  • a solubilizing agent such as lidocaine or other "caine” anesthetic to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a sealed container, such as an ampule or sachet indicating the quantity of active agent.
  • a dry lyophilized powder or water-free concentrate in a sealed container, such as an ampule or sachet indicating the quantity of active agent.
  • the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampule of sterile water for injection or saline can be provided, for example, in a kit, so that the ingredients may be mixed prior to administration.
  • the article of manufacture comprises a container and a label.
  • Suitable containers include, for example, bottles, vials, syringes and test tubes.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is effective for preventing or treating a mycobacterium-based condition or disease and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the label on or associated with the container indicates that the composition is used for treating the condition of choice.
  • the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes and package inserts with instructions for use.
  • a pharmaceutically acceptable buffer such as phosphate-buffered saline, Ringer's solution and dextrose solution.
  • buffers such as phosphate-buffered saline, Ringer's solution and dextrose solution.
  • TSE-free liquid media for culturing mycobacterial strains include Middlebrook 7H9 (Difco) or Sauton's medium (Difco) or MSM (see above), which are normally maintained at 37 0 C but in certain circumstances these organisms can also be cultured at between 3O 0 C and 4O 0 C.
  • the cultures are incubated with or without agitation at 150 oscillations per minute.
  • the growth rate of mycobacteria can be enhanced by the addition of oleic acid (0.06% v/v; Research Diagnostics Cat. No. 01257) and a surfactant (such as Tyloxapol (0.05% v/v; Research Diagnostics Cat. No.70400)).
  • TSE-free solid media for culturing mycobacterial strains includes
  • 112275/F/l maintained in gas-permeable bags to prevent desiccation of the plates and normally incubated at 37 0 C but in certain circumstances these organisms can also be incubated at between 3O 0 C and 4O 0 C.
  • the purity of mycobacterial cultures is evaluated by spreading small aliquots, typically 0.1 ml, of the culture in 10-fold serial dilutions from 10° - 10 "8 in phosphate buffered saline (herein referred to "PBS") on solid media, such as Middlebrook 7H10 at 37 0 C.
  • PBS phosphate buffered saline
  • the purity of the culture can be further assessed as described in US FDA document 21 CFR 610.12 using commercially available liquid media, such as Thioglycolate medium (Sciencelab, Cat #1891) and Soybean-Casein medium (Becton-Dickinson, Cat #: 211768).
  • All reagents used in the production of recombinant mycobacterial strains for veterinary and human applications preferably should be certified TSE-free by the manufacturer.
  • TSE-free restriction endonucleases New England Biolabs, Beverly, MA
  • T4 DNA ligase New England Biolabs
  • Taq polymerase Invitrogen, Carlsbad, CA
  • Plasmid DNA is prepared using small-scale (Qiagen Miniprep R kit, Santa Clarita, CA) or large-scale (Qiagen Maxiprep R kit, Santa Clarita, CA) plasmids DNA purification kits according to the manufacturer's protocols (Qiagen, Santa Clarita, CA).
  • Nuclease-free, molecular biology grade milli-Q water, Tris-HCl (pH 7.5), EDTA pH 8.0, IM MgCl 2 , 100% (v/v) ethanol, ultra-pure agarose, and agarose gel electrophoresis buffer are purchased from Invitrogen. Restriction endonuclease digestions, PCRs, DNA ligation reactions and agarose gel electrophoresis are conducted according to well-known procedures (127, 128).
  • Nucleotide sequencing to verify the DNA sequence of each recombinant plasmid described in the following sections is accomplished by medium- high throughput automated DNA sequencing using an ABI 8-capillary 3730 DNA Analyzer (Applied Biosystems Inc., Foster City, CA) according to the manufacturer's directions.
  • PCR primers for the amplifications are designed using Clone
  • PCR primers are purchased from commercial sources such as Sigma (St. Louis, MO) or are synthesized using an ABI model 3900 DNA synthesizer (Applied Biosystems Inc.) according to the manufacturer's directions. PCR primers are used at a concentration of 100-300 ⁇ M and annealing temperatures for the PCR reactions are determined using Clone Manager Professional Suite version 8.0 (Scientific and Educational Software Inc.).
  • thermocycler device such as the Stratagene Robocycler, model 400880 (Stratagene), and primer annealing, elongation and denaturation times in the PCRs are set according to standard procedures (128).
  • DNA fragments produced by the restriction endonuclease digestions and PCRs are analyzed by agarose gel electrophoresis using standard procedures (127, 128).
  • a positive clone is defined as one that displays the appropriate restriction endonuclease and/or PCR pattern. Plasmids identified through this procedure can be further evaluated by medium-high throughput automated DNA sequencing using an ABI 8-capillary 3730 DNA Analyzer (Applied Biosystems Inc., Foster City, CA) according to the manufacturer's directions.
  • Bacterial strains that serve as hosts and amplify recombinant plasmids such as Escherichia coli strains DH5 ⁇ and Stable2 R , are purchased from Invitrogen. Recombinant plasmids are introduced into E. coli strains by electroporation using an high- voltage electropulse device, such as the Gene Pulser (BioRad Laboratories, Hercules, CA), set at 100-200 ⁇ , 15-25 ⁇ F and 1.0-2.5 kV, as described (126). Optimal electroporation
  • 112275/F/l conditions are identified on a trial-by-error basis by determining settings that result in maximum transformation rates per meg DNA per bacterium.
