FORM 2
THE PATENTS ACT, 1970 (39 of 1970)
PROVISIONAL SPECIFICATION
(See Section 10)
A NOVEL BIOMEDICAL DEVICE FOR CANCER THERAPY
AMRITA THERAPEUTICS Ltd.,
C/O Ayurnet Health Care Ltd located, 304 Regency Plaza, Near Rahul Tower
Cross Roads, Satellite, Ahmedabad-380015, India, Indian National
The following specification describes the invention

This invention relates to biomedical device for cancer therapy.
FIELD OF THE INVENTION:
The present invention is in the field of medical devices. Particularly, the invention relates to in-dwelling therapeutic device. The therapeutic device may be stent or catheter. The invention further relates to a therapeutic device with biofilms of microorganisms useful in cancer therapy. The microorganisms may be attenuated in their virulence factors and with cloned genes encoding specific proteins with anticancer activity. The biofilm-containing devices may be encased in membranes that allow diffusion of proteins with molecular masses of 25, 50, 75 or 100 kDa but not live biofilm organisms.
Background:
Live bacteria that have been used for treating cancers, primarily in animal models, are strictly anaerobic such as Clostridia and Bifidobacteria or facultative anaerobic such as Listeria monocytogenes or Salmonella typhimurium (1, 2). Al-R strain of S. typhimurium had significant tumor regressing effect, such as that of orthotopic human pancreatic tumors, in nude mice. In addition, Al-R strain was also shown to significantly reduce the metastasis of lung and high grade osteosarcoma in nude mice as well as spinal cord gliomas (3, 4). Genetically improved avirulent mutants had also been shown to retain the tumor targeting properties (4) and will be excellent candidates for further testing in humans. However, so far, earlier phase I human clinical trials have not shown significant effects in colonization or cancer regression in metastatic melanoma, due to certain toxicity associated with injections of live bacteria in humans (5). Such injections trigger both innate and adaptive immune responses for clearance of the foreign bacteria, thereby enhancing side effects in already debilitated patients.
The reasons bacteria such as Salmonella, Listeria, Clostridia and others allow cancer regression are believed to be due to the activation of the immune system elicited by the bacteria as well as active growth of the bacteria in the hypoxic regions of the core of the tumors. Usually, virulence deletion mutants are rapidly cleared from the host

compared to the wild type. So lack of toxicity in the blood stream has a negative effect on the retention and proliferation of live bacteria in the host tumors. For example, mutants attenuated in actA and plcB genes in Listeria monocytogenes have been shown to have a reasonable safety profile in adult volunteers (5), although no tumor regression effects were studied.
Phase I human clinical trials of a live, attenuated L. monocytogenes strain in 15 patients with recurrent metastatic squamous cell carcinoma of the cervix at doses of 1X109, 3.3X109 or 1X1010 cfu showed toxicity with flu-like syndrome such as fever, chills, nausea and/or vomiting/headache (6). Such symptoms are typical of administration of IL-2, indicating the involvement of innate immune response to the i.v. infusions of live bacteria.
The use of Clostridia in cancer therapy has shown efficacy in tumor regression, but with considerable toxicity. Consequently, Clostridial strains have been used more as diagnostic agents for detection of tumors because of their high specificity for targeting tumor cells (7).
A very promising Clostridial strain, C. novyi-NT (8) was used as spores in phase I human clinical trials in 2006 (NCT 00358397, www.clinicaltrials.gov), but the study was suspended for safety issues, again demonstrating the problem of using live bacteria through the i.v. or i.m. route.
From the above discussion, it is interesting to note that most of the efforts, lasting for more than 60 years, for cancer therapy basically involve anaerobic bacteria. Yet, the most successful practical use of bacteria in cancer therapy involves the use of an aerobe, Mycobacterium bovis BCG, the vaccine strain for tuberculosis, to treat superficial urothelial carcinoma of the bladder. Live BCG cells are administered through a urethral catheter directly into an emptied bladder. The catheter is then removed, leaving the live bacteria in the bladder, which then induce an immune reaction in the bladder, leading to tumoricidal activity (9, 10). However, BCG therapy

