CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority of Singapore Patent Application No. 10201508832Q, filed 26 October 2015, the contents of which being hereby incorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION

[0002] The present invention lies in the field of biochemistry and relates to a non- viable bacterial derivative for use as a medicament and a composition comprising at least one bacterial protein for use as a medicament. The present invention also relates to a non-viable bacterial derivative of the invention or a composition of the invention for use in the treatment of cancer, a method for disturbing the integrity of the extracellular matrix (ECM) of a cell, a non-viable bacterial derivative and to a composition comprising at least one bacterial protein.

BACKGROUND OF THE INVENTION

[0003] Colorectal carcinoma, characterized by the uncontrolled growth of cells in the epithelial tissue of the large intestine, is the third most common cancer in men, second most common cancer in women around the world and the second highest leading type of cancer deaths in the United States ¹ . However, existing forms of cancer treatment are limited in their efficacy. Surgery is the first line of treatment for colorectal cancers detected in their early stage, but it is ineffective against the advanced stages of cancer ^{2 3} . The tumor microenvironment plays a key role in limiting the efficacy of other conventional forms of cancer treatment, such as chemotherapy and radiation therapy (RT). The necrotic (anoxic) core and hypoxic region are key features of the tumor microenvironment. As oxygen and the nutrient flow do not reach these parts of the tumor, their concentrations are much lower here than in normal tissues ^4,5 . RT involves the use of ionizing radiation to curb the growth of cancer cells by forming free-radical debris of DNA. Oxygen molecules react with the free-radical DNA debris to make the DNA damage permanent and bring about cell death. This makes the efficacy of RT heavily dependent on the presence of oxygen and thus, intra-tumoral hypoxia greatly curbs the effectiveness of RT in treating tumors ⁶ . Hypoxia also compromises on the efficacy of chemotherapy. There are various reasons for this. Firstly, these hypoxic tumor regions are located far away from the blood vessels, preventing the delivery of chemotherapeutic drugs to cells ^{7 8} . Secondly, some drugs such as melphalan ^{9 10} , bleomycin ¹¹ and etoposide ^{12 13} require cellular oxygen to bring about cell death and are therefore ineffective in hypoxic conditions. Finally, alkylating agents and anti-metabolite anti-cancer drugs only act against rapidly proliferating cells and because hypoxia slows down the cell-cycle, these drugs cannot effectively cause cancer cell death either ^{7 14} . The limitations of existing cancer treatment methods have led to a pressing need to explore alternative treatment methods that will overcome the hypoxic barrier of tumors and be effective in targeting cancer.

[0004] Bacterial cancer therapy has the potential to overcome these limitations and provide a viable alternative to existing treatment modalities ¹⁵ . The hypoxic conditions of the tumor microenvironment, that are a huge obstacle for RT and chemotherapy, were recognized as a potent tool for bacterial cancer therapy. This is because such conditions are perfect for the growth of anaerobic bacteria, which accumulate and proliferate in the hypoxic regions of the tumor before their natural cytotoxicity induces cancer cell death ^{16 17} . Clostridial strains have been at the center of bacterial tumor therapy since the 19 ^th century because of the ability of their spores to selectively germinate in the hypoxic cores of tumors ^{18 19} . Clostridium sporogenes, a proteolytic species, is reported to have a superior ability of tumor colonization ²⁰²¹ . Wild-type clostridial spores have been found to exert oncolytic effects on tumors ^{22 23} , clostridial spores combined with other cancer therapies were found to have an enhanced anti-cancer effect ^{24 25} , and genetically modified clostridial species have also been used in Clostridium-directed enzyme prodrug therapy (CDEPT) ¹⁶ ' ²⁰ ' ^{21 26} . Secreted bacterial products that are found in the conditioned media (CM) of bacterial cultures have been investigated and several studies have shown that they also have an anti-tumor effect ^{27 28} . Amongst clostridial products, Clostridium perfringens enterotoxin (CPE) has been studied extensively and found to interact with claudin-3 and -4 receptors that are overexpressed in many types of tumors, to trigger cancer cell death ^{29 30} . Despite these advances, clostridial cancer therapy has not gained widespread acceptance as a potential treatment method.

[0005] This is because clostridial cancer therapy has limitations of its own. The administration of this spore-forming pathogenic bacteria poses a high risk of infections and toxicity to patients after the spores have germinated. Several studies involving the administration of clostridial spores to tumor-bearing animals resulted in their death due to bacterial infections or the toxicity of the bacteria ¹⁶ ' ^{31 32} . In two recent studies C. novyi-NT spore treatment, one involving canines and the other involving canines and a human patient with a leiomysarcoma, the administered dose of the spores caused a severe case of toxicosis ^33,34 . Additionally, using anaerobic bacteria also leaves a viable rim of tumor cells which increases the chance of tumor recurrence ²² ' ^{35 37} . Another point to note is that in vitro studies done in the field of bacterial cancer therapy are performed using 2-dimensional (2D) cancer cell cultures ^{38 39} . This leads to a potential issue of reliability as these 2D cultures are not representative of in vivo tumor conditions, thus making the findings of such studies less physiologically relevant ^{40 42} .

[0006] There is a need in the art to develop a form of bacterial cancer therapy that minimizes the risk of infection and which is tested on a physiologically relevant in vitro platform, before advancing on to in vivo studies.

SUMMARY OF THE INVENTION

[0007] It is an object of the present invention to meet the above need by providing nonviable bacterial derivatives or a composition comprising at least one bacterial protein. The nonviable bacterial derivative or the composition can be efficiently used for the treatment of cancer. These therapeutic agents are non-infectious. In addition, they demonstrate significant inhibition of cancer growth in 3D cancer cell models.

[0008] Surprisingly, the present inventors have found that heat-inactivated C. sporogenes bacteria (IB) administered to colorectal cancer cells, such as CT26 and HCT116 cell lines, are able to inhibit cancer cell proliferation. Additionally, this effect was also observed using the (secreted) bacterial products of C. sporogenes in conditioned media. The anti-cancer effect was allocated to secreted proteins of the bacterium, including clostripain.

[0009] In a first aspect, the present invention is thus directed to a non-viable bacterial derivative for use as a medicament.

[00010] In various embodiments of the invention, the non-viable bacterial derivative is a heat, autoclave, ethanol, chlorine, monochloramine, chlorine dioxide or UV light inactivated bacterial cell, preferably a heat inactivated bacterial cell.

[00011] The scope of the present invention also encompasses various embodiments wherein the non-viable bacterial derivative is an inactivated Gram-positive bacterial cell.

[00012] In still further various embodiments of the invention, the non-viable bacterial derivative is an inactivated Clostridium cell, preferably an inactivated Clostridium sporogenes cell.

[00013] In a further aspect, the present invention relates to a composition comprising at least one bacterial protein for use as a medicament.

[00014] In various embodiments of the invention, at least one bacterial protein is naturally originated in a Gram-positive bacterial cell.

[00015] In various embodiments, the at least one bacterial protein is a secreted bacterial protein.

[00016] In still further various embodiments of the invention, the at least one bacterial protein is naturally originated in a Clostridium cell, preferably a Clostridium sporogenes cell.

[00017] The scope of the present invention also encompasses various embodiments wherein the composition comprises clostripain as set forth in SEQ ID NO: l or a derivative of clostripain having at least 75% sequence identity to clostripain determined overall the whole length of SEQ ID NO: l.