  • Solid media for the growth of E. coli strains can be TSE-free tryptic soy agar (Difco, Detroit, MI) and liquid media for growth of the same can be TSE-free tryptic soy broth (Difco, Detroit, MI), which are made according to the manufacturer's directions. Unless stated otherwise, all E. coli are grown at 37 0 C with gentle agitation. When appropriate, the media are supplemented with antibiotics (Sigma, St. Louis, MO). Bacterial strains are stored at -8O 0 C suspended in tryptic soy broth (Difco) containing 30% (v/v) glycerol (v/v; Sigma, St. Louis, MO) at ca. 10 9 colony-forming units (herein referred to as "cfu”) per ml.
  • cfu colony-forming units
  • Bacilligen-1010 (rBCG-FAP c ) and Bacilligen-1011 (rM. smegmati S-FAP + ), which are recombinant mycobacterial strains that constitutively express fibronectin attachment protein (GenBank Accession no. AAB71842).
  • FapB a synthetic gene is generated that encodes the Ag85A promoter (herein referred to as "P Ag 85A”; SEQ ID NO:3) functionally linked to fapB (SEQ ID NO: 12).
  • the synthetic gene, ⁇ P A & 5 A -fa ' pB, flanked by Pad sites is purchased from Picoscript (Houston, Texas) and is ligated (Example 1) into the unique Pad site in plasmid pBACIL-101 (SEQ ID NO: 13).
  • This latter plasmid is comprised of the lactose transporter LacY and ⁇ -galactosidase gene (herein referred to as "lacYZ”), which is under the control of the antigen-85B promoter (herein referred to as "P Ag 85B”) and codon optimized for expression in mycobacteria, the Kan R (GenBank accession no. U75323) and is flanked by Notl digestions sites, the E. coli plasmid origin of replication OriE (GenBank accession no. AY947541), the M. bovis genomic origin of replication (herein
  • the ligated plasmid is introduced into E coli strain Stable2 as described in Example 1.
  • An isolate harboring the recombinant plasmid designated pB ACIL- 102 i.e. pBACIL-101 ::P Ag85A -fapB
  • pB ACIL- 102 is amplified by culturing the resulting transformants in 100 ml liquid media at 37 0 C with agitation (Example 1) and the plasmid DNA is purified as described (Example 1).
  • the purified DNA is digested with Notl to remove Kan R and the large fragment encoding the Ag85A promoter functionally linked to P Ag ss A -fapB, lacYZ, OriE and OriC is purified following fractionation by agarose gel electrophoresis (Example 1).
  • the purified DNA fragment is ligated to form a closed circular DNA as described and the resulting DNA is introduced into BCG strain Danish 1331 by electroporation as described (Example 1).
  • Colonies harboring the recombinant plasmid are grown on Stauton's synthetic medium (Example 1) containing lactose in place of glycerol at 37 0 C.
  • Resulting colonies are screened by PCR (Example 1), to demonstrate the presence of the plasmid, DNA sequencing (see recombinant DNA methods above) to demonstrate that the plasmid carries the correct sequence and 2D gel electrophoresis on a fee-for- service basis at ACE BioSciences (Odense, Denmark) to demonstrate that FabB is constitutively expressed in BCG.
  • Colonies that grow on the lactose-selection medium are screened by PCR (Example 1), to demonstrate the presence of the plasmid, automated DNA sequencing (Example 1) to demonstrate that the plasmid carries the correct sequence and 2D gel electrophoresis on a fee-for-service basis at ACE BioSciences (Odense, Denmark) to demonstrate that FapB is expressed in M. smegmatis.
  • Bacilligen-1012 (rBCG-Mbl389c 1-3 6o C ) and Bacilligen-1014 (rM. smegmatis-Mbl3&9ci- 3 6o C ), which are engineered to constitutively express GGDEF domain-containing amino acids 1-360 of Mbl389c (GenBank Accession no. NP855043).
  • This truncated derivative is encoded by the complement of GenBank accession no. NC002945.3, from 1518040 to 1519911 and is shown in SEQ ID NO: 14.
  • the sequence encoding amino acids 1-360 of Mbl389c is functionally linked to P Ag 85A (SEQ ID NO:3).
  • Picoscript (Houston, Texas) encoding P Ag 85A-Mbl339c 1-3 6o and is ligated (Example 1) into the unique Pad site in plasmid pBACIL-101 (SEQ ID NO: 13).
  • the ligated plasmid is introduced into E. coli strain Stable2 as described in Example 1.
  • An isolate harboring the recombinant plasmid designated pBACIL-103 (i.e.
  • pBACIL-lOl ::PAg85A::Mbl339c 1-3 6o is amplified by culturing the resulting transformants in 100 ml liquid media at 37 0 C with agitation (Example 1) and the plasmid DNA is purified as described (Example 1).
