WO2010062982A1 - Salmonelles résistantes au co2 à ciblage tumoral - Google Patents

Salmonelles résistantes au co2 à ciblage tumoral Download PDF

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WO2010062982A1
WO2010062982A1 PCT/US2009/065970 US2009065970W WO2010062982A1 WO 2010062982 A1 WO2010062982 A1 WO 2010062982A1 US 2009065970 W US2009065970 W US 2009065970W WO 2010062982 A1 WO2010062982 A1 WO 2010062982A1
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salmonella
tumor
mutant
gene
zwf
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PCT/US2009/065970
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English (en)
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Ivan King
Verena Karsten
David Bermudes
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Vion Pharmaceuticals, Inc.
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Publication of WO2010062982A1 publication Critical patent/WO2010062982A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/255—Salmonella (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/42—Salmonella
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention is related to tumor-targeting bacteria which are modified to improve bacterial survival in C ⁇ 2 ⁇ rich and acidic environments, such as those which are often present within tumors .
  • cytotoxic peptides may exert antitumor effects via several mechanisms, including direct toxicity to tumor cells via type III secretion, in which cytotoxic peptides are injected directly into the target cell's cytoplasm; the facilitation of a potent nonspecific immune reaction; the depletion of essential nutrients; and alteration of the tumor microenvironment as a result of colonization. Additionally, immunomodulatory effects such as the stimulation of dendritic cell function or alteration of T helper cell polarization could play a role. Anaerobic bacteria's tropism for hypoxic tumor tissue allows such bacteria to exert an antitumor effect against cells that are often resistant to other forms of therapy such as chemotherapy and radiation, and the motility of said bacteria allows effective migration and dispersion through tumor tissue and potentially into distant sites.
  • Salmonella have been shown to infect and preferentially accumulate within murine tumors, achieving tumor :normal tissue ratios of ⁇ 1, 000:1 (1, 2) . Wild-type
  • Salmonella and in particular Salmonella typhimurium, produce self-limiting enteritis in most healthy adults, infects many mammalian species, and can easily be manipulated to carry therapeutic transgenes .
  • S. typhimurium exists as a facultative anaerobe, allowing it to survive in both oxygenated and hypoxic conditions; thus, it may colonize both small metastatic lesions and larger tumors.
  • S. typhimurium can also be genetically modified to reduce or eliminate pathogenicity in animals and humans .
  • a S. typhimurium strain has been attenuated by partial deletion of the msbB gene, which is responsible for addition of a terminal myristyl group to lipid A.
  • Lipopolysaccharide (LPS) derived from these lipid A mutants has a markedly diminished ability to induce tumor necrosis factor- ⁇ in isolated human monocytes, and administration of the intact organism to mice or pigs results in 14% to 33% of the tumor necrosis factor- ⁇ induction seen with wild-type S. typhimurium (3) .
  • the bacteria were further attenuated by partial deletion of the purl gene, creating a growth requirement for external sources of purines, which may be present in higher concentrations in the interstitial fluids in the tumor microenvironment .
  • VNP20009 The mutations of this attenuated form of S. typhimurium (VNP20009) were accomplished by deletions of large portions of both msbB and Purl genes, making reversion to wild- type highly unlikely. These modifications allow for significant dose escalation in mice when compared with the native organism and do not affect its tumor specificity (3) .
  • VNP20009 strain bacteria were shown to be safe in humans when administered intravenously in a phase I clinical study, the bacteria were rapidly cleared from the peripheral blood of humans and targeting to human tumors was only observed in a few patients when the bacteria were administered at the highest dose levels of 3xlO 8 cfu/m2 and lxioVm 2 (4) .
  • the present invention discloses a mutant Salmonella species capable of targeting a solid tumor when administered in vivo, comprising a mutation of a gene encoding an enzyme in the pentose-phosphate pathway.
  • the invention also discloses a method of inhibiting the growth or reducing the volume of a solid tumor cancer through the administration of an effective amount of the mutant Salmonella disclosed herein.
  • Figure 1 shows growth of bacterial strains YS873 and YS873 zwf- in ambient air supplemented with 5% CO 2 .