[00018] In a further aspect, the invention relates to a non-viable bacterial derivative of the invention or a composition of the invention for use in the treatment of cancer.

[00019] In various embodiments of the invention, the cancer is a carcinoma. In more preferred embodiments the cancer is colorectal cancer.

[00020] In a fourth aspect, the invention relates to a method for disturbing the integrity of the extracellular matrix (ECM) of a cell comprising contacting the cell comprising an extracellular matrix with the non-viable bacterial derivative of the invention or with the composition of the invention.

[00021] In various embodiments, disturbing the integrity of the extracellular matrix includes size regression of the extracellular matrix.

[00022] The scope of the present invention also encompasses various embodiments wherein the method is an in vivo method; or the method is an ex vivo method.

[00023] In a further aspect, the invention relates to a non-viable bacterial derivative, wherein the non-viable bacterial derivative comprises a heat inactivated Clostridium sporogenes cell.

[00024] In a sixth aspect, the invention relates to a composition comprising at least one bacterial protein, wherein the composition comprises clostripain as set forth in SEQ ID NO: l or a derivative of clostripain having at least 75% sequence identity to clostripain determined overall the whole length of SEQ ID NO: 1.

BRIEF DESCRIPTION OF THE DRAWINGS

[00025] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings. [00026] Figure 1 shows fluorescence image of cell viability of C. sporogenes after heat inactivation. DMAO stains bacterial cells green and EthD-III stains the non-viable bacterial cells red. (a) Bacteria before heat-treatment, (b) Bacterial cells after 80 °C heat inactivation for 2 hours. Scale bar represents 100 μπι.

[00027] Figure 2 shows effect of IB on 2D cell culture, (a) Cell proliferation with WST-1 assay of 2D culture of CT26 cells exposed to varying concentrations of inactivated bacteria. 0.1 OD = 2.4 x 10 ⁶ bacterial cells/ml. t-test is a comparison with control. (* p< 0.005) (b) Fluorescence microscopy with Calcein AM and Ethidium homodimer I (EthD-I), of cell viability of CT26 cells after they had been exposed to inactivated bacteria at varying concentrations for 24 hours. Image of cells in top left quadrant of each well. Scale bar represents 200 μπι.

[00028] Figure 3 shows effect of CM on 2D cell culture, (a) Cell proliferation with WST- 1 assay of 2D culture of CT26 exposed to 10% of Conditioned Media (CM) of C. sporogenes and Reinforced Clostridium Media (RCM). t-test is a comparison with control. (* p< 0.005) (b) Fluorescence assay of cell viability of CT26 cells after they had been exposed to 10% of RCM and CM for 24 hours. Image of cells in top left quadrant of each well. Scale bar represents 200 μπι.

[00029] Figure 4 shows H&E staining of 3D spheroids of CT26, at 72 hours. Arrows indicate necrotic regions formed within spheroid, stained only with eosin.

[00030] Figure 5 shows effect of IB on 3D spheroids, (a) Cell proliferation with WST-1 assay of CT26 and HCT116 spheroids exposed to 0.1 OD concentration of inactivated bacteria over 72 hours. 0.1 OD = 2.4xl0 ⁶ bacterial cells/ml. t-test is a comparison with control. (* p< 0.005) (b) Area of 3D spheroids after incubation with 0.1 OD of inactivated bacteria, at the 0, 24, 48 and 72 hour time points. The area (μπι ² ) of the spheroids in the images was measured using ImageJ software, t-test is a comparison with control (* p< 0.005, at all time points), (c) Effect of 0.1 OD of inactivated C. sporogenes on 3D spheroids, compared with Control. Scale bar represents 1000 μηι.

[00031] Figure 6 shows effect of CM on 3D spheroids, (a) Cell proliferation with WST-1 assay of CT26 and HCT116 spheroids exposed to 10% of Conditioned Media (CM) of C. sporogenes and Reinforced Clostridium Media (RCM). i-test is a comparison with control. (* p< 0.005). (b) Area of spheroids after incubation with 10% RCM, CM and BCM of C. sporogenes at the 0, 24, 48 and 72 hour time points. The area (μηι ² ) of the spheroids in the images was measured using ImageJ. t-test is a comparison with control (* p< 0.005, at all time points), (c) Effect of 10% CM of C. sporogenes on 3D spheroids, compared with Control, 10% RCM and 10% BCM. Scale bar represents 1000 μπι.

[00032] Figure 7 shows comparison between morphology of CT26 Control spheroid and spheroid exposed to IB after 24 hours. Images taken with scanning electron microscope (SEM). Arrows indicate elongated cells on the surface and the white arrow indicate deformation in the shape of IB -exposed spheroids.

[00033] Figure 8 shows SEM image of inactivated bacteria (IB) interacting with HCT116 cells. Black arrows indicate IB on cell surface.

[00034] Figure 9 shows HCT116 cells exposed to IB for 24 hours were stained with Annexin V/PI assay kit. Cells that have undergone apoptosis are stained green by Annexin V and those that have undergone necrosis are stained red by PI.

[00035] Figure 10 shows SDS-PAGE stained with coomassie blue of the sample. Excised protein band (indicated by dottedarrow) was sent for analysis and was positively identified as clostripain. A score of greater than 80 is regarded as significant.

[00036] Figure 11 shows IHC staining of Collagen- 1 of 3D spheroids.

[00037] Figure 12 shows IHC staining of Elastin of 3D spheroids.

DETAILED DESCRIPTION OF THE INVENTION

[00038] The present inventors surprisingly found that inactivated bacterial cells, such as heat inactivated C. sporogenes cells, or a composition comprising (secreted) bacterial proteins, such as clostripain from C. sporogenes, can be efficiently used for the treatment of cancer, such as colorectal cancer. The above agents are non-infectious. In addition, they demonstrate significant inhibition of cancer growth in 3D cancer cell models.

[00039] Therefore, in a first aspect, the present invention is thus directed to a non-viable bacterial derivative for use as a medicament.

[00040] By the term "non-viable", as used herein, is meant a population of bacteria that is not capable of replicating under any known conditions. However, it is to be understood that due to normal biological variations in a population, a small percentage of the population (i.e. 5% or less) may still be viable and thus capable of replication under suitable growing conditions in a population which is otherwise defined as non-viable.