  • the purified DNA is digested with Notl to remove Kan R and the large fragment encoding PA g 85A::Mbl339c 1-3 6o, PAgssB-focFZ, OriE and OriC is purified following fractionation in agarose (Example 1).
  • the purified DNA fragment is ligated to form a closed circular DNA as described and the resulting DNA is introduced into BCG strain Danish 1331 by electroporation as described (Example 1).
  • Colonies harboring the recombinant plasmid are grown on Stauton's synthetic medium (Example 2) containing lactose in place of glycerol at 37 0 C. Colonies that grow on this novel selection medium are screened by PCR (Example 1), to demonstrate the presence of the plasmid, DNA sequencing (see recombinant DNA methods above) to demonstrate that the plasmid carries the correct sequence and 2D gel
  • Colonies that grow on the lactose-selection medium are screened by PCR (Example 1), to demonstrate the presence of the plasmid, automated DNA sequencing (Example 1) to demonstrate that the plasmid carries the correct sequence and 2D gel electrophoresis on a fee-for-service basis at ACE BioSciences (Odense, Denmark) to demonstrate that Mbl339c 1-3 6o is over-expressed inM smegmatis.
  • Bacilligen-1015 (rBCG-CtxA + ) and Bacilligen-1016 (rM. smegmatis-CtxA + ⁇ which are engineered to constitutively express CtxA, using a similar approach as the two proceeding examples.
  • PAg8 5 A::LPAg8 5 A: 2 58 is purchased from Picoscript, Houston, Texas, and is ligated into plasmid pB ACIL-101 (SEQ ID NO: 13), as described (Example 1).
  • the resulting recombinant plasmid designated pBACIL-104 i.e. pBAdL-101 ::PA g85 A::LPAg8 5 A::CtxA
  • E. coli strain Stable2 as described in Example 1, and an isolate harboring the recombinant plasmid is
  • Colonies harboring the recombinant plasmid are grown on Stauton's synthetic medium (Example 2) containing lactose in place of glycerol at 37 0 C. Colonies that grow on this novel selection medium are screened by PCR (Example 1), to demonstrate the presence of the plasmid, DNA sequencing to demonstrate that the plasmid carries the correct sequence (Example 1) and 2D gel electrophoresis on a fee-for- service basis at ACE BioSciences (Odense, Denmark) to demonstrate that CtxA is expressed in BCG.
  • Colonies that grow on the lactose-selection medium are screened by PCR (Example 1), to demonstrate the presence of the plasmid, automated DNA sequencing (Example 1) to demonstrate that the plasmid carries the correct sequence and 2D gel electrophoresis on a fee-for-service basis at ACE BioSciences (Odense, Denmark) to demonstrate that CtxA is expressed in M. smegmatis.
  • Bacilligen-1017 (rBCG-RDl + ) and Bacilligen-1018 (rM. smegmatis-RD ⁇ + ⁇ which are
  • pBACIL-105 i.e. pBACIL-101 ::SEQ ID NO:4
  • pBACIL-105 An isolate harboring the recombinant plasmid designated pBACIL-105 (i.e. pBACIL-101 ::SEQ ID NO:4), is amplified by culturing the resulting transformants in 100 ml liquid media at 37 0 C with agitation (Example 1) and the plasmid DNA is purified as described (Example 1).
  • the purified pBACIL-105 DNA is digested with Notl to remove Kan R and the large fragment encoding SEQ ID NO:4, PA g 85B: :lacYZ, OriE and OriC is purified following fractionation in agarose (Example 1).
  • the purified DNA fragment is ligated to form a closed circular DNA as described and the resulting DNA is introduced into BCG strain Danish 1331 by electroporation as described (Example 1).
  • Colonies harboring the recombinant plasmid are grown on Stauton's synthetic medium (Example 1) containing lactose in place of glycerol at 37 0 C.
  • Colonies that grow on this novel selection medium are screened by PCR (Example 1), to demonstrate the presence of the plasmid, DNA sequencing (see recombinant DNA methods above) to demonstrate that the plasmid carries the correct sequence and 2D gel electrophoresis on a fee-f or- service basis at ACE BioSciences (Odense, Denmark) to demonstrate that ESAT-6 and CFPlO are expressed by the rBCG strain, Bacilligen-1017 (rBCG-RDl + ).
  • Colonies that grow on the lactose-selection medium are screened by PCR (Example 1), to demonstrate the presence of the plasmid, automated DNA sequencing (Example 1) to demonstrate that the plasmid carries the correct sequence and 2D gel electrophoresis on a fee-for-service basis at ACE BioSciences (Odense, Denmark) to demonstrate that ESAT-6 and CFPlO are expressed in xM. Smegmatis strain, Bacilligen-1018 (rM. smegmatis-KDl + ).
  • Bacilligen-1019 (rBCG-NS3 + ) and Bacilligen-1020 (rM. smegmatis-NS3 + ), which are engineered to constitutively express the proteolytic domain of NS3, using a similar approach as in the proceeding examples.
  • macrophages are less effective than DCs at promoting the development of CD4 + and CD8 + T cell responses (41). Therefore, induction of apoptosis in macrophages provides a delivery mechanism through which antigens from cells infected with mycobacteria are transferred to DCs leading to the induction of strong effector T cells (40, 41). Furthermore, macrophages undergoing apoptosis are more effective at killing mycobacteria than activated macrophages and macrophages undergoing necrosis (42), suggesting that mycobacteria that promote apoptosis will display an improved safety profile as anticancer immunotherapeutics.