  • Bacterial growth was determined by inoculating bacteria in 3 ml LB broth tubes, which were placed in a 37°C shaker set at 250 rpm in air or air/5% CO 2 . O. D.600 was measured every 60 minutes.
  • Bacterial culture was diluted and plated onto MSB or LB agar plates to calculate the number of colony forming units (C. F. U.) per ml.
  • YS873 grew well in air, but not in the presence of 5% CO 2 .
  • Zwf mutant (mutation in the zwf gene) grew well in both conditions. The results suggest that the zwf mutation suppresses the sensitivity of msbB mutants to CO 2 .
  • Figure 2 shows the pentose phosphate pathway.
  • Figure 3 shows the growth of msbB mutants and zwf mutants on LB, MSB, LB-O, and LB-O sucrose-supplemented media plates in the presence of 0.33% gluconate in ambient air and 5% CO 2 .
  • Figure 4 shows the efficacy of CO 2 -resistant tumor- targeted msbB mutant Salmonella clones and CO 2 -sensitive msbB tumor-targeted mutant Salmonella clones against B16-F10 tumors in C57/B16 mice.
  • VNP20009 is a CO 2 -sensitive control.
  • the CO 2 - resistant clones designated C32.2, 14.2, and C37.2 have Tn5 insertions causing loss of function in the zwf gene.
  • Figure 5 shows the growth rates of YS873 clones and YS873 zwf- clones in acidic pH.
  • Salmonella or “Salmonella species” refers to all Salmonella species, including Salmonella typhi, Salmonella choleraesuis , and Salmonella enteritidis . Serotypes of Salmonella are also encompassed herein, for example, typhimurium, a subgroup of Salmonella enteritidis , commonly referred to as Salmonella typhimurium.
  • strain designations VNP20009 and YS1646 are used interchangeably and each refer to the strain deposited with the American Type Culture Collection and assigned Accession No. 202165.
  • targeting is defined as preferential attachment to, infection of, and/or ability to remain viable in the target cell .
  • attenuation refers to one or more modifications so that a microorganism or vector is less pathogenic. The end result of attenuation is that the risk of toxicity as well as other side effects is decreased when the microorganism or vector is administered to the patient. As herein used, “attenuation” also refers to the modification of a microorganism vector so that a lower titer of that derived microorganism vector can be administered to a patient and still achieve comparable results as if one had administered a higher titer of the parental microorganism vector.
  • solid tumor refers to an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors may be benign (non-cancerous), or malignant
  • solid tumors include, but are not limited to, sarcomas, carcinomas, lymphomas, germ line tumors, tumors of the central nervous system, breast cancer, prostate cancer, cervical cancer, uterine cancer, lung cancer, ovarian cancer, testicular cancer, thyroid cancer, astrocytoma, glioma, pancreatic cancer, stomach cancer, liver cancer, colon cancer, and melanoma.
  • Salmonella bacteria can live almost everywhere, including tissues which are badly supplied with blood and concomitantly have hardly any oxygen supply. It is precisely these areas that are scarcely reachable in a cancerous ulcer using common cancer therapies: chemotherapeutics cannot be transported to an area where there is no blood flow. Furthermore, radiation therapy is not efficacious in low oxygen environments, such as the hypoxic areas in the tumor.
  • Bacteria such as Salmonella are causative agents of diseases and toxic events in humans and animals .
  • One such toxic event that can be caused by Salmonella is sepsis, which is a serious problem because of the high mortality rate associated with the onset of septic shock. Therefore, to allow the safe use of Salmonella vectors in the present invention, the bacterial vectors such as Salmonella are attenuated in their virulence for causing disease.
  • Attenuation in addition to its traditional definition in which a microorganism vector is modified so that the microorganism vector is less pathogenic, is intended to include also the modification of a microorganism vector so that a lower titer of that derived microorganism vector can be administered to a patient and still achieve comparable results as if one had administered a higher titer of the parental microorganism vector.
  • the end result serves to reduce the risk of toxic shock or other side effects due to administration of the bacterial vector to the patient.