[00041] The terms "bacteria" or "bacterial", as used herein include, but are not limited to, Gram positive and Gram negative bacteria. The term "bacteria" can include, but are not limited to, species of the genera Abiotrophia, Achromobacter, Acidaminococcus, Acidovorax, Acinetobacter, Actinobacillus, Actinobaculum, Actinomadura, Actinomyces, Aerococcus, Aeromonas, Afipia, Agrobacterium, Alcaligenes, Alloiococcus, Alter omonas, Amycolata, Amycolatopsis, Anaerobospirillum, Anaerorhabdus, Arachnia, Arcanobacterium, Arcobacter, Arthrobacter, Atopobium, Aureobacterium, Bacteroides, Balneatrix, Bartonella, Bergeyella, Bifidobacterium, Bilophila Branhamella, Borrelia, Bordetella, Brachyspira, Brevibacillus, Brevibacterium, Brevundimonas, Brucella, Burkholderia, Buttiauxella, Butyrivibrio, Calymmatobacterium, Campylobacter, Capnocytophaga, Cardiobacterium, Catonella, Cedecea, Cellulomonas, Centipeda, Chlamydia, Chlamydophila, Chromobacterium, Chyseobacterium, Chryseomonas, Citrobacter, Clostridium, Collinsella, Comamonas, Corynebacterium, Coxiella, Cryptobacterium, Delftia, Dermabacter, Dermatophilus, Desulfomonas, Desulfovibrio, Dialister, Dichelobacter, Dolosicoccus, Dolosigranulum, Edwardsiella, Eggerthella, Ehrlichia, Eikenella, Empedobacter, Enterobacter, Enterococcus, Erwinia, Erysipelothrix, Escherichia, Eubacterium, Ewingella, Exiguobacterium, Facklamia, Filifactor, Flavimonas, Flavobacterium, Francisella, Fusobacteriumi, Gardnerella, Gemella, Globicatella, Gordona, Haemophilus, Hafnia, Helicobacter, Helococcus, Holdemania Ignavigranum, Johnsonella, Kingella, Klebsiella, Kocuria, Koserella, Kurthia, Kytococcus, Lactobacillus, Lactococcus, Lautropia, Leclercia, Legionella, Leminorella, Leptospira, Leptotrichia, Leuconostoc, Listeria, Listonella, Megasphaera, Methylobacterium, Microbacterium, Micrococcus, Mitsuokella, Mobiluncus, Moellerella, Moraxella, Morganella, Mycobacterium, Mycoplasma, Myroides, Neisseria, Nocardia, Nocardiopsis, Ochrobactrum, Oeskovia, Oligella, Orientia, Paenibacillus, Pantoea, Parachlamydia, Pasteurella, Pediococcus, Peptococcus, Peptostreptococcus, Photobacterium, Photorhabdus, Plesiomonas, Porphyrimonas, Prevotella, Propionibacterium, Proteus, Providencia, Pseudomonas, Pseudonocardia, Pseudoramibacter, Psychrobacter, Rahnella, Ralstonia, Rhodococcus, Rickettsia Rochalimaea Roseomonas, Rothia, Ruminococcus, Salmonella, Selenomonas, Serpulina, Serratia, Shewenella, Shigella, Simkania, Slackia, Sphingobacterium, Sphingomonas, Spirillum, Staphylococcus, Stenotrophomonas, Stomatococcus, Streptobacillus, Streptococcus, Streptomyces, Succinivibrio, Sutterella, Suttonella, Tatumella, Tissierella, Trabulsiella, Treponema, Tropheryma, Tsakamurella, Turicella, Ureaplasma, Vagococcus, Veillonella, Vibrio, Weeksella, Wolinella, Xanthomonas, Xenorhabdus, Yersinia, and Yokenella. Examples of bacterial species include, but are not limited to, Clostridium sporogenes, Mycobacterium tuberculosis, M. bovis, M. typhimurium, M. bovis strain BCG, BCG substrains, M. avium, M. intracellular, M. africanum, M. kansasii, M. marinum, M. ulcerans, M. avium subspecies paratuberculosis, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus equi, Streptococcus pyogenes, Streptococcus agalactiae, Listeria monocytogenes, Listeria ivanovii, Bacillus anthracis, B. subtilis, Nocardia asteroides, Streptococcus viridans group, Actinomyces israelii, Propionibacterium acnes, Clostridium tetani, Clostridium botulinum, Pseudomonas aeruginosa, Vibrio cholera, Actinobacillus pleuropneumoniae, Pasteurella haemolytica, Pasteurella multocida, Legionella pneumophila, Salmonella typhi, Brucella abortus, Chlamydi trachomatis, Chlamydia psittaci, Coxiella burnetii, Escherichia coli, Neiserria meningitidis, Neiserria gonorrhea, Haemophilus influenzae, Haemophilus ducreyi, Yersinia pestis, Yersinia enterolitica, Escherichia coli, E. hirae, Brucella abortus, Burkholderia cepacia, Burkholderia pseudomallei, Francisella tularensis, Bacteroides fragilis, Fudobascterium nucleatum, and Cowdria ruminantium, or any strain or variant thereof.

[00042] The term "derivative", as used herein, relates to a bacterial cell that is not viable, meaning that it has not the capability to replicate, but still contains most of its natural components, such as proteins, peptides, amino acids, sugars, fats, nucleic acids etc. In various embodiments, the derivative contains at least 40% of the natural components contained in a viable cell. In more preferred embodiments, the derivative contains at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% of the natural components contained in a viable cell.

[00043] In various embodiments of the invention, the non-viable bacterial derivative is a heat, autoclave, ethanol, chlorine, monochloramine, chlorine dioxide or UV light inactivated bacterial cell, preferably a heat inactivated bacterial cell.

[00044] Several different methods to inactivate bacterial cells are well-known in the art. Chlorine, monochloramine, chlorine dioxide, and ultraviolet light inactivation is, for example, described by Rose and Rice (Rose LJ and Rice EW, J Water Health. 2014 Dec;12(4):618-33). Smelt and Brul, for example, describe heat inaction of bacteria (Smelt and Brul, Critical reviews in food science and nutrition 54(10): 1371-85, 2014). The skilled person is also well aware of the fact that inactivation parameters, such as temperature, time, intensity etc., may need to be adapted depending on the given bacterial genera, taxon, species or strain desired to be inactivated.

[00045] The term "bacterial cell", as used herein, refers to a prokaryotic organism, i.e. a bacterium. Bacteria can be classified based on their biochemical and microbiological properties as well as their morphology. These classification criteria are well known in the art. The bacterial cell referred to in accordance with the present disclosure is preferably from the genus of Clostridium.

[00046] The scope of the present invention also encompasses various embodiments wherein the non-viable bacterial derivative is an inactivated Gram-positive bacterial cell.

[00047] As used herein, the term "Gram-positive" means bacteria that retain the color or the crystal violet stain in the Gram stain. This is characteristic of bacteria that have a cell wall composed of a thick layer of a particular substance (called peplidologlycan). The Gram-positive bacteria include Clostridium, staphylococci ("staph"), streptococci ("strep"), pneumococci, and the bacterium responsible for diphtheria (Cornynebacterium diphtheriae) and anthrax (Bacillus anthracis), among others. [00048] In still further various embodiments of the invention, the non-viable bacterial derivative is an inactivated Clostridium cell, preferably an inactivated Clostridium sporogenes cell.

[00049] The term "Clostridium" , as used herein, relates to a genus of Gram-positive bacteria, which includes several significant human pathogens, including the causative agent of botulism and an important cause of diarrhea, Clostridium difficile. They are obligate anaerobes capable of producing endospores. The normal, reproducing cells of Clostridium, called the vegetative form, are rod-shaped, which gives them their name spindle. Clostridium endospores have a distinct bowling pin or bottle shape, distinguishing them from other bacterial endospores, which are usually ovoid in shape. Clostridium species inhabit soils and the intestinal tract of animals, including humans. Clostridium is a normal inhabitant of the healthy lower reproductive tract of women.

