Patent Application
20180333439
The present invention relates to inactivated or attenuated pathogenic bacteria, compositions comprising the bacteria, and use thereof for treating cancers such as urinary and prostate cancers.
Bladder cancer is the fourth most common cancer in men both in Europe and USA. The high and rising incidence rates in patients over 65 years suggest that the burden of this cancer will increase with population aging.
Use of bacteria to enhance immunity and treat cancer was pioneered in 1891 by William Coley, who successfully treated sarcomas with extracts of pyogenic bacteria (Coley, W. B. 1910, Proc R Soc Med. 3, 1-48).
The Mycobacterium bovis Bacillus Calmette-Guerin (BCG) vaccine is a standard treatment for superficial bladder cancer together with chemotherapy. BCG-induced tumor elimination involves a local immune response with massive release of cytokines and a delayed type immune response predominantly through Thl cells.
Natural Killer (NK) cells are cells of the innate immune system that kill tumor and viral-infected cells Killing by NK cells is mediated mainly through the Natural Cytotoxic Receptors (NCRs) which recognize ligands present on the surface of target cells. The expression levels of the NCR ligands on bladder tumors can predict the effectiveness of BCG treatment on bladder cancer in humans (Yutkin et al. J Urol 178, 2660-2664). In a murine model, successful BCG treatment of bladder tumor was found to be NK-cells dependent and was completely ineffective in NK-deficient mice (Brandau et al., Int J Cancer 2001, 92, 697-702). BCG therapy for non-muscle invasive bladder cancer reduces the risk of recurrence by 32% and the odds of tumor progression by 27%. However, immunotherapy with BCG is greatly hampered by its toxicity and severe adverse reactions that result in discontinuous treatment in 10-58% of the patients. Though attenuated, BCG is still a virulent microbe and its inoculation in the patient's bladder often leads to serious life threatening infections. BCG can infect the lungs, liver, bone, and the circulatory system. This infection is hard to treat due to the slow response of BCG to antibiotics. In addition, BCG is a tedious (high maintenance), slow growing microbe (doubling time of 23 h).
Urinary tract infection (UTI), caused by urinary pathogenic Escherichia coli (UPEC) is a very common disease and approximately 50% of women will have at least one episode of UTI at some point of their lives. Natural Killer (NK) cells recognize various pathogens, however, their involvement in UPEC-mediated UTI is practically unknown. Lately, the inventors of the present invention showed that UPEC adhere to NK cells primarily via their type I fimbriae and kill NK cells by the hemolysinA toxin. In the absence of hemolysinA, NK cells directly respond to the bacteria and secrete TNF-α which results in reduction of bacterial burden in the infected bladders (Gur et al., 2013. Cell Host Microbe 14, 664-674).
WO 2005/120560 discloses methods of treating cancers of a specific organ, tissue or cell in a subject by administering an antigen of one or more pathogenic bacterial species that are pathogenic in the specific organ, tissue or cell.
EP Pat. No. 1765391 discloses a preparation of killed bacteria of one or more pathogenic bacterial species, or a cell wall extract, a cell membrane extract, a whole cell extract, or an isolated antigen thereof for use in treating a cancer in a specific organ or tissue in a subject.
Radford et al. (Gene Therapy, 2002, 9:1455-1463) describes that immunization of mice by direct injection of recombinant E. coli expressing listeriolysin O (tumor antigen) and ovalbumin (OVA) provides an anti-tumor response, resulting in complete protection of 75% of mice which are challenged with OVA-expressing melanoma cell line (B16-OVA).
There is an unmet need for additional approaches with improved effectiveness for treating cancer, having fewer or no side effects.
The present invention is directed to specific types of E. coli bacteria for treating urogenital cancer. The present invention in some embodiments provides mutated hemolysin deficient bacterial strains derived from urinary pathogenic Escherichia coli and compositions comprising same for treating cancer. The present invention in some embodiments further provides inactivated urinary pathogenic E. coli for treating cancer. In particular, the bacteria disclosed herein are useful for attenuating or inhibiting tumor growth and even inducing tumor regression. The present invention further provides methods of treating cancer that comprise administering mutated or inactivated pathogenic E. coli directly to the site of the tumor.
The present invention is based in part on the unexpected discovery that treating bladder tumor bearing mice with hemolysin mutated E. coli strains significantly improves the survival of the mice. In addition, pathogenic E. coli bacterial strains that were inactivated by formaldehyde showed highly potent anti-bladder cancer activity. Furthermore, the bacterial strains of E. coli when directly administered into the bladder, showed greatly superior anti-cancer activity when compared to Mycobacterium bovis Bacillus Calmette-Guerin (BCG) and Helicobacter pylori. It is now disclosed that treatment with bacteria according to the principles of the invention markedly reduced the tumor burdens in mice bearing bladder cancer. Unexpectedly, although the treatments started after the mice already had visible tumors, the tumors regressed and, remarkably, most tumors were eliminated within several weeks.