  • Such attenuated bacteria can be isolated by means of a number of techniques.
  • Attenuation can be achieved by the deletion or disruption of DNA sequences which encode for virulence factors that insure survival of the bacteria in the host cell, especially macrophages and neutrophils.
  • deletion or disruption techniques are well known in the art and include, for example, homologous recombination, chemical mutagenesis, radiation mutagenesis, or transposon mutagenesis.
  • Salmonella virulence factors whose deletion would result in attenuation are well known in the art.
  • the bacterial vectors of the present invention are tumor targeted, i.e. the bacteria preferentially attaches to, infects, and/or remains viable in a tumor or tumor cell versus a normal tissue, non- tumor or non-tumor cell .
  • Suitable methods for obtaining attenuated tumor-targeted bacteria are well known in the art.
  • Attenuated bacterial vectors are highly specific and super- infective: the difference between the number of infecting bacteria found at the target tumor or tumor cell as compared to the non-cancerous counterparts becomes larger and larger as the dilution of the microorganism culture is increased such that lower titers of microorganism vectors can be used with positive results .
  • the stability of the attenuated phenotype is important so that the strain does not revert to a more virulent phenotype during the course of treatment of a patient.
  • Such stability can be obtained, for example, by providing that the virulence gene is disrupted by deletion or other non-reverting mutations on the chromosomal level rather than epistatically .
  • Another method of insuring the attenuated phenotype is to engineer the bacteria such that it is attenuated in more than one manner, e.g., a mutation in the pathway for lipid A production, such as the msbB mutation and one or more mutations to auxotrophy for one or more nutrients or metabolites, such as uracil biosynthesis, purine biosynthesis, and arginine biosynthesis as known in the art.
  • a mutation in the pathway for lipid A production such as the msbB mutation and one or more mutations to auxotrophy for one or more nutrients or metabolites, such as uracil biosynthesis, purine biosynthesis, and arginine biosynthesis as known in the art.
  • MsbB Salmonella mutants (Salmonella with a deletion in the msbB gene) do not grow well in the presence of 5% CO 2 ( Figure 1); however, it is possible that the CO 2 sensitivity could be suppressed by a second mutation.
  • the present invention discloses that the growth of msbB Salmonella mutants is highly inhibited in a 5% CO 2 atmosphere in LB media as well as under low pH conditions when compared to wild-type Salmonella .
  • several CC> 2 -resistant clones were selected from msbB Salmonella with Tn5 transposon insertion. Three clones with three different mutations were mapped and all were shown to contain the Tn5 marker in the zwf gene, which encodes the enzyme glucose-6-phosphate-dehydrogenase .
  • U.S. Patent 6,190,657 describes tumor-targeted bacterial strains which may further contain a plasmid encoding the Herpes Simplex Virus thymidine kinase gene (pTK-Sec3) , a plasmid encoding the E. coli cytosine deaminase gene (pCD- Secl) , or the human p450 oxidoreductase gene (pSP-SAD4-5) ;
  • U.S. patent 6,685,935 further enumerates other suicide genes.
  • the presently disclosed bacteria may be similarly modified to include such enumerated suicide genes .
  • U.S. Patent 7,452,531 describes attenuated tumor- targeted bacteria comprising one or more nucleic acid molecules encoding one or more primary effector molecules.
  • at least one of the primary effector molecules is a mammalian anti-angiogenic polypeptide.
  • the claims of the patent enumerate various promoters that may be utilized with the claimed bacteria.
  • the CC> 2 -resistant bacteria of the present disclosure may be modified so that it may further comprise one or more nucleic acid molecules encoding one or more primary effector molecules, as well as, optionally, nucleic acid molecules encoding one or more secondary effector molecules as taught in the ⁇ 531 patent.
  • the primary effector molecules can be derived from any known organism, including, but not limited to, animals, plants, bacteria, fungi, and protista, or viruses.
  • primary effector molecules include, but are not limited to, members of the TNF family, anti-angiogenic factors, cytotoxic polypeptides or peptides, tumor inhibitory enzymes, and functional fragments thereof.