  • 112275/F/l strain that causes the endosome to become leaky (24); however, this example provides the materials and methods to construct novel rBCG and rM.
  • smegmatis strains that harbor an RDS encoding a genetic fusion between the LP Ag 85A (SEQ ID NO:2), the intercellular trafficking domain of VP22 (SEQ ID NO: 15; herein referred to a "VP22i TD "), which spans amino acids 81-195, and the proteolytic domain of NS3 Pr (SEQ ID NO:8); encoded by base pairs 6469-7039 of West Nile virus isolate Mex03 (GenBank Accession no. AY660002), which spans amino acids 1-190.
  • Picoscript (Houston, Texas) and are optimized for expression in mycobacteria by using the preferred codon bias of this genus (SEQ ID NO:8; (69, 70)).
  • Secretion of the NS3 Pr by the recombinant mycobacteria of the present invention is accomplished by generating a genetic fusion between DNA encoding LP Ag 85A (SEQ ID NO:2) and DNA encoding the PAP.
  • Transport of NS3 Pr from the endosome to the cytoplasmic compartment of the host cell is accomplished by inserting DNA encoding the ITP human herpes virus VP22 amino acids 81-195 (herein referred to as "VP22 8 i-i 9 5"; SEQ ID NO: 15) between DNA encoding LPA g 85A and NS3p r .
  • NS3p r activity is enhanced by separating the fusion partners VP22 8 i-i95 and NS3 Pr with DNA encoding a flexible linker, as known in the art, including, for example, one containing a furin degradation motif (herein referred to as "FLf ur "; i.e Serine-Glycine-Glycine-Glycine-Glycine-Arginine-Threonine- Lysine-Arginine-Glycine-Glycine-Glycine-Glycine-Glycine-Serine; SEQ ID NO: 11).
  • FLf ur furin degradation motif
  • DNA encoding the genetic fusion is functionally linked to PA g 85A (SEQ ID NO:3).
  • Synthetic DNA encoding recombinant gene P Ag85A -LP Ag85A ::VP22i TD ::FL fur ::NS3p r is purchased from Picoscript (Houston, Texas) and is ligated (Example 1) into plasmid pB ACIL-101 (SEQ ID NO: 13). The ligated plasmid is introduced mto E. coli strain Stable2 as described in Example
  • pBACIL-106 An isolate harboring the recombinant plasmid designated pBACIL-106 (i.e. pBACIL- 101 ::P Ag85 A -LP Ag85A ::VP22i ⁇ D ::FL fur ::NS3p r ), is amplified by culturing the resulting transformants in 100 ml liquid media at 37 0 C with agitation (Example 1) and the plasmid DNA is purified as described (Example 1).
  • pBACIL-106 i.e. pBACIL- 101 ::P Ag85 A -LP Ag85A ::VP22i ⁇ D ::FL fur ::NS3p r
  • the purified DNA is digested with restriction endonuclease Notl (New England Biolabs) to remove Kan R and the large fragment encoding PA g 85A-LPAg85A::VP22i ⁇ D::FLf ur ::NS3p r , PA g 85B: .lacYZ, OriE and OriC is purified following fractionation in agarose (Example 1).
  • the purified DNA fragment is ligated to form closed circular DNA as described (Example 1) and the resulting DNA is introduced into BCG strain Danish 1331 by electroporation as described (Example 2).
  • Colonies harboring the recombinant plasmid are grown on Stauton's synthetic medium (Example 2) containing lactose in place of glycerol at 37 0 C. Colonies that grow on this novel selection medium are screened by PCR using forward primer: 5'-GGCGTGTTGTGGGACACTCCCTCA-S ' (SEQ ID NO: 16) and reverse primer 5'-GATCTGTTTTTTCCTCAGCATCTC-S ' (SEQ ID NO: 17) (Example 1) to demonstrate the presence of the plasmid, DNA sequencing (See recombinant DNA methods above) to demonstrate that the plasmid carries the correct sequence and 2D gel electrophoresis on a fee-for-service basis at ACE BioSciences (Odense, Denmark) to demonstrate that NS3 Pr is expressed in BCG.
  • Stauton's synthetic medium Example 2D gel electrophoresis on a fee-for-service basis at ACE BioSciences (Odense, Denmark) to
  • Colonies that grow on the lactose-selection medium are screened by PCR (Example 1), to demonstrate the presence of the plasmid, automated DNA sequencing (Example 1) to demonstrate that the plasmid carries the correct sequence and 2D gel electrophoresis on a fee-for-service basis at ACE BioSciences (Odense, Denmark) to demonstrate that NS3 Pr is expressed in M. smegmatis.
  • Bacilligen-1021 (rBCG FAP c -NS3 Pr + -RDl + ), Bacilligen-1022 (rBCG FAP C - Mbl389c 1-360 C - RDl + ) and Bacilligen-1023 (rM. smegmatis FAP c -Mbl389c 1-3 6o C ), which are engineered to constitutively express the proteolytic domain of NS3, using a similar approach as the proceeding examples.