Without being bound by any particular theory or mechanism of action, the anti-cancer effect of the hemolysin deficient bacteria may be due to decreased cytotoxicity towards NK cells. Urinary pathogenic Escherichia coli, when inoculated into the bladder recruit natural killer cells to the bladder, adhere to the NK cells and kill them. When administered into the bladder, the hemolysin deficient bacteria of the invention recruit the NK (and T cells) to the bladder but do not kill, or kill with less efficiency, the NK cells. It is now disclosed for the first time that a mutation in the hemolysin gene hlyA is sufficient for eliciting an improved anti-cancer activity of uropathogenic E. coli (UPEC) harboring the mutation.
The present invention further discloses that strains of uropathogenic E. coli (UPEC), and enteropathogenic E. coli (EPEC) inactivated by formaldehyde, exhibits a highly potent anti-bladder cancer activity. Unexpectedly, the formaldehyde inactivated UPEC and EPEC had highly efficient anti-cancer effect on mice bearing visible and large tumors. The survival rate of these mice was remarkably high.
The present invention further provides advantageous methods of treating cancer comprising intravesicular administration of bacteria. The therapeutic activity of the bacteria was highly potent when administered directly into the bladder of mouse models of bladder cancer.
According to one aspect, the present invention provides a mutant hemolysin deficient bacterial strain derived from a pathogenic strain of E. coli or component thereof for use in treating a cancer of the genitourinary system.
According to some embodiments, the hemolysin deficient bacterial strain is a urinary or enteropathogenic mutated strain of E. coli. According to certain embodiments, the mutated bacterial strain is derived from uropathogenic E. coli (UPEC). According to other embodiments, the mutated bacterial strain is derived from enteropathogenic E. coli (EPEC).
According to some embodiments, the mutant hemolysin deficient E. coli bacterial strain or a component thereof is for use in treating urogenitary cancer by intravesicular administration. According to exemplary embodiments, the hemolysin deficient bacterial strain or component thereof is for local administration at or in proximity to the cancer site.
According to some embodiments, the mutated bacterial strain has a mutation in the hly gene.
According to some embodiments, the hly gene is selected from the group consisting of: hlyA, hlyC, and hlyD. Each possibility represents a separate embodiment of the invention.
According to some embodiments, the mutant hemolysin deficient bacterial strain has a reduced amount of hemolysin by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% compared to the wild type. Each possibility represents a separate embodiment of the invention.
According to some embodiments, the mutant hemolysin deficient bacterial strain has hemolysin having reduced activity by at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to the wild type. Each possibility represents a separate embodiment of the invention.
According to some embodiments, the mutant hemolysin deficient bacterial strain is attenuated.
According to additional aspect, the present invention provides an inactivated uropathogenic Escherichia coli strain CFT073 or component thereof for use in treating a cancer of the genitourinary system. According to some embodiments, the treating comprises intravesicular administration.
According to additional aspect, the present invention provides an inactivated pathogenic bacterial strain of E. coli or a component thereof for use in treating urogenitary cancer by intravesicular administration. According to exemplary embodiments, the inactivated pathogenic bacterial strain or component thereof is for local administration at or in proximity to the cancer site.
According to some embodiments, the inactivated pathogenic bacterial strain is uropathogenic E. coli (UPEC). According to other embodiments, the inactivated pathogenic bacterial strain is enteropathogenic E. coli (EPEC).
According to additional aspect, the present invention provides an E. coli bacterial strain for use in treating a cancer of the genitourinary system, wherein the bacterial strain has type I fimbria, the bacterial strain is selected from the group consisting of: (i) a mutant hemolysin deficient bacterial strain derived from a pathogenic strain of E. coli; (ii) an inactivated UPEC; and (iii) an inactivated EPEC. Each possibility represents a separate embodiment of the invention.
According to some embodiments, the UPEC strain is CFT073.
According to additional aspect, the present invention provides an E. coli bacterial strain for use in treating a cancer of the genitourinary system, wherein the bacterial strain overexpresses type I fimbria.
According to some embodiments, the cancer of the genitourinary system is selected from the group consisting of: bladder cancer, prostate cancer, kidney cancer and urethral cancer. Each possibility represents a separate embodiment of the invention. According to certain embodiments, the cancer of the genitourinary system is a bladder cancer. According to additional exemplary embodiments, the cancer is non-muscle-invasive urothelial carcinoma of the bladder.
According to some embodiments, the bacterial component is selected from the group consisting of a protein, a membrane and a cell wall fraction. Each possibility represents a separate embodiment of the invention. According to certain embodiments, the component is Type 1 fimbria. According to other embodiments, the component comprises Lipopolysaccharides (LPS).
According to some embodiments, the bacterial strain has type I fimbria.
According to some embodiments, the bacterial strain or component thereof is for use in combination with an anticancer agent.
According to some embodiments, the bacterial strain or component thereof is for use in combination with another type of pathogenic bacterial strain in treating a cancer of the genitourinary system.