for bladder cancer is associated with a variety of complications, ranging from minor cystitis to life-threatening BCG sepsis, occurring in up to 90% of patients (11, 12).
As of today, there are no live bacteria, other than M. bovis BCG which has a very limited application only in superficial bladder cancer therapy, that have gone past phase I/II human clinical trials successfully and none is on the horizon, given the toxicity associated with intravenous injections of live bacteria in the blood stream evoking strong immune reaction. Attenuated bacteria, with or without cloned genes, get cleared by the immune system quickly, thereby reducing their effectiveness and staying power in the tumor vicinity.
A major problem in the application of live bacteria in cancer therapy (1, 2) is a lack of understanding of how bacteria actually cause tumor regression. Spectacular antineoplastic effect, when C. novyi-NT was used along with mitomycin C and dolastatin-10, instead of just mitomycin C and dolastatin-10, certainly points out to the active involvement of bacteria in tumor regression and lysis (13). Similarly, the exquisite targeting of tumor and growth in the core of the tumors is known to be a hallmark of Salmonella'*s ability to cause tumor shrinkage (2, 4, 14). However, such therapies are often known as immunotherapy because the tumor regression is believed to be due to bacteria's preferential growth in the hypoxic core of the tumor, thereby depriving the tumor cells of nutrients, and eliciting immune action that causes tumor regression.
Do certain bacteria actually fight tumors by elaborating anticancer agents, rather than simply targeting tumors or invoking a strong immune response? Indeed, an enzyme arginine deiminase (ADI) produced by Mycoplasma arginini has been shown as early as in 1990 to have anti-tumor activity (15). Since then, many studies including phase I/II human clinical studies, have been performed with ADI and a polyethylene glycol (PEG)-conjugated ADI, termed ADI-PEG20, in patients with hepatocellular carcinoma and malignant melanoma (16). In general, such results have shown modest anticancer effect of ADI-PEG20 in such patients with tolerable side effects and the clinical trials are continuing with additional patient recruitment (16). ADI action is believed to be due

to depletion of arginine in such cancer cells as hepatocellular carcinoma, melanoma or renal cell carcinoma which do not express arginino-succinate synthetase in vivo,
The successful use of a bacterial protein, ADI, rather than live bacteria in human clinical trials raises an interesting question: do other bacteria produce similar proteins or small molecule compounds to fight cancer? Arginine deiminase is not unique to Mycoplasma, including M. arginini. The ADI from Pseudomonas aeruginosa, an aerobic opportunistic pathogen, has also been shown to have anticancer activity against a range of cancers such as fibrosarcoma, breast and ovarian cancers (17). Most interestingly, a 17 kDa truncated N-terminal part of ADI, called Pa-CARD because it harbors a caspase recruitment domain (CARD), has even higher anticancer activity than ADI in such cancers and in liquid-borne cancers such as chronic myeloid leukemia (CML) or acute myeloid leukemia (AML) cell lines (17, 18).
Pseudomonas aeruginosa, not previously known or tried in any live cell anticancer drug development, not only produces ADI, but another protein with significant anticancer activity called azurin (19). Azurin has not only anticancer activity against a range of cancers, but also strong growth suppressing activity against viruses such as the HIV/AIDS virus HIV-1 or parasites such as the malarial parasite Plasmodium falciparum or the toxoplasmosis parasite Toxoplasma gondii (19). Most interestingly, azurin, which is an intracellular periplasmic protein, is secreted by P. aeruginosa when P. aeruginosa cells are exposed to cancer cells, suggesting that azurin is a weapon that P. aeruginosa uses to keep cancer cells in check. Pseudomonas aeruginosa is a biofilm-forming extracellular pathogen that prefers to establish long term residence in human tissues without causing much harm to the host normal cells but becomes very protective of the host as its habitat and has developed promiscuous protein weapons to target other invaders of the human body such as cancers, viruses and parasites (19). A similar azurin-like protein, termed Laz, is produced by gonococci/meningococci which also retains such activity against cancers, HIV-1, P. falciparum and T, gondii (19). A 28 amino acid peptide derived from azurin, azurin 50-77 termed p28, has no toxicity or imrnunogenicity in animals, including non-human primates (20). In phase I human clinical trials in Chicago, p28 (NSC 745104, www.clinicaltrials.gov) has shown no side