[00050] Clostridium contains around 100 species that include common free-living bacteria, as well as important pathogens. For example, the main species responsible for disease in humans are: Clostridium botulinum, which produces botulinum toxin in food or wounds and can cause botulism; Clostridium difficile, which can flourish when other gut flora bacteria are killed during antibiotic therapy, leading to superinfection and potentially fatal pseudomembranous colitis; Clostridium perfringens, which causes a wide range of symptoms, from food poisoning to cellulitis, fasciitis, and gas gangrene; Clostridium tetani, which causes tetanus; Clostridium sordellii, which causes a fatal infection in exceptionally rare cases after medical abortions. Bacillus and Clostridium are often described as Gram-variable, because they show an increasing number of Gram-negative cells as the culture ages.

[00051] Clostridium is in the division Firmicutes. Clostridium grows in anaerobic conditions, forms bottle-shaped endospores, does not form the enzyme catalase. Glycolysis and fermentation of pyruvic acid by Clostridia yield the end products butyric acid, butanol, acetone, isopropanol, and carbon dioxide.

[00052] The term "Clostridium sporogenes", as used herein, relates to a species of Gram- positive bacteria that belongs to the genus Clostridium. Like other strains of Clostridium, it is an anaerobic, rod-shaped bacterium that produces oval, subterminal endospores and is commonly found in soil. Unlike Clostridium botulinum, it does not produce the botulinum neurotoxins. In colonized animals, it has a mutualistic rather than pathogenic interaction with the host.

[00053] In a further aspect, the present invention relates to a composition comprising at least one bacterial protein for use as a medicament.

[00054] "At least one", as used herein, relates to one or more, in particular 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

[00055] The term "protein", as used herein, relates to one or more associated polypeptides, wherein the polypeptides consist of amino acids coupled by peptide (amide) bonds. The term polypeptide refers to a polymeric compound comprised of covalently linked amino acid residues. The amino acids are preferably the 20 naturally occurring amino acids glycine, alanine, valine, leucine, isoleucine, phenylalanine, cysteine, methionine, proline, serine, threonine, glutamine, asparagine, aspartic acid, glutamic acid, histidine, lysine, arginine, tyrosine and tryptophan.

[00056] The term "gene product", as used in the present invention, relates to a biochemical material, either RNA or protein, resulting from expression of a gene. Moreover, the proteins may form complexes with other proteins via covalent and non-covalent bonds.

[00057] In various embodiments, the at least one bacterial protein is a secreted bacterial protein.

[00058] The term "secretion" or "secreted", as interchangeably used herein, refer to translocation of a polypeptide or protein, specifically a protein of interest such as clostripain, across both the plasma membrane and the cell wall of a host cell. The secreted protein may be either part of the cell membrane as a membrane-bound protein that is anchored within the cell wall, or released as soluble protein to the cell supernatant.

[00059] In various embodiments of the invention, the at least one bacterial protein is naturally originated in a Gram-positive bacterial cell.

[00060] The term "natural", as used herein, means any compound, such as a protein, or form of matter that exists in or is derived from plants, animals, and/or other microorganisms as opposed to compounds or forms of matter that are artificial, synthetic and/or made by chemical synthesis or proteins that are not expressed by their natural origin organism. Such expression includes, but is not limited to heterologous expression.

[00061] "Origin", as used herein, refers to a cell, tissue or organism capable of expressing a protein of interest that naturally occurs in said cell, tissue or organism.

[00062] In still further various embodiments of the invention, the at least one bacterial protein is naturally originated in a Clostridium cell, preferably a Clostridium sporogenes cell.

[00063] The scope of the present invention also encompasses various embodiments wherein the composition comprises clostripain as set forth in SEQ ID NO: l or a derivative of clostripain having at least 75% sequence identity to clostripain determined overall the whole length of SEQ ID NO: l.

[00064] The term "clostripain", as used herein, relates to a protein having the enzyme commission number EC 3.4.22.8 (also known as clostridiopeptidase B, Clostridium histolyticum proteinase B, alpha-clostridipain, clostridiopeptidase, Endoproteinase Arg-C). Clostripain is a proteinase that cleaves proteins on the carboxyl peptide bond of arginine. The isoelectric point of the enzyme is 4.8-4.9 (at 8°C), and optimum pH is 7.4-7.8 (against a-benzoyl-arginine ethyl ester). The composition of the enzyme is indicated to be of two chains of relative molecular mass 45,000 and 12,500.

[00065] Preferably at least one protein of the composition of the invention has an amino acid sequence that has at least about 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least about 99% sequence identity to the sequence of SEQ ID NO: 1.

[00066] Methods for the alignment of sequences for comparison are well known in the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. The BLAST algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10) calculates percent sequence identity or similarity or homology and performs a statistical analysis of the identity or similarity or homology between the two sequences. The software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (NCBI). [00067] The sequence identity is determined over the whole length of the sequence set forth in SEQ ID NO: l. This means that even if the protein of interest contains parts having a higher degree of homology to SEQ ID NO: l, the overall length of SEQ ID NO: l is relevant to determine the degree of sequence identity.

[00068] In a further aspect, the invention relates to a non-viable bacterial derivative of the invention or a composition of the invention for use in the treatment of cancer.

[00069] In various embodiments of the invention, the cancer is a carcinoma. In more preferred embodiments the cancer is colorectal cancer.

[00070] The term "cancer," as used herein, refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Exemplary cancers include: carcinoma, melanoma, sarcoma, lymphoma, leukemia, germ cell tumor, and blastoma. More particular examples of such cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma, multiple myeloma and B-cell lymphoma, brain, as well as head and neck cancer, and associated metastases.

[00071] The term "carcinoma", as used herein, refers to an invasive malignant tumor consisting of transformed epithelial cells or transformed cells of unknown histogenesis, but which possess specific molecular or histological characteristics that are associated with epithelial cells, such as the production of cytokeratins or intercellular bridges. Exemplary carcinomas of the present application include colorectal cancer, ovarian cancer, vaginal cancer, cervical cancer, uterine cancer, prostate cancer, anal cancer, rectal cancer, colon cancer, stomach cancer, pancreatic cancer, insulinoma, adenocarcinoma, adenosquamous carcinoma, neuroendocrine tumor, breast cancer, lung cancer, esophageal cancer, oral cancer, brain cancer, medulloblastoma, neuroectodermal tumor, glioma, pituitary cancer, and bone cancer.

[00072] The term "lymphoma", as used herein, is a cancer of lymphatic cells of the immune system. Lymphomas typically present as a solid tumor. Exemplary lymphomas include: small lymphocytic lymphoma, lymphoplasmacytic lymphoma, Waldenstrom macroglobulinemia, splenic marginal zone lymphoma, plasmacytoma, extranodal marginal zone B cell lymphoma, MALT lymphoma, nodal marginal zone B cell lymphoma (NMZL), follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, Burkitt lymphoma, B cell chronic lymphocytic lymphoma, classical Hodgkin lymphoma, nodular lymphocyte-predominant Hodgkin lymphoma, adult T cell lymphoma, nasal type extranodal NK/T cell lymphoma, enteropathy-type T cell lymphoma, hepatosplenic T cell lymphoma, blastic NK cell lymphoma, mycosis fungoide, Sezary syndrome, primary cutaneous CD30- positive T cell lymphoproliferative disorders, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T cell lymphoma, unspecified peripheral T cell lymphoma, and anaplastic large cell lymphoma. Exemplary forms of classical Hodgkin lymphoma including: nodular sclerosis, mixed cellularity, lymphocyte -rich, and lymphocyte-depleted or not depleted.