The bacterial strain can be administered per se or as a composition comprising the bacteria. According to some embodiments, the bacterial strain is for weekly administration. According to certain embodiments, the bacterial strain is for weekly administration at a unit dosage form comprising from 107 to 1012 bacterial cells. According to certain embodiments, the bacterial strain is for weekly administration at a unit dosage form comprising from 107 to 1012 for a period of at least 2 weeks.
The present invention further provides a mutant hemolysin deficient bacterial strain derived from uropathogenic Escherichia coli (UPEC), being a mutant of Escherichia coli CFT073, wherein the mutant has an insertion in the hemolysin toxin gene, and wherein the mutant is selected from the group consisting of: C93 having an insertion in the hlyA gene, E49 having an insertion in hlyC and D57 having an insertion in hylD. According to some embodiments, the mutant bacterial strain is C93 having an insertion in the hlyA gene.
According to additional aspect, the present invention provides a pharmaceutical composition comprising the bacterial strain and a pharmaceutical acceptable carrier.
According to some embodiments, the pharmaceutical composition is formulated for intravesicular administration.
The present invention further provides a method of treating a cancer of the genitourinary system, the method comprising administering to a subject in need thereof a mutant hemolysin deficient bacterial strain derived from a pathogenic strain of E. coli bacterial strain or component thereof.
The present invention further provides a method of treating a cancer of the genitourinary system, the method comprising administering to a subject in need thereof an inactivated Escherichia coli uropathogenic strain or component thereof. According to some embodiments the inactivated strain is CFT073. According to particular embodiments the inactivation is by formaldehyde.
The present invention further provides a method of treating bladder cancer, the method comprising intravesicularly administering to a subject in need thereof an inactivated pathogenic E. coli. According to some embodiments, the administered inactivated E. coli has type I fimbria.
Other objects, features and advantages of the present invention will become clear from the following description, examples and drawings.
The present invention provides inactivated pathogenic bacterial strains and use thereof for treating urogenital cancers. The present invention further discloses methods of treating cancers of the genitourinary system using a mutated hemolysin deficient bacterial strain. In addition, methods of treating cancer that comprise the step of directly administering of a mutated or inactivated pathogenic bacterial strain to the cancer site strain are provided.
The methods of the present invention are advantageous over previously known methods for using bacteria in that their ability to depress cells of the immune system is reduced or neutralized. The bacteria of the invention serve as potent and effective agents that recruit the immune system against cancerous cells. The bacteria of the invention may serve as a universal therapy for bladder cancer patients. The therapy does not depend on the adaptive immune system, requiring an early exposure to the bacteria. Without wishing to be bound by any theory regarding the mechanism of action, the bacteria of the invention act locally and recruit immune cells and/or immune related proteins into the cancer site. Moreover, the bacteria of the invention, when intravesicularly administered to mice, do not necessarily activate a systemic immune response, hence having minimal side effects. In addition, the bacteria of the invention proved to be efficient when the bacteria are inactivated. Overall, the bacterial strains disclosed herein seem very promising as an efficacious medication for treating a variety of cancers without causing undesired side effects.
The inventors of the present application have shown that urinary pathogenic Escherichia coli adhere to human and murine natural killer (NK) cells primarily through their type I fimbriae and kill NK cells via their hemolysinA toxin. Three strains of urinary pathogenic E. coli, mutated in the hemolysinA operon and termed ‘C93’ (mutated in hlyA), ‘E49’ (mutated in hlyC), and ‘D57’ (mutated in hlyD) were prepared. The C93 mutated E. coli strain was found to improve the survival of mouse models of bladder cancer. The hlyA gene encodes for the hemolysinA toxin structural protein. In some embodiments hlyA accession number is AE014075.1. The hlyC encodes for the hemolysin-activating lysine-acyltransferase and in some embodiments hlyC Gene ID: 1789689. The hlyD encodes for hemolysin translocator protein. According to some embodiments, the hemolysin is hemolysinA toxin.
The term “hemolysin deficient” refers to a bacterium that completely lacks hemolysin or has a reduced amount of hemolysin by at least 30%, 40%, 50%, 60%, 70%, 80% or 90% compared to the wild type bacterial strain. The term “hemolysin deficient” further refers to a mutated or functionally inactive form of the hemolysin, wherein the mutated form has a reduced activity by at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to the wild type form. The wild type form of the bacterial strain has active hemolysin.
The term “pathogenic bacterial strain” refers to any bacterial strain that can cause infection.
The terms “urinary pathogenic” and “uropathogenic” are used herein interchangeably.
The term “bacterial strain component” refers to a component of the bacterial strain that is capable of exhibiting an anti-cancer activity in a similar manner as the bacterial strain. Non limiting examples for the component are protein, membrane, and cell wall fraction. According to some embodiments, the bacterial component is an immune stimulating component.
According to certain embodiments, the component is type I fimbria. Type 1 fimbriae are adhesion organelles expressed by many Gram-negative bacteria. They facilitate adherence to mucosal surfaces, particularly to the bladder surface, and inflammatory cells, and contribute to the pathogenesis of E. coli in the urinary tract.
The term “has type I fimbria” refers to a bacterial strain that has an active or functioning type I fimbria.