effects in 10 advanced cancer patients where no drug is working and who have an average life expectancy of 8 weeks. p28 has shown significant beneficial effect in such patients in a dose dependent manner without demonstrating any side effects.
The therapeutic efficacy of azurin in mouse xenograft models, and the lack of side effects of the p28 peptide with anticancer activity in both animal and phase I human clinical trials raises an important question: can live cells of P. aeruginosa fight cancer similar to the anaerobic or facultative anaerobic bacteria such as Clostridia, Salmonella, etc, as mentioned earlier? As a known pathogen, P. aeruginosa, even attenuated strains lacking its major virulence factors such as Exotoxin A, elastase, and other toxins such as ExoS, ExoT and ExoU, that are injected in host cells by a type III secretion mechanism, will evoke strong immune response resulting in considerable toxicity when given intravenously. A unique feature of many pathogenic bacteria such as P. aeruginosa, Streptococcus and many others is their ability to form biofilms which allows surface adhesion of such bacteria on biotic or abiotic surfaces and acquisition of enough nutrients to allow a slow mode of growth (21, 22). Indeed, bio film formation of P. aeruginosa in both the lungs of cystic fibrosis patients and in-dwelling medical devices is well known for its pathogenesis, allowing the biofilm bacteria to resist both immune attack and antibiotic treatment (21, 22). It is also conceivable that P. aeruginosa biofilms in an in-dwelling medical device such as a catheter or stent inserted near a tumor can keep the tumor growth in check through elaboration of weapons such as azurin. This approach can contribute to effective cancer therapy, alone or in combination with common anticancer drugs.
Since azurin is a small 14 kDa protein secreted in presence of cancer cells, are there other pathogenic bacteria with long term residence in human bodies that can secrete small (less than 25 to 30 kDa) protein with anticancer activity? Having access to such bacterial protein weapons, with inhibitory activity against a range of cancers, viruses, parasites and pathogenic bacteria, perhaps even multiply drug resistant ones, will not only provide us with promiscuous, multi-disease-targeting drugs, but will provide a general principle of method development for isolation of such potential drugs. Indeed, we have recently described the isolation of a 17 kDa protein, MPT63, produced and

secreted by Mycobacterium tuberculosis and M. bovis BCG, and a 30 amino acid peptide derived from MPT63, termed MB30, that, similar to azurin-p28, have strong anticancer activity against a range of cancers such as bladder, colon, etc (23). Similar to azurin, MPT63 demonstrates promiscuity by strongly inhibiting the growth of the HIV/AIDS virus HIV-1. The production of such a protein weapon by M. bovis BCG, widely used in the treatment of superficial bladder cancer as mentioned earlier (9, 10), clearly suggests that bacterial regression of cancer is not just due to growth inside tumors or induction of an immune response, as is widely believed (24), but is due to active participation by the microorganisms in secreting or exposing on the surface various protein or other weapons to suppress invasion and growth of a variety of invaders of the human body including cancers, viruses, parasites and pathogenic bacteria/fungi. It is thus quite possible that anaerobic/facultative anaerobic bacteria such as Clostridia, Salmonella, etc, mentioned earlier, may produce and secrete similar weapons to fight cancers and other invaders. This kind of an approach will allow them to be used as biofilms on catheters/stents or other in-dwelling devices and inserted in the vicinity of the tumor, singly or in combination with other in-dwelling medical devices harboring other types of bacteria as biofilms. The protein weapons can also be isolated and used as diagnostic agents and/or therapeutic compounds. The in-dwelling devices containing the biofilms of P. aeruginosa, M. tuberculosis/M. bovis, Clostridia, Salmonella, etc, either wild type or attenuated by mutations/deletions in various genes contributing to their virulence, can be used as is, or preferably encased by membrane filters with pore sizes that allow diffusion of only molecules with a mass of 25 kDa or 40 kDa. For example, a catheter with attenuated P. aeruginosa harboring deletions in exotoxin A, elastase (lasA/lasB) and type III secretion systems (but with or without hyperexpression of the azurin gene), and encased by a membrane filter with a cut-off molecular mass of 25 kDa will allow secretion and diffusion of azurin to reach the tumor but not any residual toxins with molecular mass higher than 25 kDa. In none of the cases, the biofilm bacteria can escape from the catheter to reach the blood stream but remains very close to the tumor to sense its presence. Such an approach greatly reduces any toxicity associated with an immune response, as is normally observed with live bacteria given orally, intravenously or by other means. Multiple catheters with