  • TNF family members include, but are not limited to, tumor necrosis factor-a (TNF- ⁇ ) , tumor necrosis factor- ⁇ (TNF- ⁇ ) , TNF- ⁇ -related apoptosis-inducing ligand (TRAIL) , TNF- ⁇ -related activation-induced cytokine (TRANCE) , TNF- ⁇ -related weak inducer of apoptosis (TWEAK), CD40 ligand (CD40L) , LT- ⁇ , LT- ⁇ , OX40L, CD40L, FasL, CD27L, CD30L, 4 -IBBL, APRIL, LIGHT, TLl, TNFSF16, TNFSFI7, and AITR-L.
  • TNF- ⁇ tumor necrosis factor-a
  • TNF- ⁇ tumor necrosis factor- ⁇
  • TRANCE TNF- ⁇ -related activation-induced cytokine
  • the primary effector molecules can be anti-angiogenic factors or functional fragments thereof.
  • anti-angiogenic factors include, but are not limited to, endostatin, angiostatin, apomigren, anti-angiogenic antithrombin III, the 29 kDa N-terminal and a 40 kDa C-terminal proteolytic fragments of fibronectin, a uPA receptor antagonist, the 16 kDa proteolytic fragment of prolactin, the 7.8 kDa proteolytic fragment of platelet factor-4, the anti- angiogenic 24 amino acid fragment of platelet factor-4, the anti-angiogenic factor designated 13.40, the anti-angiogenic 22 amino acid peptide fragment of thrombospondin I, the anti- angiogenic 20 amino acid peptide fragment of SPARC, RGD and NGR containing peptides, the small anti-angiogenic peptides of laminin, fibronectin, procollagen and EGF, and peptide
  • the primary effector molecules can be cytotoxic polypeptides or peptides, or functional fragments thereof.
  • a cytotoxic polypeptide or peptide is cytotoxic or cytostatic to a cell, for example, by inhibiting cell growth through interference with protein synthesis or through disruption of the cell cycle. Such a product may act by cleaving rRNA or ribonucleoprotein, inhibiting an elongation factor, cleaving mRNA, or by other mechanisms that reduce protein synthesis to a level such that the cell cannot survive. Examples of cytotoxic polypeptides are well known in the art.
  • the primary effector molecules can be tumor inhibitory enzymes or functional fragments thereof.
  • tumor inhibitory enzymes are well known in the art, e.g. methionase, asparaginase, lipase, phospholipase, protease, ribonuclease, DNAase, and glycosidase.
  • the primary effector molecule is co-expressed in the bacterial vector with a secondary effector molecule.
  • the secondary effector molecule provides additional therapeutic value and/or facilitates the release of the contents of the modified bacterial vector (which expresses at least one primary effector molecule and optionally one or more secondary effector molecules) into the surrounding environment.
  • the secondary effector molecule provides an additive or synergistic, cytostatic, or cytotoxic effect on a tumor, e.g., in addition to that provided by the primary effector molecule (s) .
  • a secondary effector molecule functions as an additional therapeutic factor and/or a release factor.
  • the secondary effector molecule can be preferentially or specifically activated or expressed at the desired site, i.e. at the site of the tumor.
  • the secondary effector molecule can serve two functions, i.e. promote the release of the bacterial cell contents (e.g., by promoting bacterial cell lysis or quasi lysis) and provide therapeutic value (e.g., by cytotoxicity to the tumor cells) .
  • the cytotoxicity of the secondary effector molecule can be mediated by the patient's immune system; accordingly such a secondary effector molecule can function as an immunomodulator .
  • the secondary effector molecule is proteinaceous or a nucleic acid molecule.
  • the nucleic acid molecule can be double-stranded or single- stranded DNA or double-stranded or single-stranded RNA, as well as triplex nucleic acid molecules .
  • the nucleic acid molecule can function as a ribozyme, or antisense nucleic acid, etc.
  • the attenuated tumor-targeted bacteria of the present invention can be used in conjunction with other well-known cancer therapies to treat a solid cancer tumor.