According to some embodiments, the bacterial strain is a mutant strain that overexpresses type I fimbria. As used herein, the term “overexpress” refers to a bacterium that is genetically modified to produce type I fimbria in an amount that is greater than the amount that is produced in an unmodified bacterium. Overexpression can be achieved by mutating the protein to produce a more active form or a form that is resistant to inhibition, by removing inhibitors, or adding activators, and the like. Overexpression can also be achieved by removing repressors, adding multiple copies of the gene to the cell, or up-regulating the endogenous gene, and the like. The type I fimbria overexpression may be produced as described in Chelsea Lane et al. (Journal Of Bacteriology, 2007, Vol. 189, No. 15. p. 5523-5533).
According to certain embodiment, the component comprises Lipopolysaccharides (LPS). Lipopolysaccharides, also known as lipoglycans and endotoxins, are large molecules consisting of a lipid and a polysaccharide composed of 0-antigen, outer core and inner core joined by a covalent bond
The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
The terms “urogenital cancer” and “cancer of the genitourinary system” are used herein interchangeably and refer to any cancer of the genital or urinary system. Non-limiting examples include bladder cancer, kidney cancer, urethral cancer and prostate cancer.
As used herein, the term “subject” refers to any animal, such as a mammal like a dog, cat, livestock, and preferably a human
The term “treating” a subject for a disease, as used herein, is intended to encompass curing, as well as ameliorating at least one symptom of the disease.
The term “intravesicular” or “intravescial distillation” are used herein interchangeably and refer to a therapy method, in which the drug is inserted directly to the bladder, preferably by catheter.
The term “mutant” as used herein refers to the bacteria of the invention that have been mutated one or more times in, for example, one or more of the hly genes or any other genes that participate in the hemolysin synthesis or expression pathway.
According to some embodiments, the bacterial strain is attenuated or inactivated.
According to some embodiments, the present invention provides attenuated pathogenic bacteria. The term “attenuated,” as used herein, describes the diminution in the ability of the bacteria to cause disease in an animal as a whole, e.g., as measured by the LD.sub.50 of the bacteria. More specifically, the pathogenic characteristics of the attenuated bacteria strain have been lessened compared with wild-type bacteria, although the attenuated bacteria are capable of growth and maintenance in culture. An attenuated strain of bacteria according to the present invention is thus one which does not kill an animal to which it is administered, or is one which kills the animal only when the number of bacteria administered is vastly greater than the number of wild type non-attenuated bacteria which would be required to kill the same animal
According to other embodiments, the bacteria of the invention are inactivated. The term inactivated bacterial strain designates “not live” or “killed” bacteria.
According to some embodiments, the bacterial strain is inactivated by exposure to a fixative compound. According to some embodiments, the bacterial strain is inactivated by a compound selected from the group consisting of: formaldehyde, paraformaldehye, glutaraldehyde, and derivatives thereof. Each possibility represents a separate embodiment of the invention.
Without being bound by any theory or mechanism, it is speculated that formaldehyde inactivation is superior to other inactivation methods in preserving type I fimbria function. It is speculated that the type I fimbria enable the bacterial strain to attach to the bladder wall and not be washed out.
The hemolysin deficient bacteria may be generated by any method as known in the art. A diverse variety of different mechanisms for generating such bacteria are described herein. According to some embodiments, genes that are involved in hemolysin synthesis are mutated. According to exemplary embodiments, the genes are mutated so as to reduce or eliminate their expression. According to additional embodiments, the hemolysin function is altered by providing inhibitors.
According to some embodiments, the hemolysin deficient bacterial strain is a mutated bacterial strain. The term “mutant” as used herein refers to an amino acid, DNA or RNA sequence that is differs from wild type sequence. The mutants of the invention include insertion, deletion and/or substitution of nucleotides in the DNA of the bacterial strain, with the proviso that the mutant/s affects the expression and/or activity of hemolysin. The term “mutant” also refers to any modification of DNA or protein that affects the expression and/or activity of hemolysin.
The term “insertion mutation” refers to the addition of one or more nucleotide base pairs into a DNA sequence.
According to some embodiments, the bacterial strain has a deletion mutation. It is preferred that mutations in the above described genes be introduced as deletions since this will preclude reversion mutations and enhance the safety of the strains containing them. It is preferred to use a scar-less mutation, which refers to a mutation that leaves minimum exogenous sequence (“scar”). In particular, it is important to delete the resistance marker used for the deletion.
According to some embodiments, the hemolysin deficient bacterial strain is a stable hemolysin-A deleted (AhlyA) UPEC mutant.
According to certain embodiments, the hemolysin deficient bacteria have only a single type of hly mutation, as described above. In yet other embodiments, the bacteria have two or more of the specific hly mutations.