  • Strain Bacilligen-1021 is constructed by introducing a derivative of pBACIL-101 designated pBACIL-107, which carries synthetic sequences that express a FAP C , NS3 Pr + and RDl + phenotype in BCG. Plasmid pBACIL-107 is assembled using the cloning schematic shown in Figure 2. A small sequence encoding the Mycobacterial consensus ribosomal binding site (SEQ ID NO: 19 herein referred to as "RBS”) is inserted upstream of the sequence encoding LP Ag85A ::VP22i ⁇ D ::FL fur ::NS3p r by PCR-directed insertional mutagenesis (Example 1). The sequence encoding
  • RBS::LP Ag85A ::VP22i ⁇ D ::FL fur ::NS3p r is then digested with Ascl (New England Biolabs, Cat. No. R0558S) and joined to Ascl-digested DNA encoding P Ag 85A-FAP ( Figure 2).
  • the resulting chimeric fragment is purified following agarose gel electrophoresis (Example 1) and digested with Pad (New England Biolabs, Cat. No. R0547S) and Fsel (New England Biolabs, Cat. No. R0588S).
  • DNA encoding RDl + (SEQ ID NO:4) is amplified from plasmid pBACIL-105 (Example 5) by PCR so as to insert Fsel and Pad sites at the 5' and 3' ends, respectively. This PCR-generated fragment is digested with Pad and Fsel. Finally, pBACIL-101 is digested with Pad and equimolar amounts of the three digested DNA preparations are introduced into a ligation reaction, resulting in the generation of pBACIL-107 ( Figure 2).
  • the ligated plasmid is introduced into E. coli strain Stable2 as described in Example 1.
  • An isolate harboring the recombinant plasmid designated pB ACIL- 106 i.e. pBACIL-101 ::P Ag85A -NS3p r
  • pB ACIL- 106 is amplified by culturing the resulting transformants in 100 ml liquid media at 37 0 C with agitation (Example 1) and the plasmid DNA is purified as described (Example 1).
  • the purified DNA is digested with restriction endonuclease Notl (New England Biolabs) to remove Kan R and the large fragment encoding PA g 85A-FAP-RBS- NS3p r -RDl + , PA g 85B: :lacYZ, OriE and OriC is purified following fractionation in agarose (Example 1).
  • the purified DNA fragment is ligated to form closed circular DNA as described (Example 1) and the resulting DNA is introduced into BCG strain Danish 1331 by electroporation as described (Example 2).
  • Colonies harboring the recombinant plasmid are grown on Stauton's synthetic medium (Example 2) containing lactose in place of glycerol at 37 0 C. Colonies that grow on this novel selection medium are screened by PCR using forward primer: 5'- GGCGTGTTGTGGGAC ACTCCCTC A-3' (SEQ ID NO: 16) and reverse primer 5'-GATCTGTTTTTTCCTCAGCATCTC-S ' (SEQ ID NO: 17) (Example 1) to demonstrate the presence of plasmid pBACIL-107, DNA sequencing (see recombinant DNA methods above) to demonstrate that the plasmid carries the correct sequence and 2D gel electrophoresis on a fee-for-service basis at ACE BioSciences (Odense, Denmark) to demonstrate that FAP, NS3p r , ESAT-6 and CFPlO are expressed in the resulting rBCG strains Bacilligen-1021.
  • Stauton's synthetic medium
  • Strain Bacilligen-1022 is constructed by introducing a derivative of pBACIL-101 designated pBACIL-108, which carries synthetic sequences that express a FAP C (SEQ ID NO:9), Mbl389c 1-36 o + (SEQ ID NO: 14) and RDl + (SEQ ID NO:4) phenotype in BCG.
  • Plasmid pBACIL-108 is assembled using the cloning schematic shown in figure 3.
  • a small sequence encoding the Mycobacterial consensus RBS (SEQ ID NO: 19) is inserted upstream of the sequence encoding Mbl389c 1-3 6o by PCR-directed insertional mutagenesis
  • the ligated plasmid is introduced into E. coli strain Stable2 as described in Example 1.
  • An isolate harboring the recombinant plasmid designated pB ACIL- 108, is amplified by culturing the resulting transformants in 100 ml liquid media at 37 0 C with agitation (Example 1) and the plasmid DNA is purified as described (Example 1).
  • the purified DNA is digested with restriction endonuclease Notl (New England Biolabs) to remove Kan R and the large fragment encoding P Ag85A -FAP-RBS-Mbl389c 1-36 o-RDl + , PA g 85B: :lacYZ, OriE and OriC is purified following fractionation in agarose (Example 1).
  • the purified DNA fragment is ligated to form closed circular DNA as described (Example 1) and the resulting DNA is introduced into BCG strain Danish 1331 by electroporation as described (Example 2).