multiple different bacterial biofilms will produce a synergistic effect without concomitant side effects, in absence or in presence of treatments with other drugs.
OBJECTIVES OF THE INVENTION:
The main objective of the present invention is to provide a biomedical device for cancer therapy
Specifically the objective of the invention is to provide an in-dwelling therapeutic device.
The other objective is to provide a therapeutic device that may be a stent or a catheter.
Yet another objective is to provide a therapeutic device with biofilms of microorganisms useful in cancer therapy.
The device may be made of any physiologically acceptable material comprising plastic, ceramic, wood, metal, polymer (natural or synthetic) or hydrogel.
Still another objective is to provide a therapeutic device containing live Pseudomonas aeruginosa biofilms. Pseudomonas aeruginosa may be wild type, or virulence compromised or mutated to reduce toxin and/or cloned with the azurin gene under a strong promoter to hyperexpress this gene.
The mutations may be in genes encoding type III secretion and Exotoxin A and the elastase genes las A/las B that are important virulence factors for P. aeruginosa. For other biofilms comprising of other bacteria such as Salmonella, Clostridia, M. bovis BCG, etc, in the in-dwelling device, appropriate attenuated strains may be used.

Yet another objective is to insert a therapeutic device at the tumor site wherein the biofilm microorganisms be encased in a membrane surrounding the device. The membrane may be provided with pore sizes capable of preventing diffusion of macromolecules with masses greater than 25 KDa to 40 kDa. Such membranes with defined pore sizes will prevent the release of any bacteria from the biofilm to the blood stream. It is to be noted that while biofilm bacteria are resistant to antibiotics or immune attack, in the absence of membrane encasing, any released bacteria from the device will be susceptible to antibiotics or immune attack, facilitating their clearance.
SUMMARY OF THE INVENTION:
Accordingly the present invention provides a therapeutic device containing live microorganisms, either wild type or attenuated, for cancer therapy.
In one of the embodiment, the device may be a stent or catheter or any other similar means that can be implanted at tumor site.
In other embodiment, the device could be made of any physiologically acceptable material that can facilitate growth of microorganisms, such as plastic, ceramic, wood, metal, polymer (natural or synthetic) or hydrogel.
In yet other embodiment, the therapeutic device may harbor biofilms of organisms such as Pseudomonas aeruginosa. Pseudomonas aeruginosa may be wild type, or virulence compromised or mutated to eliminate toxin production, and with cloned azurin gene under a strong promoter to hyperproduce this anticancer protein.
The mutations may be in genes encoding type III secretion and Exotoxin A and the elastase genes las A/las B.
In yet another embodiment, the therapeutic device may contain microorganisms encased in a membrane and the membrane may be provided with pore sizes capable of preventing diffusion of macromolecules of various sizes such as 25 kDa to 40 kDa.

The biofilms may be composed of different sets of bacteria such as Salmonella, Clostridia, M. bovis BCG, etc. Such medical devices can be used in conjunction with other traditional anticancer drug treatment.
DETAILED DESCRIPTION:
There are emerging reports on the use of live and virulence attenuated bacteria for the treatment of cancers. Few bacterial species such as Salmonella, Clostridia, Mycobacterium bovis, when injected intravenously, intramuscularly or other means, have the ability to enter into the human tumors and allow tumor regression. Mycobacterium bovis BCG is used in the treatment of superficial bladder cancer and is thought to induce cancer regression through activation of the immune system.
However, there is no report on the use of live wild type or attenuated Pseudomonas aeruginosa for the treatment of cancers.
In prior art, the organisms being administered parenterally via injections or orally pose problem in reaching and targeting the poorly vascularized tumors, which have no or low level oxygen. Additionally, eradication of the disseminated/migrated organisms to other parts of the body is another problem. Further, these pathogenic organisms, even when attenuated, evoke strong immune response, leading to toxicity problems.
The therapeutic device of the present invention having confinement of microorganisms encased in the membrane of the device eliminates the problem of dissemination in other parts of the body or blood stream and toxicity development. Further the right choice of microorganism, attenuated Pseudomonas aeruginosa with mutations/deletions in various toxin genes helps in solving certain problems of accumulation of toxins. The devices with bacterial biofilms without any membrane encasement do not pose any problems because any bacteria released from the biofilm will be susceptible to antibiotics or immune attack, while the biofilm bacteria will remain resistant.