The hemolysin deficient bacterial strain may have attenuating mutations that improve their safety and suitability for human treatment. The aro genes are required in the biosynthesis of aromatic amino acids and the metabolic intermediate chorismate. Mutations in aro genes are attenuating because the mutants cannot synthesize chorismate, which is required in turn to synthesize folate (not produced by mammals), the major methyl donor in many biosynthetic reactions. Chorismate is also required for the synthesis of enterobactin, a protein involved in the in vivo acquisition of iron. OmpF and OmpC are outer membrane porins that mediate osmoregulation in E. coli. Live Enterotoxigenic Escherichia coli (ETEC) attenuated by deletions of the genes coding for aroC, ompC and ompF, and the genes for the enterotoxins heat-labile enterotoxin (LT), heat-stable enterotoxin (ST) and Heat-stable enterotoxin 1 (EAST), is used as a commercially available oral vaccine known as ACAM2017 (Acambis plc, Cambridge, UK) that was recently shown to be safe for use even in HIV infected humans. Therefore, it would be useful to attenuate the bacteria by mutating the above described genes. According to some embodiments, the hemolysin deficient bacterial strain is further mutated in at least one gene selected from the group consisting of aroC, ompC and ompF genes. Each possibility represents a separate embodiment of the invention.
According to additional aspect, the present invention provides a mutated hemolysin deficient bacterial strain involved in bladder infection or component thereof for use in treating a cancer of the urinary system.
According to additional aspect, the present invention provides a hemolysin deficient E. coli strain or component thereof for use in treating a cancer of the urinary system or prostate cancer. According to some embodiments, the hemolysin deficient E. coli strain or component thereof is for use in treating bladder cancer.
According to additional aspect, the present invention provides an inactivated uropathogenic E. coli (UPEC) for use in treating a cancer of the urogenital system.
According to additional aspect, the present invention provides an inactivated enteropathogenic E. coli for use in treating a cancer of the urogenital system.
According to another aspect, the present invention provides a method of treating a cancer of the urogenital system in a subject in need thereof, comprising directly administering to the bladder of said subject a mutant hemolysin deficient bacterial strain or component thereof. According to some embodiments, the bacterial strain is E. coli. According to certain embodiments, the bacterial strain is UPEC. According to other embodiments, the bacterial strain is EPEC.
According to some embodiments, the bacterial strain exhibits at least one biological activity selected from the group consisting of: stimulating the antitumor cytolytic activity of lymphocytes and/or NK cells; recruiting T-cells and neutrophils; stimulating proliferation of NK cells; and inducing regression of established tumors and/or of primary solid tumors. Each possibility represents a separate embodiment of the invention.
According to additional aspect, the present invention provides a method of treating a bladder cancer, the method comprising directly administering to the bladder of a subject in need thereof an inactivated Escherichia coli strain CFT073 or component thereof. According to some embodiments, the method comprises a local administration at or in proximity to the cancer site.
According to additional aspect, the present invention provides a method of treating a cancer of the urogenital system in a subject in need thereof, comprising administerin a E. coli bacterial strain, said bacterial strain overexpresses type I fimbria.
According to Additional aspect, the present invention provides a pathogenic E. coli bacterial strain for use in treating a cancer of the genitourinary system, wherein the bacterial strain overexpresses type I fimbria.
According to Additional aspect, the present invention provides a pathogenic E. coli bacterial strain for use in treating a bladder cancer, wherein the bacterial strain overexpresses type I fimbria.
According to some embodiments, the bacterial strain is inactivated. According to certain embodiments, the bacterial strain is inactivated by formaldehyde.
Administration
The bacteria of the invention can be administered using any known practice. In some embodiments, the bacterial strain is administered to the patient parenterally. According to some embodiments, parenteral administration is administration intravesicularly, intravenously, intramuscularly, intraperitoneally, intradermally, buccally, transdermally, intradermally, intraviterally or subcutaneously. Each possibility represents a separate embodiment of the invention.
According to exemplary embodiments, the bacterial strain of the invention is intravesicularly administered. In certain embodiments, the bacterial strain is administered directly, or adjacent to the site of the cancer.
The present invention provides in some embodiments pharmaceutical compositions comprising the bacterial strain or component thereof and a pharmaceutically acceptable carrier, excipient or diluent.
The term “pharmaceutical composition” as used herein refers to any composition comprising at least one pharmaceutically active ingredient, formulated such that it facilitates accessibility of the active ingredient to the target organ.
The compositions of the invention may include the whole bacteria, or may include extracts and/or components such as cell wall or cell membrane extracts that exhibits the anticancer activity according to the invention.
The compositions of the invention may be administered alone or in combination with other compounds, and in the present of an adjuvant, buffer or carrier. The buffer or carrier include, but not limited to, water for injection or physiological saline.
According to some embodiments, the bacterial strain or component thereof is for use in combination with an anticancer agent.
According to some embodiments, the composition is formulated for intravesicular administration. According to certain embodiments, the bacterial strain or composition comprising the bacterial strain is provided in a dosage form. According to some embodiments, the dosage form is administered in an amount selected from 1 ng/kg to 1 g/kg, 10 ng/kg to 0.1 g/kg, 50 ng/kg to 10 mg/kg, 0.1 μg/kg to 1 mg/kg, and 0.2 μg/kg to 0.2 mg/kg body. Each possibility represents a separate embodiment of the invention. It may be appropriate to administer the required dose as two, three, four or more sub-doses at appropriate intervals throughout the day.