  • Colonies harboring the recombinant plasmid are grown on Stauton's synthetic medium (Example 2) containing lactose in place of glycerol at 37 0 C. Colonies that grow on this novel selection medium are screened by PCR using forward primer: 5'- GGCGTGTTGTGGGAC ACTCCCTCA-3' (SEQ ID NO: 10) and reverse primer 5'- GATCTGTTTTTTCCTC AGC ATCTC-3' (SEQ ID NO: 17) (Example 1) to demonstrate the presence of plasmid pBACIL-108 [ ⁇ Kan R ], DNA sequencing (See recombinant DNA methods above) to demonstrate that the plasmid carries the correct sequence and 2D gel electrophoresis on a fee-for-service basis at ACE BioSciences (Odense, Denmark) to
  • Strain Bacilligen-1023 is constructed by introducing a derivative of pBACrL-101 designated pBACIL-109, which carries synthetic sequences that express a FAP C (SEQ ID NO: 8) and Mbl389c 1-360 + (SEQ ID NO: 14) phenotype in M. smegmatis.
  • Plasmid pBACIL-109 is assembled using the cloning schematic shown in Figure 4.
  • a small sequence encoding the mycobacterial consensus RBS (SEQ ID NO: 19) is inserted upstream of the sequence encoding Mbl389c 1-3 6o by PCR-directed insertional mutagenesis (Example 1).
  • the ligated plasmid is introduced into E. coli strain Stable2 as described in Example 1.
  • An isolate harboring recombinant plasmid pBACIL-109 is amplified by culturing the resulting transformants in 100 ml liquid media at 37 0 C with agitation (Example 1) and the plasmid DNA is purified as described (Example 1).
  • the purified DNA is digested with restriction endonuclease Notl (New England Biolabs) to remove Kan R and the large fragment encoding PA g 85A-FAP-RBS-Mbl389c 1-3 6o, PA g 85B:J «c7Z, OriE and OriC is purified following fractionation in agarose (Example 1).
  • the purified DNA fragment is ligated to form closed circular DNA as described (Example 1) and the resulting DNA is introduced into M. smegmatis (ATCC#12051; American Type Culture Collection, Manassas, VA) by electroporation as described (Example 2).
  • Colonies harboring the recombinant plasmid are grown on Stauton's synthetic medium (Example 2) containing lactose in place of
  • Colonies that grow on this novel selection medium are screened by PCR using forward primer: 5'- GGCGTGTTGTGGGACACTCCCTCA-3' (SEQ ID NO: 16) and reverse primer 5'-GATCTGTTTTTTCCTCAGCATCTC-S ' (SEQ ID NO: 17) (Example 1) to demonstrate the presence of plasmid pBACIL-109 [ ⁇ Kan R ], DNA sequencing (See recombinant DNA methods above) to demonstrate that the plasmid carries the correct sequence and 2D gel electrophoresis on a fee-for-service basis at ACE BioSciences (Odense, Denmark) to demonstrate that FAP and Mb 1389C 1-36 O are expressed in the resulting rM. smegmatis strains Bacilligen-1023.
  • the safety, toxicity and potency of recombinant mycobacterial strains are evaluated according to the guidelines in 21 CFR 610, which include: (i) general safety test; (ii) stringent safety test in immunocompetent mice; (iii) guinea pig safety test; and (iv) acute and chronic toxicity tests, as described below. (i) General safety test
  • the bacteria are harvested by centrifugation (5000 x g for 10 min) and washed in 100 ml PBS.
  • Inoculation suspensions are prepared by resuspending the washed bacteria in normal saline (0.85% w/v NaCl) containing 10% glycerol and 0.05% (v/v) tyloxapol to a density of 5 x 10 7 cfu per ml.
  • Groups of eight BALB/c mice are inoculated intraperitoneally with 100 ⁇ l of inoculation suspensions containing 5 x 10 6 cfu of the recombinant mycobacterial strain of interest and the analogous parental strain, as shown in the table below.
  • Thee animals are monitored for general health and body weight for 14 days post infection. Similar to animals that receive BCG, animals that receive the recombinant mycobacterial strains remain healthy, and do not lose weight or display overt signs of disease during the observation period.
  • the bacteria are harvested by centrifugation (5000 x g for 10 min) and washed in 100 ml PBS.
  • Inoculation suspensions are prepared by resuspending the washed bacteria in normal saline (0.85% w/v NaCl) containing 10% glycerol and 0.05% (v/v) tyloxapol to a density of 2 x 10 7 cfu per ml.
  • Groups of 15 healthy BALB/c mice are infected intravenously with 100 ⁇ l of the inoculation suspensions containing 2xlO 6 viable recombinant mycobacteria and parental strains as shown in the table below.
  • mice in each group are sacrificed and the cfu numbers in the spleen, lung and liver homogenates are analyzed to ensure each animal receives an equivalent infection dose.
  • week 4 8, 12, and 16 post infection, 3 mice in each group are sacrificed and cfu numbers in spleen, live and lung homogenates are obtained to assess the in vivo growth of the recombinant mycobacterial strains as compared to the parental strains.