[00073] The term "sarcoma", as used herein, is a cancer that arises from transformed cells in one of a number of tissues that develop from embryonic mesoderm. Thus, sarcomas include tumors of bone, cartilage, fat, muscle, vascular, and hematopoietic tissues. For example, osteosarcoma arises from bone, chondrosarcoma arises from cartilage, liposarcoma arises from fat, and leiomyosarcoma arises from smooth muscle. Exemplary sarcomas include: Askin's tumor, botryodies, chondrosarcoma, Ewing's-PNET, malignant Hemangioendothelioma, malignant Schwannoma, osteosarcoma, soft tissue sarcomas. Subclases of soft tissue sarcomas include: alveolar soft part sarcoma, angiosarcoma, cystosarcoma phyllodes, dermatofibrosarcomadesmoid tumor, desmoplastic small round cell tumor, epithelioid sarcomaextraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, hemangiopericytoma, hemangiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcomal, lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma, and synovial sarcoma.

[00074] The term "leukemia", as used herein, is a cancer of the blood or bone marrow characterized by an abnormal increase of white blood cells. Leukemia is a broad term covering a spectrum of diseases. In turn, it is part of the even broader group of diseases called hematological neoplasms. Leukemia is subdivided into a variety of large groups; the first division is between acute and chronic forms of leukemia. Acute leukemia is characterized by a rapid increase in the numbers of immature blood cells. Crowding due to such cells makes the bone marrow unable to produce healthy blood cells. Chronic leukemia is characterized by the excessive buildup of relatively mature, but still abnormal, white blood cells. Typically taking months or years to progress, the cells are produced at a much higher rate than normal cells, resulting in many abnormal white blood cells in the blood. Leukemia is also subdivided by the blood cells affected. This split divides leukemias into lymphoblastic or lymphocytic leukemias and myeloid or myelogenous leukemias. In lymphoblastic or lymphocytic leukemias, the cancerous change takes place in a type of marrow cell that normally goes on to form lymphocytes. In myeloid or myelogenous leukemias, the cancerous change takes place in a type of marrow cell that normally goes on to form red blood cells, some other types of white cells, and platelets. Combining these two classifications provides a total of four main categories. Within each of these four main categories, there are typically several subcategories. There are also rare types outside of this classification scheme. Exemplary leukemias include: acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), hairy cell leukemia (HCL), T-cell prolymphocytic leukemia, large granular lymphocytic leukemia, juvenile myelomonocytic leukemia, B-cell prolymphocytic leukemia, Burkitt leukemia, and adult T-cell leukemia.

[00075] The term "melanoma", as used herein, is a cancer or malignant tumor of melanocytes. Melanocytes are cells that produce the dark pigment, melanin, which is responsible for the color of skin. They predominantly occur in skin, but are also found in other parts of the body, including the bowel and the eye. Melanoma is divided into the following stereotypes and subtypes: lentigo maligna, lentigo maligna melanoma, superficial spreading melanoma, acral lentiginous melanoma, mucosal melanoma, nodular melanoma, polypoid melanoma, desmoplastic melanoma, amelanotic melanoma, soft-tissue melanoma, melanoma with small nevus-like cells, melanoma with features of a Spitz nevus, and uveal melanoma.

[00076] The term "germ cell tumor (GCT)" as used herein is a neoplasm derived from germ cells. Germ cell tumors can be cancerous or non-cancerous tumors. Germ cells normally occur inside the gonads (ovary and testis). Germ cell tumors that originate outside the gonads may be birth defects resulting from errors during development of the embryo. Germ cell tumors are broadly divided in two classes: germinomatous or seminomatous and nongerminomatous or nonseminomatous germ cell tumors. Exemplary germinomatous or seminomatous germ cell tumors include: germinoma, dysgerminoma, and seminoma. Exemplary nongerminomatous or nonseminomatous germ cell tumors include: Embryonal carcinoma, endodermal sinus tumor or yolk sac tumor (EST, YST), choriocarcinoma, mature teratoma, dermoid cyst, immature teratoma, teratoma with malignant transformation, polyembryoma, gonadoblastoma, and mixed GCT.

[00077] "Colorectal cancer," as used herein, refers to a disease characterized by unchecked proliferation of cells of the large intestine, including cells of the colon or rectum. Colorectal cancer typically originates in epithelial cells of the large intestine with intestinal crypt stem cells being a likely cell of origin. Genetic mutations that result in carcinogenesis include mutations in members of the Wnt signaling pathway such as β-catenin, APC, AXIN1, AXIN2, TCF7L2, and NKD1, members of the TGF-β cell signaling pathway such as TGF-βΙ and SMAD family members, proteins that regulate the balance between cell proliferation and cell death such as TP53, and other proteins such as DCC. Proliferation of the epithelial cells that carry the causative mutation or mutations can result in invasive growth into the muscle layers and through the bowel wall. Symptoms of colorectal cancer typically include rectal bleeding, anemia, constipation, blood in the stool, weight loss, fever, loss of appetite, and nausea or vomiting. The vast majority of colorectal cancer tumors can be classified as adenocarcinomas while lymphomas and squamous cell carcinomas are observed in a smaller subset of cases. Accordingly, the term "colorectal tumor," as used herein, refers to any abnormal malignant growth of tissue associated with cells originating in the large intestine or colorectal cancer pathology. [00078] In a fourth aspect, the invention relates to a method for disturbing the integrity of the extracellular matrix (ECM) of a cell comprising contacting the cell comprising an extracellular matrix with the non-viable bacterial derivative of the invention or with the composition of the invention.

[00079] The phrase "extracellular matrix (ECM)", as used herein, refers to a complex network of materials produced and secreted by cells into the surrounding extracellular space and/or medium and which typically together with the cells impart its mechanical and structural properties. Generally, the ECM includes fibrous elements (particularly collagen, elastin, or reticulin), cell adhesion polypeptides (e.g., fibronectin, laminin and adhesive glycoproteins), and space-filling molecules [usually glycosaminoglycans (GAG), proteoglycans]. A tissue-of- interest (e.g., pancreas, myocardium, colon tissue, rectal tissue) may be derived from an autologous or non-autologous tissue (e.g., allogeneic or even xenogeneic tissue, due to non- immunogenicity of the resultant decellularized matrix). The tissue can be obtained from a subject [e.g., an animal, preferably a mammal, such as a pig, monkey or chimpanzee, or alternatively, a human being. In various embodiments, the whole tissue can be used, however, in alternative embodiments the tissue may be cut e.g. sliced. Such tissue segments can be of various dimensions, depending on the original tissue used and the desired application. In the context of ECM, the term "integrity" means that the ECM has a structure, physical properties and/or enzymatic activity comparable to an identical ECM in its natural tissue. In various embodiments, the term "disturbed integrity" refers to one or more of the above mentioned parameters (structure, physical properties, enzymatic activity and/or etc.) that demonstrates 90% or less, 80% or less, 75% or less, 70% or less 65% or less, 60% or less, 55% or less or 50% or less of the amount of the given parameter compared to the identical ECM in its natural tissue.