According to some embodiments, the treatment period is at least 1 week, at least 2 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, least 6 months, or at least 1 year. In some embodiments the treatment period is from 1 day to 1 week, from 1 week to 4 weeks, from 1 month to 3 months, from 3 months to 6 months, from 6 months to 1 year, or for over a year. Each possibility represents a separate embodiment of the invention.
According to some embodiments, from 106 to 1012 microorganisms total may be administered to the patient in a given dosage form. According to certain embodiments, from 107 to 1010 microorganisms total may be administered to the patient in a given dosage form. According to additional embodiments, an effective amount may be provided in an amount of from 0.5 ml to 40 ml of the bacterial composition having from 106 to 1011 bacteria per ml.
Any of the preparations described herein may be administered as a single treatment or as multiple treatments, such as once a week for several weeks, or the composition may be administered intermittently according to a set schedule, e.g., once weekly, once monthly, or when the patient relapses from the primary illness.
Hemolysin Deficient Mutants:
Construction of UPEC CFT073 (deposited under ATCC®700928™) Transposon Library: EZ::TnKan transposomes (Gur et al., Cell Host Microbe. 2013 Dec. 11; 14(6):664-74) were purified from the pMODKan plasmid according to manufacturer's instructions and electroporated into UPEC CFT073 bacteria harboring a GFP-expressing pCM18 plasmid. The library was screened to identify the genes involved in NK cells killing by incubating the mutants for 3 hours with NK cells at 37° C. More than 1000 mutated clones were screened. Three mutants were identified, having an intact adhesion ability, but impaired killing activity. To determine the transposon location genomic DNA was purified from the selected clones (GenElute, Sigma) and restricted with HpaI, MfeI, and NdeI (New England Biolabs) which do not cleave within the inserted transposon. The resulting fragments were then self-ligated (TaKaRa) and used as a template for inverse PCR (Herculase II, Agilent) for amplification of the transposons' flanking sequences. The generated sequences were blasted against the CFT073 genome to determine the location of the insertion mutation. The disrupted genes of the three mutants were identified and were found to reside in the hlyA operon that encodes for the hemolysinA toxin: ‘C93’ (mutated in hlyA), ‘E49’ (mutated in hlyC), and ‘D57’ (mutated in hlyD) (Gur et al., Cell Host Microbe. 2013 Dec. 11; 14(6):664-74).
Transitional Cell Cancer (TCC):
MB49 bladder cancer cells line, derived from C57/B16 mice were injected/implanted intravesically into female mice using a protocol described by Günther et al. (Cancer Research, 2834-2837, 59 Jun. 1, 1999). Briefly, female mice (10-12 mice per group), 8-10 weeks age were anesthetized with Ketamine/Xalazine. An area of about 1 cm2 on the back of the animal was shaved and then the mice were placed on a ground plate of the cautery unit. The urinary bladder was catheterized with Teflon catheter 25-gauge and a soft tipped guide wire 24-gauge (0.7×19 mm) was inserted until it touched the bladder wall. The guide wire was attached to the cautery unit and monopolar coagulation current was applied for 5 seconds at the lowest power setting. After removal of the wire, 50 μl of DMEM MB49 tumor cell suspension (30000 tumor cells per mouse) were instilled. Then, the catheter was clamped and left in the bladder for 2-3 hours while the mouse is anaesthetized. MB49-luc stably transfected with luciferase were used, enabling the monitoring of the tumors with CCCD camera.
Intravesical Treatment:
Two days following tumor cells implementation, the mice were randomized into 6 groups and received various treatments through intravesicl instillation once a week. The bacteria or PBS were injected through catheters inserted into the bladders.
Group 1: Treatment with PBS. The first intravesicl instillation was 3 days following TCC inoculation, than every week (5 injections total).
Group 2: Treatment with PBS as described for group 1 and anti-NK1.1 (mAb PK136, by intraperitoneal injection) twice a week (on day 1, on day 4 and then every week for 6 weeks).
Group 3: Treatment with 50 μl containing 109/ml C93 bacteria. The first intravesicl instillation was 3 days following TCC inoculation, than every week (5 injections total).
Group 4: Treatment with 50 μl containing 109/ml C93 bacteria as described for group 3 and anti-NK1.1 (mAb PK136) twice a week (on day 1, on day 4 and then every week for 6 weeks).
Group 5: Treatment with an Onco-Tice® BCG strain (2-8×108 CFU per vial). The vial was reconstituted in 3 ml of normal saline and 0.05 ml were injected per mouse. The first intravesicl instillation was 3 days following TCC inoculation, than every week (5 injections total).
Group 6: Treatment with an Onco-Tice® BCG strain as described above in combination with anti-NK1.1 (mAb PK136) twice a week (on day 1, on day 4 and then every week for 6 weeks).