  • the attenuated tumor-targeted bacteria of the present invention can be used in conjunction with chemotherapeutic agent (s), or radiation therapy (e.g., gamma radiation or x-ray radiation) .
  • chemotherapeutic agent s
  • radiation therapy e.g., gamma radiation or x-ray radiation
  • the present invention includes the sequential or concomitant administration of an anti-cancer agent or therapy and the attenuated tumor-targeted bacteria disclosed herein.
  • the effects of the combined uses of the anti-cancer agent/therapy and the attenuated tumor-targeted bacteria of the present invention can be additive or synergistic.
  • the anti-cancer agent/therapy and the attenuated tumor-targeted bacteria of the present invention have different sites of action. Such a combination provides an improved therapy based on the dual action of these therapeutics whether the combination is synergistic or additive.
  • the novel combinational therapy of the present invention yields improved efficacy over either agent used as a single-agent therapy.
  • the present invention provides a mutant Salmonella species capable of targeting a tumor when administered in vivo, wherein said Salmonella comprises a plurality of gene alterations comprising (i) alteration of a gene encoding an enzyme in the pentose-phosphate pathway, and (ii) alteration of one or more genes that results in attenuation of the Salmonella.
  • the genes can be altered by insertion, deletion, mutation, or point mutation.
  • the gene for the attenuation is the msbB gene.
  • a gene e.g. zwf
  • encoding glucose-6-phosphate dehydrogenase in the pentose-phosphate pathway is altered.
  • the mutant Salmonella of the present invention can be used to target a solid tumor.
  • the mutant Salmonella of the present invention can be Salmonella typhi, Salmonella choleraesuis , or Salmonella enteritidis .
  • the mutant Salmonella disclosed herein are able to grow better in acidic or C ⁇ 2 ⁇ rich environments as compared to Salmonella without the alteration of a gene encoding an enzyme in the pentose- phosphate pathway as disclosed herein.
  • the present invention also provides a method of inhibiting the growth or reducing the volume of a solid tumor cancer, comprising the step of administering an effective amount of the mutant Salmonella disclosed herein to a subject having a solid tumor cancer.
  • the method further comprises a second anti-tumor treatment (such as surgery, radiation therapy or chemotherapy) .
  • the mutant Salmonella sp. and the second anti-tumor treatment can be administered sequentially or concurrently.
  • Salmonella P22 transductions were performed by the method of Davis et al . (7), except that LB-O plates supplemented with the appropriate antibiotic were used. EGTA was not added to the antibiotic plates for transductions.
  • a BioRad Gene Pulser was used for electroporation with the following settings: 2.5 kV, 1000 ohms and 25 ⁇ FD for transformation of YSl and 1.7 kV, 186 ohms and 25 ⁇ FD were used for YS873, YS1646, and ATCC 14028 (8) .
  • a library of transposons in YS1646 was made using the EZ: :TN ⁇ Kan-2> insertion kit from Epicentre (Madison, WI) . Selection was made for CO 2 resistant colonies by plating dilutions of the resultant bacteria on LB-Kan plates and incubating said plates in 5% CO 2 . One of the resistant clones was picked and a P22 lysate was generated and transduced to a non-suppressed strain (YS873) and purified for kanamycin resistance under non-CC> 2 conditions in order to separate spontaneous mutants from Tn5-based suppressors. Transposon- associated Tn5 insertions were identified by replica plating in air and CO 2 . Mapping of the insertion sites was performed by using the GenomeWalkerTM kit (Clonetech, Mountain View, CA) according to the manufacturer's instructions.
  • Zwf encodes glucose-6-phosphate- dehydrogenase, an enzyme of the pentose-phosphate-pathway (PPP) .
  • PPP pentose-phosphate-pathway
  • zwf converts glucose-6-phosphate, from glycolysis, to gluconate-6-phosphate and to generate NADPH, ribose and CO 2 ( Figure 2) .
  • a non-polar deletion in zwf is generated by constructing a pCVD442 vector capable of deleting the entire zwf coding region by homologous recombination with the Salmonella chromosome (9) .