[00080] The term "contacting", as used herein, refers generally to providing access of one component, reagent, analyte or sample to another. For example, contacting can involve mixing the non-viable bacterial derivative of the invention or the composition of the invention with cell comprising an extracellular matrix (ECM) or a sample comprising a plurality of said cells and the extracellular matrix (ECM). This reaction may comprise one or more components, reagents, analytes or samples, such as dimethyl sulfoxide (DMSO) or a detergent, which facilitates mixing, interaction, uptake, or other physical or chemical phenomenon advantageous to the contact between cell/sample and bacterial derivative/composition.

[00081] In various embodiments, disturbing the integrity of the extracellular matrix includes size regression of the extracellular matrix.

[00082] As used herein, the terms "regression" and "inhibition" comprehend arresting or retarding the growth of a cell or a cellular component, such as ECM, as compared to a reference cell or cellular component. The reference cell or cellular component may be from a healthy person or a cancer patient, which has not been treated with the non-viable bacterial derivative of the invention or the composition of the invention

[00083] The scope of the present invention also encompasses various embodiments wherein the method is an in vivo method; or the method is an ex vivo method.

[00084] The term "ex vivo", as used herein, refers to an environment outside of a patient. Accordingly, a sample taken from a patient which is cultured or investigated is considered ex vivo. The term "m vivo", as used herein, refers to a region within a body of an animal or patient, for example, within a bodily cavity, for example of a human body.

[00085] In a further aspect, the invention relates to a non-viable bacterial derivative, wherein the non-viable bacterial derivative comprises a heat inactivated Clostridium sporogenes cell.

[00086] Methods for heat inactivation are well-known to the skilled person. In one embodiment, the bacterial culture is harvested and heat-inactivated by placing it in an 80 °C water bath for 2 hours.

[00087] In a sixth aspect, the invention relates to a composition comprising at least one bacterial protein, wherein the composition comprises clostripain as set forth in SEQ ID NO: l or a derivative of clostripain having at least 75% sequence identity to clostripain determined overall the whole length of SEQ ID NO: 1.

[00088] Methods to prepare compositions comprising at least one bacterial protein are well-known in the art. In one embodiment, the composition is a conditioned media (CM). The conditioned media may be prepared by centrifuging a media-bacteria mixture to pellet the bacteria. The media may be Reinforced Clostridial Media (RCM). The supernatant can be collected and filtered. The result is defined as conditioned media (CM).

EXAMPLES

Materials and Methods

2D Cancer Cell Culture

[00065] The CT26 murine colorectal cancer cells (ATCC CRL-2638) and HCT116 human colorectal cancer cells (ATCC CCL-247) were purchased from the American Type Culture Collection. The complete growth medium used for the CT26 cell culture was the Roswell Park Memorial Institute (RPMI) culture medium (Hyclone, USA) and that for the HCT116 cell culture was McCoy's 5A medium (Sigma-Aldrich, USA), supplemented with 10% Foetal Bovine Serum (FBS) (Gibco, USA) and 1% Penicillin-streptomycin (Gibco, USA). The cells were cultured in flasks (Corning, USA) in their respective complete growth media, incubated in a humidified atmosphere at 37°C, 5% C0 ₂ . They were grown to 70-80% confluence before being passaged. The cells were seeded in 96-well tissue culture plates at a density of 5000 cells/well, and were cultured overnight to allow them to adhere to the bottom of the wells in monolayers before being used for the experiments.

3D Spheroid Culture

[00066] The CT26 and HCT116 cells were cultured in round-bottomed, ultra-low adhesion 96-well plates (Corning, USA). The complete growth medium used for the 3D spheroid culture was the RPMI culture medium (Hyclone, USA) for CT26 cells and McCoy's 5A culture medium (Sigma-Aldrich, USA) for HCT116 cells, supplemented with 10% (FBS) (Gibco, USA) and 1% Penicillin-streptomycin (Gibco, USA). 200 μΐ of the complete growth medium was added to each well and 1500 cells were seeded in each well. The cells were spun at 986 g for 10 minutes using a plate centrifuge (Thermo Scientific) to facilitate their collection at the bottom of the wells. The 3D spheroid culture was incubated at 37 °C, 5% CO2 for a period of 3 days to allow the development of stable spheroids with necrotic regions that simulate in vivo tumors. After this 3 day growth period, the spheroids were used for the experiments. Bacterial Culture and Inactivation

[00067] The C. sporogenes spores (ATCC 13732) were purchased from the American Type Culture Collection. 15 μΐ of the spore suspension was added to 15 ml of Reinforced Clostridial Media (RCM) (Oxoid, England), in petri dishes. An anaerobic chamber was set-up following the protocol of the GasPak™ EZ Anaerobic Container System (Becton, Dickinson and Company, USA). The bacterial culture was placed in the anaerobic chamber and incubated at 37 °C, 5% C0 ₂ for 3 days. The bacterial culture was then harvested from the anaerobic chamber and heat-inactivated by placing it in an 80 °C water bath for 2 hours. After heat- inactivation, the optical density of the bacterial suspension was measured using UV-vis spectrophotometer (VWR, USA) quantify the amount of bacteria present in a 1ml sample of the culture at 600 nm ⁴³ . The culture was centrifuged at 2500 g for 20 minutes ²⁸ to pellet the bacteria. After the supernatant was discarded, the inactivated bacterial pellet was re-suspended in complete growth media of cancer cells for use in the experiments.

Bacterial Viability Assay

[00068] The heat-inactivated C. sporogenes bacteria were subjected to a bacterial viability test to confirm the effectiveness of the heat-inactivation process. The Viability/Cytotoxicity Assay kit for Bacterial Live & Dead Cells (Biotium, USA), was used to stain the IB using fluorescent nucleic acid dyes following the manufacturer's protocol. DMAO is a green dye that stains both live and dead bacteria while Ethidium Homodimer-III (EthD-III) is a red dye that only stains dead bacteria. Images were obtained using a fluorescence microscope (Olympus, Japan).

Preparation of Conditioned Media and Boiled Conditioned Media

[00069] The spores were cultured in RCM using the same protocol as described above. After 3 days, the bacterial culture was removed from the anaerobic chamber and centrifuged at 2500 g for 20 minutes ²⁸ to pellet the bacteria. The supernatant was collected and filtered using a 0.20 μπι filter. The result is defined as conditioned media (CM). The CM was placed in a water bath at 100 °C for 30 minutes to obtain boiled conditioned media (BCM). Cell Proliferation Assay

[00070] The inactivated bacteria (IB) suspension in complete growth medium was serially diluted to concentrations of 0.1, 0.2, 0.3, 0.4, 0.6 and 0.8 OD (measured at 600 nm). The CM sample was prepared as 10% of CM in complete growth media (10% CM). For the 2D cell culture, cells were grown in 96 well-plates overnight at a seeding density of 5000 cells/well. They were then incubated at 37 °C, 5% C0 ₂ with 200 μΐ of the 10% CM test samples and IB test samples of varying concentration over a 72 hour period. For the 3D spheroid culture, 3 day old spheroids were incubated with 0.1 OD IB, 10% CM and 10% BCM test samples over a 72 hour period. In both 2D and 3D experiments, two controls were maintained where cells or spheroids were incubated with 200 μΐ of complete growth media and with 200 μΐ of 10% RCM. At each 24 hour time point, the test samples in each well were replaced with fresh RPMI media. The 3D spheroids were re-suspended into a single cell suspension. Cell Proliferation Reagent WST-1 (Roche, USA), was added to each well in a 1 : 10 ratio and the cells were incubated for 2 hours under culture conditions. The absorbance of the samples was measured using a microplate reader (BioRad, USA) at 430 nm with a reference wavelength of 650 nm. The absorbance value of each well is directly proportional to the number of viable cells.