  • Recombinant mycobacterial strains are expected to display similar or less growth to that of the parental strains, (iii) Guinea pig safety test
  • Each strain is grown in 1.0 L liquid cultures as described (Example 1); the bacteria are harvested by centrifugation (5000 x g for 10 min) and washed in 1 L PBS. Inoculation suspensions are prepared by resuspending the washed bacteria in normal saline (0.85% w/v NaCl) containing 10% glycerol and 0.05% (v/v) tyloxapol to a density of 5 x 10 9 cfu per ml. Groups of 8 guinea pigs are inoculated intramuscularly with 100 ⁇ l of the inoculation suspensions containing 5 x 10 8 cfu (i.e. 100 x of human dose) of the recombinant mycobacterial and parental strain as shown in the table below.
  • Guinea pigs immunized with either the parental or recombinant strains are euthanized at various intervals after inoculation, after which cfu counts of the recombinant mycobacterial strains and parental strains are determined in lung, spleen, and regional (inguinal) lymph node homogenates.
  • Each strain is grown in 1.0 L liquid cultures as described (Example 1); the bacteria are harvested by centrifugation (5000 x g for 10 min) and washed in 1 L PBS. Inoculation suspensions are prepared by resuspending the washed bacteria in normal saline (0.85% w/v NaCl) containing 10% glycerol and 0.05% (v/v) tyloxapol to a density of 5 x 10 7 (standard dose), 2 x 10 8 (high dose) and 1.25 x 10 7 (low dose) cfu per ml.
  • mice of 16 guinea pigs are inoculated intradermally with 100 ⁇ l of the inoculation suspensions containing 5 x 10 6 cfu (i.e. 1 x human dose), 2 x 10 7 cfu (i.e. 4 x human dose) and 1.25 x 10 6 cfu (0.25 x human dose) of recombinant mycobacterial and parental strains or saline respectively as shown in the table below.
  • syngeneic tumor model transplantation of carcinogen-induced bladder cancer in syngeneic, immunocompetent mice (6-9).
  • syngeneic murine bladder tumor model seems to be the most appropriate model because of the chance to study the local tumor in an immunocompetent host.
  • Syngeneic tumor cells can be implanted either subcutaneously (heterotopic tumor) or intravesically (orthotopic).
  • each strain is grown in 1.0 L liquid cultures as described (Example 1); the bacteria are harvested by centrifugation (5000 x g for 10 min) and washed in 1 L PBS. Inoculation suspensions are prepared by resuspending the washed bacteria in normal saline (0.85% w/v NaCl) containing 10% glycerol and 0.05% (v/v) tyloxapol to a density of 1 x 10 7 cfu per ml.
  • mice Female C57B1/6 mice, 6-8 weeks old, each weighing 17 g, are purchased from Charles River (Maine) and maintained at an animal facility for 1 week prior to use. The mice are housed five per cage, in a limited access area at a room temperature of 20+1 0 C and a humidity of 50+10%, with food and water ad libitum.
  • Tumor cells used in this study are derived from the 7,12- dimethylbenzanthracene-induced murine bladder cancer MB49 (12). The cells are maintained in in vitro culture (DMEM, 10% FCS, and 1% w/v penicillin/streptomycin at 37°C and 5% CO 2 ). Tumor cells are harvested by trypsinization and suspended in DMEM without L- glutamine, FCS, and antibiotics. Viability is determined by trypan blue exclusion, and only tumor cell suspensions with 90% viable cells are used for tumor implantation. The concentrations of the tumor cell suspensions used for implantation are adjusted to 2 x 10 6 cells/ml.
  • Intravesical tumor implantation is performed as described by Soloway and Masters (8, 13) and Shapiro et al. (14) for the MBT-2 model and by Hudson et al. (7) for the MB49 model. Briefly, after a short ether inhalation anesthesia, the mice received an i.p. injection of diluted sodium pentobarbital (6 mg/ml) for general anesthesia of a single dose of 0.06 mg/g body weight. After shaving areas of 1 cm 2 on the backs of the mice, a 24-gauge Teflon i.v.
  • the guide wire is attached to the cautery unit (Elektrotom 500; Gebruder Martin, Tuttlingen, Germany), and a monopolar coagulation is applied for 5 s at the lowest setting (5 W). After removal of the guide wire, 0.05 ml of the tumor cell suspension is instilled. Unlike the conventional cautery unit (Elektrotom 500; Gebruder Martin, Tuttlingen, Germany), and a monopolar coagulation is applied for 5 s at the lowest setting (5 W). After removal of the guide wire, 0.05 ml of the tumor cell suspension is instilled. Unlike the conventional cautery unit (Elektrotom 500; Gebruder Martin, Tuttlingen, Germany), and a monopolar coagulation is applied for 5 s at the lowest setting (5 W). After removal of the guide wire, 0.05 ml of the tumor cell suspension is instilled. Unlike the conventional cautery unit (Elektrotom 500; Gebruder Martin, Tuttlingen, Germany), and
  • the animals are randomized into groups with 15 animals each to receive PBS or BCG/rBCG/rM smegmatis therapy as shown in the table below. Intravesical instillations of PBS, BCG, rBCG and rM. smegmatis are performed on days 1, 8, 15, and 22 after tumor implantation by the technique described above in a volume of 0.05 ml containing 5 x 10 6 cfu.
  • the bacteria are harvested by centrifugation and washed 3 times in 10 ml PBS containing 0.05% tyloxapol.