  • Primers for PCR are designed such that they would generate one product immediately upstream of the 5' ATG start codon and a separate product immediately downstream of the 3' stop codon of the zwf coding region. The two separate products are then ligated sequentially into the pCVD442 vector.
  • the primers also contain internal multiple restriction endonuclease sites in order to facilitate cloning of DNA fragments into the ⁇ zwf for stable chromosomal integration without antibiotic resistance.
  • the zwf non-polar deletion clones are selected for their capability of growing in the presence of 5% CO 2 , preferentially accumulating in tumors, and inhibiting tumor growth in vivo, according to established experimental protocol. Using similar methods described herein, Karsten et al (10) ., has generated non-polar clones capable of surviving in 5% CO 2 .
  • Zwf catalyzes the first step of the pentose phosphate pathway (PPP) .
  • PPP pentose phosphate pathway
  • the PPP produces NADPH for anabolic pathways and the molecules generated by this pathway serve as building blocks for nucleotides, sugars, amino acids, and vitamins (11) .
  • Zwf catalyzes the conversion of glucose- 6-phosphate to gluconate-6-phosphate .
  • Gluconate-6-phosphate can also be formed from gluconate by gluconate kinase (12), which bypasses the PPP' s requirement for Zwf ( Figure 2) .
  • the addition of gluconate to media thereby allows for the production of gluconate-6-phosphate in the absence of zwf.
  • the enzyme gluconate-6-phosphate-dehydrogenase then decarboxylates gluconate-6-phosphate, converting it from a 6-carbon to a 5- carbon (ribulose-5-phosphate) sugar and releasing CO 2 gas.
  • Solid tumor models were obtained by subcutaneous injection of B16-F10 tumor cells (murine melanoma) in the right hind flank of C57B/6 mice.
  • B16-F10 tumor cells murine melanoma
  • Tumor size was determined from measurements obtained with electronic calipers along three axes: length (L) , width (W) , and height (H) .
  • the volume of the tumor was calculated using the following formula: (LxWxH) /2.
  • Figure 4 shows that all the bacterial clones were capable of inhibiting the growth of B16-F10 tumor and the CO 2 - resistant clones showed better tumor inhibition than the CO 2 - sensitive clone VNP20009.
  • YS873 and YS873zwf were grown in buffered LB broth at pH 7.6, or 6.6 in air. As shown in Figure 5, YS873zwf grew normally in LB broth at both pH 7.6 and 6.6. In contrast, the growth of YS873 was significantly decreased when the pH of LB was 6.6. A loss-of-function mutation in zwf allows for YS873 to grow well in LB broth at a pH of 6.6.

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Abstract

La présente invention porte sur des salmonelles mutantes msbB résistantes au CO2, comprenant une mutation d'un gène codant pour une enzyme dans la voie des pentose-phosphates. Dans un mode de réalisation, la mutation est dans le gène zwf codant le glucose-6-déshydrogénase. L'invention porte également sur un procédé d'utilisation des salmonelles mutantes msbB résistantes au CO2 afin d'empêcher la croissance ou afin de réduire le volume d'une tumeur solide.
PCT/US2009/065970 2008-11-26 2009-11-25 Salmonelles résistantes au co2 à ciblage tumoral WO2010062982A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050255088A1 (en) * 1997-09-10 2005-11-17 Vion Pharmaceuticals, Inc. Genetically modified tumor-targeted bacteria with reduced virulence

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050255088A1 (en) * 1997-09-10 2005-11-17 Vion Pharmaceuticals, Inc. Genetically modified tumor-targeted bacteria with reduced virulence

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LUNDBERG ET AL.: "Glucose 6-Phosphate Dehydrogenase Is Required for Salmonella typhimurium Virulence and Resistance to Reactive Oxygen and Nitrogen Intermediates.", INFECTION AND IMMUNITY, vol. 67, no. 1, January 1999 (1999-01-01), pages 436 - 438 *
PLATT ET AL.: "Antitumour effects of genetically engineered Salmonella in combination with radiation.", EUROPEAN JOURNAL OF CANCER, vol. 36, no. ISS.18, December 2000 (2000-12-01), pages 2397 - 2402 *

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