The mice were examined daily on the first week and then 3 times a week. The tumor progression was monitored weekly using CCCD imaging. For Luciferase assay/CCCD imaging in vivo the mice were anesthetized with Isofluran. The luciferin was injected IP in an amount of 125 μg/g body weight and the imaging was performed in 5-10 minutes.
All mice were sacrificed on day 62 after tumor implementation using ketamin/xylasin anesthesia with subsequent neck dislocation.
Experimental Procedure:
Day −1: Examining MB49 tumor cell line for luciferase and NK1.1 injection (100 microgram per mouse) for mice of groups 2, 4, and 6.
Day 0: tumor implementation.
Day 3: Treatment with PBS, C93, or BCG as described above and NK1.1 injection (100 microgram per mouse) for mice of groups 2, 4, and 6.
For the remaining 5.5 weeks after tumor implantation:
Results:
To examine the effect of hemolysin deficient bacteria on cancer, bladder cancer was induced in mice, and the survival rate and tumor size of mice treated with C93 cells were measured. C57BL mice (10 mice per group), were inoculated in the bladder with MB49 bladder carcinoma cells. As described above the mice were treated with PBS, C93, or BCG, with or without NK1.1 mAb, antibodies that deplete NK cells.
As shown in
UPEC CFT073 were exposed to formaldehyde (4% in PBS) overnight, washed three times and brought to OD=1 (approximately 109 cells/me.
To test whether all bacteria were killed by the formaldehyde treatment, 100 μl of the OD=1 suspension were plated in 1:1, 1:10, and 1:100 dilutions and incubated overnight at 37° C. No growth was detected, confirming bacterial inactivation.
The amount of residual formaldehyde in the bacterial preparation was measured using the Formaldehyde VACUettes Kit (Catalog No. K-4605D) chemetrics (VA. USA) and found to be 0-5 ppm (0.0005%) (n=3).
Next, the effect of formaldehyde inactivated bacteria on the survival rate of mouse models of bladder cancer was examined MB49 bladder carcinoma cells were inoculated into the bladder of C57BL mice. Treatments started ten days after cancer inoculation. Mice were treated weekly (days marked with arrows;
To further and examine the effect of inactivation by formaldehyde, the survival rate of mouse models of bladder cancer treated with UPEC or EPEC which were inactivated by formaldehyde was examined MB49 bladder carcinoma cells were inoculated into the bladder of C57BL mice. The mice were treated weekly with 3×108 cells/mouse UPEC (CFT073 FA), EPEC (EPEC FA), and Helicobacter pylori, starting day 10 after TCC inoculation.
As shown in
The ability of UPEC bacteria to recruit immune cells was examined. 1×108 of formaldehyde inactivated UPEC CFT073 or Helicobacter pylori were inoculated into the bladder of mice. Two days later the mice were sacrificed and NK and T cells were quantified by flow cytometry. As showed in
Next, the cytokins/chemokines response profile following bacterial inoculation was examined 1×108 of formaldehyde-inactivated (FA) UPEC CFT073, E. coli XL1 blue (laboratory strain of E. coli), Helicobater pylori or BCG were inoculated into the bladders of each mouse. After 2 days the mice were sacrificed and bladder cytokines/chemokines were quantified using a cytokine array. Remarkably, the amounts of several cytokines and other immune related molecules, such as IL-1ra and sICAM-1, were significantly higher in bladders inoculated with formaldehyde inactivated (FA) UPEC when compared to inoculation with other types of bacteria (
Overall, these results suggest that inactivated UPEC induce a local immune response.
Next, a comparison between formaldehyde inactivation and heat shock inactivation was performed. Mice bearing MB49 bladder carcinoma cells were treated weekly with formaldehyde inactivated UPEC strain CFT073 or C93 mutant, and CFT073 or C93 strains that were killed by heat. The procedure was performed as described in example 1. Control mice received PBS.
To examine the effect of route of administration, mice were inoculated intravesicularly or subcutaneously with 108 formaldehyde inactivated UPEC CFT073 (N=5 per group). After 48 hours bladders were harvested and Peripheral Blood Lymphocytes (PBLs) were extracted from the bladders and analyzed using cell cytometry.
As shown in
Bacterial Membrane and Membrane Protein Extraction:
Bacterial cells are grown overnight and washed in PBS and resuspended in PBS supplemented with 3 mM PMSF (Sigma-Aldrich, Germany) A French press (8,000 psi×3 times) is used to disrupt the cells, and unbroken cells are removed by sedimentation at 10,000×g at 4° C. for 10 minutes.
Alternatively, cells are disrupted using glass beads. Cell suspensions are placed in 2 ml microfuge tubes containing 600 μl of glass beads (106 μm, Sigma). Cells are then disrupted two to four times using the FastPrep cell disruptor (Bio 101, Savant Instruments, Inc., NY, USA) at a speed of 6 m/s for 45 s, with ice-cooling between disruptions. The lysates are centrifuged at 10,000×g for 5 min
The supernatant is collected and subjected to high-speed centrifugation (150,000×g at 4° C. for 1 hour), and the resulting pellet containing the cell walls is washed twice, resuspended in sodium phosphate buffer and kept at −80° C. until used. To determine proteins amount and integrity, a sample of each pelleted membrane is boiled in SDS-PAGE sample buffer for 10 minutes, and fractioned using SDS-PAGE (12% and 4%) to visualize protein bands.