  • the Bacilligen-1021 bacilli are resuspended in PBS containing 0.05% tyloxapol, 10% glycerol (Sigma, St Louis MO) at a density of 5 x 10 6 CFU/ml and stored at -8O 0 C.
  • mice are given a total of 3 doses of vaccine at 0, 14 and 60 days and the immune response to hemagglutinin is measured by ELISA using sera collected from the tail vein of individual mice at 10 day intervals, as described (130). The neutralization of influenza virus is measured in the collected 80 days after the first vaccination, as described (131).
  • Bacilligen-1021 has the capacity to substantially increase the magnitude and potency of the humoral response to hemagglutinin and therefore possesses useful adjuvant properties.
  • BCG Bacillus calmette-guerin
  • a conditioned dendritic cell can be a temporal bridge between a CD4+ T-helper and a T-killer cell. Nature 393:474.
  • Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs. Nature 392:86.
  • Salmonella virulence factor SipB induces activation and release of IL- 18 in human dendritic cells. J Leukoc Biol 72:743.
  • GGDEF domain is homologous to adenylyl cyclase. Proteins 42:210.

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Abstract

Cette invention concerne des souches de Mycobacterium génétiquement modifiées qui sont utiles comme agents immunothérapeutiques anti-cancers. Les souches de Mycobacterium génétiquement modifiées présentent de préférence au moins une modification génétique conduisant à une adhérence tissulaire accrue, qui peut être combinée avec des modifications génétiques qui augmentent l'immunopuissance et conduisent à une production d'adjuvants. L'invention concerne également des compositions et des procédés pour le développement desdites souches de Mycobacterium, et des compositions pharmaceutiques et des procédés pour l'utilisation desdites souches de Mycobacterium comme agents thérapeutiques anti-cancer.
PCT/US2007/086335 2006-12-04 2007-12-04 Nouvelles microbactéries immunothérapeutiques, formulations pharmaceutiques et utilisation de celle-ci WO2008140598A2 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010081026A1 (fr) * 2009-01-08 2010-07-15 Albert Einstein College Of Medicine Of Yeshiva University, A Division Of Yeshiva University Vaccins bactériens avec des glycolipides du type céramide associés à la paroi cellulaire et leurs utilisations
US9371352B2 (en) 2013-02-08 2016-06-21 Vaccinex, Inc. Modified glycolipids and methods of making and using the same
US9809654B2 (en) 2002-09-27 2017-11-07 Vaccinex, Inc. Targeted CD1d molecules
WO2022203308A1 (fr) * 2021-03-22 2022-09-29 클립스비엔씨 주식회사 Nouvelle souche recombinée de mycobacterium smegmatis et son utilisation
WO2023150848A1 (fr) * 2022-02-11 2023-08-17 Instituto Butantan Compositions comprenant des souches de mycobacterium recombinantes, leurs utilisations et méthodes pour la prévention et/ou le traitement de cancer

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE60236573D1 (de) * 2002-04-05 2010-07-15 Pasteur Institut Identifizierung der Virulenz-assoziierten Regionen RD1 und RD5, die die Entwicklung von verbesserten Impfstoffen mit M. bovis BCG und M. microti ermöglicht
DE602006015180D1 (de) * 2006-07-25 2010-08-12 Pasteur Institut Rekombinanter Mycobakteriumstamm, der ein FAP Protein aus Mycobakterium unter der Kontrolle eines Promotors, der unter hypoxischen Konditionen aktiv ist, exprimiert, und dessen Verwendung zur Tumortherapie

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9809654B2 (en) 2002-09-27 2017-11-07 Vaccinex, Inc. Targeted CD1d molecules
WO2010081026A1 (fr) * 2009-01-08 2010-07-15 Albert Einstein College Of Medicine Of Yeshiva University, A Division Of Yeshiva University Vaccins bactériens avec des glycolipides du type céramide associés à la paroi cellulaire et leurs utilisations
CN102325875A (zh) * 2009-01-08 2012-01-18 阿尔伯爱因斯坦医科叶希瓦大学,叶希瓦大学分部 具有细胞壁结合神经酰胺类糖脂的细菌疫苗及其应用
US9139809B2 (en) 2009-01-08 2015-09-22 Albert Einstein College Of Medicine Of Yeshiva University Bacterial vaccines with cell wall-associated ceramide-like glycolipids and uses thereof
CN102325875B (zh) * 2009-01-08 2018-04-10 阿尔伯爱因斯坦医学有限公司 具有细胞壁结合神经酰胺类糖脂的细菌疫苗及其应用
US9371352B2 (en) 2013-02-08 2016-06-21 Vaccinex, Inc. Modified glycolipids and methods of making and using the same
US10111950B2 (en) 2013-02-08 2018-10-30 Vaccinex, Inc. Modified glycolipids and methods of making and using the same
WO2022203308A1 (fr) * 2021-03-22 2022-09-29 클립스비엔씨 주식회사 Nouvelle souche recombinée de mycobacterium smegmatis et son utilisation
WO2023150848A1 (fr) * 2022-02-11 2023-08-17 Instituto Butantan Compositions comprenant des souches de mycobacterium recombinantes, leurs utilisations et méthodes pour la prévention et/ou le traitement de cancer

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