Cell Viability Assay

[00071] Cells, grown overnight in 96 well-plates at a seeding density of 5000 cells/well, were incubated at 37 °C, 5% C0 ₂ with IB test samples at varying concentrations of 0.1, 0.2, 0.3, 0.4, 0.6, 0.8 OD and 10% CM over a 72 hour period. LIVE/DEAD ® Viability/Cytotoxicity Kit for mammalian cells (Invitrogen, USA), containing Calcein AM and Ethidium homodimer -I (EthD-I) was used to determine the cell viability assay of the 2D cell culture. At each 24 hour time point, the test samples were removed and the wells were washed with PBS. 100 μΐ of the fluorescence dye working solution was added to each well and the microplate was incubated in darkness at room temperature for 30 minutes. A fluorescence microscope (Olympus, Japan) was used to take the fluorescence images of each well.

Spheroid Area Measurement

[00072] 3 day old spheroids were incubated at 37 °C, 5% C0 ₂ with 0.1 OD IB, 10% CM and 10% BCM over a 72 hour period. At each 24 hour time point, the spheroids were imaged using a bright-field microscope (Olympus, Japan) and the diameter was measured using the Image J software.

Histology

[00073] 3D spheroids were initially grown for 3 days and were incubated in the presence/ absence of IB for an additional 3 days. The spheroids were then harvested and fixed in 10% paraformaldehyde (PFA). They were then dehydrated with a series of increasing concentrations of ethanol before being embedded in paraffin. The embedded samples were sectioned to a thickness of 5.0 μηι and mounted onto polysine slides. The sections were then stained with haematoxylin and eosin and imaged using a bright-field microscope.

SEM Imaging

[00074] 3 day old spheroids grown in complete growth media and 0.1 OD of IB for another 72 hours were fixed in PFA and dehydrated with a series of increasing ethanol concentrations. Once the samples were completely dehydrated, they were coated with platinum for 60 seconds with a 20 mA coating current and imaged with a Scanning Electron Microscope (JEOL, Japan).

Statistical Analysis and Data Processing

[00075] All experiments were performed in triplicate, for statistical significance. All data were expressed as Mean ± Standard Deviation of three separate experiments. Statistical analysis was conducted using two-tailed Student's t-test.

Example 1: Heat-Inactivation kills bacterial cells

[00076] C. sporogenes were heat-inactivated to minimize the risk of infection. Live bacteria and the heat-inactivated bacteria were tested with the bacterial viability assay, using DMAO and Ethidium homodimer-III (EthD-III) dyes. DMAO can permeate cell membranes of both live and dead bacteria, but EthD-III can only permeate dead bacterial cells. As shown in Fig. la, live vegetative bacteria was stained green with DMAO. After heat-inactivation, the bacteria in the sample got stained red with EthD-III, indicating that the heat treatment is effective (Fig. lb). Example 2: Inactivated C. sporo genes and its conditioned media inhibit colorectal cancer cells in 2D culture

[00077] The 2D monolayer cultures of CT26 and HCT116 cells were exposed to varying concentrations of IB and the cell proliferation rate was expressed as a percentage of the control. At all the concentrations of IB, there was a significant decrease in the cell proliferation rates. It is important to note that even the lowest concentration of IB (0.1 OD) inhibits the cell proliferation of the CT26 cells to 37.0% of the control after just 24 hours. At the 48 and 72 hour time -point, the cell proliferation of the sample is reduced to 7.5% and 6.3% respectively (Fig. 2a). On the other hand, when exposed to an eight times higher concentration of IB (0.8 OD), the proliferation of cells decreased significantly to 13.1%, 3.0% and 2.0% at 24, 48 and 72 hours respectively. Correspondingly, the viability of the cells decreased in a dose dependent manner. This is validated by the fluorescence images demonstrating that an increase in IB concentrations leads to decrease in cell viability (Fig. 2b). Further data shows that 0.1 OD of IB reduces the cell proliferation of HCT116 cells to 97.0%, 77.0% and 44.3% at 24, 48 and 72 hours respectively. Additionally, 0.8 OD of IB reduces HCT116 cell proliferation to 40.4%, 1.49% and 0.63% at 24, 48 and 72 hours respectively.

[00078] After establishing the effect of IB on cells, the bacteria-free CM was studied in comparison to RCM, which is the un-inoculated bacterial media (Fig. 3). Cell proliferation of CT26 cells exposed to 10% CM decreased to 10.8% of the control in the first 24 hours, after which it further decreased to 4.5% (48 hours) and 2.4% (72 hours). 10% CM inhibits the cell proliferation of HCT116 to 55.9%, 30.6% and 7.4% at 24, 48 and 72 hours respectively. In comparison, the cell proliferation rate of CT26 cells exposed to 10% RCM is 76.0% of the control at the first time point and it decreases to 68.1% (48 hours) and 67.9% (72 hours) later (Fig. 3a) and that of HCT116 is 83.2% at 24 hours, 72.8% at 48 hours and 75.4% at 72 hours. The fluorescence images of the cells, support the cell proliferation results (Fig. 3b). They show that the amount of viable cells remaining after exposure to CM is much lower than those exposed to RCM. Example 3: Inactivated C. sporo genes and its conditioned media inhibit colorectal cancer cells in 3D culture

[00079] After having examined these non-viable derivatives on a 2D monolayer culture, its effects on a 3D spheroid culture were studied. H&E staining was conducted on CT26 spheroids grown for 72 hours and an absence of haematoxylin was observed in some regions of the spheroids. This indicates a lack of nuclei in the region, suggesting that the cells in this region were necrotic (Fig. 4).

[00080] The spheroids were exposed to IB (0.1 OD) over a period of 72 hours. The cell proliferation was measured using the WST-1 assay at 24 hour intervals (Fig. 5a). At each time point, a decrease in the cell proliferation was observed. At 72 hours, the cell proliferation of CT26 spheroids reduces to 57.3% and that of HCT116 reduces to 26.2%. These values are is higher than the cell proliferation of cells in the 2D model exposed to the same concentration of IB. As anticipated, the size of the control spheroids increases significantly between each time point, over the entire duration of the study. 0.1 OD IB appears to stunt the spheroid growth, with the increase in the area of the spheroids being marginal at each time point (Fig. 5b). Over time, there is a significant difference in the area of the spheroids and at the end of 72 hours, the CT26 spheroids exposed to IB (3.25 x 10 ⁵ μπι ² ) are 25% smaller than the control spheroids (4.34 x 10 ⁵ μπι ² ) and the HCT116 spheroids exposed to IB (1.60 x 10 ⁵ μπι ² ) are 44% smaller than the control (2.85 x 10 ⁵ μπι ² ). The inhibitory effect of higher concentrations of IB (0.4 OD and 0.8 OD) on CT26 and HCT116 spheroids is greater than that of 0.1 OD IB. 0.4 OD IB causes a 32% decrease in CT26 spheroid area and a 50% decrease in HCT116 spheroid area. 0.8 OD IB causes a 33% and 60% decrease in CT26 and HCT116 spheroid area respectively. Moreover, it is interesting to note that despite the increase in size, the shape of the control spheroids is maintained throughout the study, while the shape of the CT26 spheroids exposed to IB starts to get deformed after the 48 hour time point (Fig. 5c).