Outer Membrane Vesicle Preparation:
Stationary cultures are harvested by centrifugation at 10,000×g for 20 min at 4° C. Culture supernatants are collected and filtered through a 0.2 μm filter (Whatman Schleicher & Schuell). Cell-free supernatants are centrifuged at 100,000×g for 2 hr. The supernatant is discarded and the pellet containing the vesicles is washed twice with Tris-buffered saline (TBS, 0.05 M Tris-HCl [pH 7.8], 0.1 M NaCl) by centrifugation at 100,000×g. The pellet is stored at −20° C. until further use.
Bacterial Lysate:
Bacterial cells are grown overnight, washed in PBS and resuspended in PBS supplemented with 3 mM PMSF (Sigma-Aldrich, Germany)
Cell suspensions are placed in 2 ml microfuge tubes containing 600 μl of glass beads (106 μm, Sigma). Cells are disrupted two to four times using the FastPrep cell disruptor (Bio 101, Savant Instruments, Inc., NY, USA) at a speed of 6 m/s for 45 s, with ice-cooling between disruptions. The lysates are centrifuged at 10,000×g for 5 min and the supernatant fluid is collected.
The bacterial lysate bacterial protein extract and membrane fraction are administered to mouse model of bladder cancer as describe in the previous examples. The survival rate and tumor size are examined
A clinical study is designed for assessing the effect of the bacterial strains on bladder cancer patients. The primary objective is to assess efficacy and safety of the formaldehyde inactivated Uropathogenic E. coli CFT073 given as six intravesical instillations of 1010 inactivated-CFU (or cells) per instillation. The primary efficacy objective is to determine the ability of the treatment to prevent the tumor recurrence after 6 weekly intravesical instillations of the tested product. The recurrence is assessed 8-10 weeks following commencement of the treatment. The primary safety objective is to determine adverse effects induced by the tested product. Secondary objectives include determining the long term (one year) freedom of the cancer recurrence and time to tumor recurrence in patients free of tumor who did not recur after 8-10 weeks.
Study Design:
Subject recruitment—The patients are recruited through the urologic oncology clinic in Hadassah Medical Center, Israel.
Subject inclusion criteria—Patients that underwent complete transurethral excision of recurrent, intermediate to high risk for recurrence non-muscle invasive bladder cancer (NMIBC), and have a history of at least one course of adjuvant immunotherapy or chemotherapy intravesical treatment prior to this recurrence.
Subject Exclusion Criteria:
i. Pregnant or breast feeding women
ii. T1 High grade urothelial carcinoma of bladder
iii. Concomitant upper tract urothelial carcinoma
iv. TCC in bladder diverticulum or prostatic urethra
v. Muscle invasive UC
vi. Metastatic UC
vii. Karnofsky's performance status <60
viii. Known immunodeficiency status
ix. Active urinary tract infection
x. other active malignancy
The patients that consented to participate in the study start the treatment within 4-5 weeks after a Transurethral resection of the bladder tumour (TURBT). The patients are treated with weekly intravesical instillation of formaldehyde-inactivated UPEC for 6 weeks. The product is given through 12 French Tiemann urethral catheter and patients are asked to refrain from voiding for 2 hours.
Subject Follow-Up:
Urine culture, urinalysis, cystoscopy and urine cytology tests are performed 2 months following the last intravesical treatment and every 4 months for 2 years thereafter.
aHematology includes CBC count, differentials, and platelets.
bBlood chemistries include: 1) liver function tests: ALT/SGPT, AST/SGOT, total bilirubin, alkaline phosphatase, albumin, and total protein, 2) renal function tests: creatinine and BUN, and 3) electrolytes: calcium, potassium, sodium, and chloride.
cOnly females of child-bearing potential will have a pregnancy test. A serum pregnancy test will be performed at the start of screening and a urine pregnancy test will be performed within 7 days prior to the start of treatment.
dAEs will be assessed at each study visit before any study procedures are performed as well as after study procedures are performed.
Formulation and Preparation:
Fresh colonies of CFT073 are grown in LB medium overnight. 1010 CFU of bacteria are pelleted by centrifugation at 4° C., 6000 rpm for 5 minutes and washed twice with 30 ml of PBS. The pellet is re-suspended in 10 ml 4% formaldehyde and then the bacteria are incubated overnight at 4° C. for inactivation. The inactivated bacteria are pelleted by centrifugation at the same conditions, washed three times in 30 ml PBS, re-suspended in PBS, and brought to a concentration of 109 cells/ml (OD=1, 600 nm). The final product is labeled as CFT-FA (formaldehyde inactivated UPEC CFT073).
The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IL2015/051085 | 11/10/2015 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62077483 | Nov 2014 | US | |
| 62088747 | Dec 2014 | US |