The applicant had recently described the production of anti-cancer proteins, or peptides derived from them, such as azurin, arginine deiminase (ADI), Pa-CARD and azurin-p28 that allow tumor regression both in vitro and in vivo in mice (1, 17, 18, 19). Although, as described earlier, live bacteria such as Clostridia, Salmonella, etc., have been shown to allow tumor regression, there is no report on the use of live or attenuated Pseudomonas aeruginosa for the treatment of cancers. Pseudomonas aeruginosa is known to form biofilms on the abiotic or biotic surfaces and can grow under microaerophilic conditions. The use of a stent, catheter or other similar therapeutic device that contains biofilm bacteria of the wild type or virulence compromised P. aeruginosa strains may provide potential and viable treatment of cancers in humans. The stent, catheter, hydrogel or other similar therapeutic device containing P. aeruginosa is administered in the human body at the site of the tumors or near to the tumors where the anticancer proteins or small molecules secreted by the bacterium allow regression of solid tumors. The use of wild type or virulence compromised P. aeruginosa is an excellent way to treat a variety of human cancers. For example, a patient with prostate cancer may have a catheter containing P. aeruginosa biofilms in the urovesicular region near the tumor. While the P. aeruginosa persists in the catheter, the patient's condition will be relatively stable with the tumor either stabilized or undergoing shrinkage. Additional therapy with anticancer drugs will help in the complete elimination of the tumor.
Further, it is clear that such a stent, catheter, or similar therapeutic device with P. aeruginosa also may be encased in membranes with pore sizes that allow diffusion of only 25 Kda to 40 kDa macromolecules. Because the P. aeruginosa toxins such as Exotoxin A or ExoS/T are larger than 40 Kda, the live cells of P. aeruginosa or their released toxins cannot come out of the catheter to enter the tumors or surrounding tissues, leaving the release of low molecular weight anticancer proteins such as azurin to fight cancer.
Here, we focus on the use of plastic, ceramic, wooden or metal stent, catheter, or similar therapeutic device containing a biofilm of the live bacterium Pseudomonas aeruginosa that is either wild type or genetically modified to eliminate its pathogenicity and to be inserted at the site of the tumors to allow tumor regression in cancer patients.

The strain of the P. aeruginosa in the biofilm could be laboratory or clinical isolates of wild-type bacteria or laboratory-derived mutants of such bacteria defective in the production of toxins such as Exotoxin A, Elastase or toxins elaborated through the type III secretion system (ExoS, ExoT, ExoU, etc). P. aeruginosa may have cloned azurin gene under a strong promoter to hyperexpress azurin gene with its signal sequence to allow secretion of the azurin.
Other biofilm-forming bacteria such as Streptococcus, Staphylococcus, Mycobacterium bovis BCG, etc, also may be used in place of P. aeruginosa.
The cancers to be treated by the live cells of P. aeruginosa or other biofilm-forming bacteria as part of catheters or stent surfaces are prostate, melanoma, sarcoma, breast, lung, ovarian, kidney, cervical, liver, bladder, colon, pancreas or other tumor types. The use of catheter/stent or similar therapeutic device containing P. aeruginosa or other bacterial biofilms inserted at the site of the tumor(s) is a treatment modality that can be given singly or in conjunction with other treatments such as radiation, chemotherapy or the emerging treatments with bacterial proteins or peptides given via intravenous/intra muscular routes.
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