[00081] The effect of CM on the spheroids was investigated similarly. RCM caused a slight decrease in the cell proliferation across all time points. On the other hand, the inhibitive effect of CM is significant, with the cell proliferation of CT26 spheroids decreasing to 20.0% and that of HCT116 spheroids decreasing to 17.0% after 72 hours (Fig. 6a). At the end of the study, the area of the CM exposed CT26 spheroids is 38% smaller than the area of the control spheroids, while the area of CM exposed HCT116 spheroids is 53% smaller than the control spheroids (Fig. 6b). CM induces a slight regression in the CT26 spheroids from 2.73 x 10 ⁵ μπι ² at 48 hours to 2.67 x 10 ⁵ μπι ² at 72 hours. Additionally, CM causes an overall regression of the HCT116 spheroids from 1.54 x 10 ⁵ μπι ² at 0 hours to 1.33 x 10 ⁵ μπι ² at 72 hours. For RCM exposed spheroids, the area, although smaller than the control, was still larger than the spheroids exposed to CM. This inhibitive effect of CM is also reflected the morphology of the spheroids (Fig. 6c). CM causes deformation of the spheroids, stunting of the spheroid size initially and then its regression. In contrast, RCM maintained overall spheroid shape as compared to CM exposed spheroids.

Example 4: Heat-sensitive proteins in C. sporogenes Conditioned Media responsible for inhibiting CT26 cells

[00082] In an attempt to determine if proteins are involved in the previous observations, CM was boiled to denature the proteins and obtain boiled conditioned media (BCM). 3D spheroids were exposed to BCM in a 72 hour study. By the end of the study, it was observed that BCM has a considerably lesser inhibitive effect on cell proliferation (64.7% for CT26 spheroids, and 63.7% for HCT116 spheroids) than CM (20.0% for CT26 spheroids and 17.0% for HCT116 spheroids) (Fig. 6a). The area of the CT26 and HCT116 spheroids exposed to BCM is consistently higher than that of the spheroids exposed to CM. Furthermore, the area of BCM exposed spheroids is similar to that of the area of spheroids exposed to RCM (Fig. 6b). The growth of the spheroids exposed to BCM is stunted, although their shape is maintained. Comparatively, spheroids exposed to CM regress in size and also lose their shape (Fig. 6c).

Example 5: Inactivated C. sporogenes break down the ECM in 3D spheroids

[00083] To further characterize the role of IB in inhibiting cancer cell growth, the morphology of the 3D spheroids was examined. It was found that the cells on the periphery of the control CT26 spheroids were circular in shape, packed together with tight cell contacts and thickly covered by the ECM. On the other hand, on the periphery of the spheroids exposed to IB, the cells were elongated in shape, loosely packed and the ECM appeared to be thinner. The spherical morphology of the control spheroid was not observed in the IB spheroid, which appeared deformed (Fig. 7). This is in agreement with Fig. 5c, where the IB-exposed CT26 spheroids appeared to have lost is morphological integrity at 72 hours.

Example 6: Interaction of inactivated C. sporogenes and colorectal cancer cells in 2D culture

[00084] Our published work has shown that the administration of IB to the 3D spheroids inhibits their growth. The heat-inactivation of the bacteria renders it incapable of replication, metabolic activity and secretion of bacterial products. Therefore, it is likely that the membrane surface of the inactivated bacteria (IB) contains the key factors responsible for the inhibition of the cancer cells. Studying the physical interaction between the IB and cancer cells is crucial to understanding the underlying mechanism of inhibition.

[00085] It was hypothesized that the IB physically interacts with the cancer cells and this interaction between them enables the inhibitive effect that is observed. A Scanning Electron Microscope (SEM) allows the visualization of the surface structure and interaction of the cancer cells and IB. A 2D monolayer of HCT116 cells was cultured and exposed to IB for 24 hours before conducting SEM imaging of the sample. Figure 8 shows the results of this preliminary study. The image shows that the IB adheres to the surface of the HCT116 cells, as indicated by the black arrows. This confirms the notion of the physical interaction between the IB and cancer cells.

Example 7: Cell Death Analysis of Colorectal Cancer Cells

[00086] The type of cell death that cancer cells undergo when exposed to IB is a starting point for understanding the mechanism of IB-induced inhibition of cancer cells. To study the nature of the cell death of the cancer cells, an Annexin V/PI assay was conducted. This assay was used to understand the type of cell death that the cancer cells go through when exposed to IB. The Annexin V stains the apoptotic cells and PI stains the necrotic cells. Figure 9 shows the preliminary result of the Annexin V/PI assay conducted on HCT116 cells that were exposed to IB. Following this, flow cytometry will then be used to quantify the number of each type of cells to obtain a more conclusive understanding of the nature of cell death that is induced by the IB.

Example 8: Identification and isolation of secreted bacterial products in CM that inhibit cancer cells and elucidation of the underlying mechanism of inhibition

[00087] The above data demonstrates that the conditioned media (CM) is a significant inhibitory effect on the growth of the 3D spheroids of colorectal cancer cells. The key components in the CM that are responsible for this inhibitory effect have to be isolated and identified before the potential of CM as a therapeutic agent can be fully realized. The above results also indicate that denaturing the proteins in CM causes a significant reduction in the inhibition effect of the CM. This indicates that the bacterial proteins present are playing a role in exerting an anti-cancer effect. In order to identify the proteins in CM, SDS-Page gel electrophoresis was conducted to separate the proteins in CM according to their molecular weight. Figure 10 shows the protein bands that were obtained.

[00088] Mass spectroscopy was used for the identification of the proteins. The expressed proteins are subjected to in-gel digestion and MALDI-TOF-TOF analysis ^[17] . Protein identification was performed using the Mascot proGram (Matrix Science, UK) (http://www.matrixscience.com). To date, there has only been a few reports on the proteins (enzymes) secreted by C. sporogenes ^{[18 19]} . From the various bands obtained from gel electrophoresis, one band has been identified as the proteolytic enzyme clostripain (Figure 10).

Example 9: Effect of Bacterial Products on Extracellular Matrix (ECM)

[00089] As C. sporogenes is a proteolytic bacteria, CM contains various proteases. Thus, it is likely that the proteases target the ECM proteins in the 3D spheroids, thereby causing a regression in their size. To investigate this, IHC staining of spheroids exposed to CM will be conducted. A preliminary study examined the effect of CM was conducted to examine the presence of ECM proteins such as collagen (Figure 11) and elastin (Figure 12). The results show that the collagen- 1 and elastin content in the spheroids does not change much after exposure to CM and IB. [00090] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject-matter from the genus, regardless of whether or not the excised material is specifically recited herein. Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[00091] One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. Further, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The compositions, methods, procedures, treatments, molecules and specific compounds described herein are presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention are defined by the scope of the claims. The listing or discussion of a previously published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[00092] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein.

Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. The word "comprise" or variations such as "comprises" or

"comprising" will accordingly be understood to imply the inclusion of a stated integer or groups of integers but not the exclusion of any other integer or group of integers. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by exemplary embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[00093] The content of all documents and patent documents cited herein is incorporated by reference in their entirety.

References

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