Modified microorganisms and uses thereof

Abstract

The present disclosure provides a genetically modified Clostridium bacterium, methods of producing said genetically modified Clostridium bacterium, populations comprising said genetically modified Clostridium bacteria, and spores obtained from said genetically modified Clostridium bacterium. Also provided is a microorganism modified to express a heterologous protein and / or to reduce or prevent expression of a gene encoding a toxin, a spore derived from said modified microorganism, or a composition comprising said modified microorganism or said spore, for use in a method of treating or preventing a cancer. Further provided is a Clostridium septicum bacterium for use in a method of medical treatment or prophylaxis.

Description

[0001] Modified microorganisms and uses thereof

[0002] This application claims priority from SG 10202401624U filed 7 June 2024, the contents and elements of which are herein incorporated by reference for all purposes.

[0003] Technical Field

[0004] The present disclosure relates to the fields of microbiology and immunotherapy.

[0005] Background

[0006] Despite advancements in cancer therapy, hypoxic tumours are still refractory to treatment, resulting in poor prognoses due to the poor penetration of drugs, metabolic reprogramming, and cellular quiescence (1). While the utility of oncolytic bacteria in humans was first reported more than a century ago (2), the use of anaerobic bacteria to target tumour hypoxia has only gained traction recently. Currently, most research is focused on three bacteria, namely Salmonella, Listeria and Clostridium.

[0007] Among these three candidates, Clostridium is the only one which targets tumour hypoxia. This selectivity arises at two levels. Firstly, Clostridium lacks oxygen scavenging proteins, which means that it is strictly anaerobic and cannot survive the well-oxygenated tissues outside the tumour (3). Secondly, Clostridium spores require reducing conditions to germinate, so spores administered intravenously will only germinate and colonize hypoxic tumour tissues (4). Currently, the most extensively studied oncolytic Clostridium is Clostridium novyi-NT. Spores of C. novyi-NT were found to be non-toxic and safe for intravenous administration (4) and are currently undergoing various human (5) and veterinary (6,7) clinical trials.

[0008] Although the use of Clostridium to target hypoxia has been well-documented, there is a gap of scientific knowledge concerning how Clostridium might modulate the anti-tumour immune response. For example, there is evidence that the presence of Fusobacterium nucleatum in colorectal tumours decreases the efficacy of PD-1 blockade. Further, while Salmonella and Listeria have been well-studied as platforms for delivering immunomodulatory payloads, there is a dearth of such investigation with Clostridium.

[0009] C. septicum very rarely infects humans. It has an annual incidence between 0.4 and 1 per million (8) and accounts for merely 1.3% of all Clostridium infections (9). Although C. septicum infection is a low probability event, the rare patient who presents with a C. septicum infection has a -80% chance of also having an accompanying malignancy (9-11). In comparison, Clostridium perfringens infections are linked to an accompanying malignancy only 4% of the time. The high predictive value of C. septicum for cancer suggests that C. septicum’s host tropism may be more specific for tumours than other Clostridium species. In select cases, C. septicum has been reported to infect and debulk metastatic tumours (12,13).

[0010] Summary

[0011] In a first aspect, the present disclosure provides a Clostridium bacterium, wherein the bacterium is genetically modified. In some embodiments, the bacterium is modified to express a heterologous protein. In some embodiments, the heterologous protein is an antigen. In some embodiments, the antigen is a tumour- associated antigen. In some embodiments, the antigen is a tumour neoantigen, comprises an immunodominant epitope and / or comprises a mimotope.

[0012] In some embodiments, the bacterium is modified to express a plurality of heterologous proteins.

[0013] In some embodiments, the bacterium is modified to reduce or prevent expression of a gene encoding a toxin. In some embodiments, the gene encodes an alpha toxin. In some embodiments, the gene is csa.

[0014] In some embodiments, the bacterium is of a species selected from Clostridium septicum and Clostridium novyi. In some embodiments, the bacterium is of the species Clostridium septicum.

[0015] The present disclosure also provides a method of producing the Clostridium bacterium according to the present disclosure.

[0016] The present disclosure also provides a Clostridium bacterium obtainable by a method according to the present disclosure.

[0017] The present disclosure also provides an isolated Clostridium bacterium according to the present disclosure.

[0018] The present disclosure also provides a population comprising the Clostridium bacterium according to the present disclosure.

[0019] The present disclosure also provides Clostridium spores obtained from the Clostridium bacterium according to the present disclosure.

[0020] The present disclosure also provides a composition comprising the Clostridium bacterium or the Clostridium spores according to the present disclosure.

[0021] The present disclosure also provides a modified microorganism, spores derived from a modified microorganism, or a composition comprising a modified microorganism or spores derived from a modified microorganism, for use in a method of treating or preventing a cancer, wherein the modified microorganism is modified to express a heterologous protein and / or to reduce or prevent expression of a gene encoding a toxin.

[0022] The present disclosure also provides use of a modified microorganism, spores derived from a modified microorganism, or a composition comprising a modified microorganism or spores derived from a modified microorganism, in the manufacture of a medicament for treating or preventing a cancer, wherein the modified microorganism is modified to express a heterologous protein and / or to reduce or prevent expression of a gene encoding a toxin.

[0023] The present disclosure also provides a method of treating or preventing a cancer, comprising administering to a subject a therapeutically- or prophylactically-effective amount of a modified microorganism, spores derived from a modified microorganism, or a composition comprising a modified microorganism or spores derived from a modified microorganism, wherein the modified microorganism is modified to express a heterologous protein and / or to reduce or prevent expression of a gene encoding a toxin.

[0024] In some embodiments, the heterologous protein is an antigen. In some embodiments, the antigen is a tumour-associated antigen. In some embodiments, the antigen is a tumour neoantigen, comprises an immunodominant epitope and / or comprises a mimotope.

[0025] In some embodiments, the modified microorganism is modified to express a plurality of heterologous proteins.

[0026] In some embodiments, the gene encoding a toxin encodes an alpha toxin. In some embodiments, the gene is csa.

[0027] In some embodiments, the modified microorganism is a Clostridium bacterium. In some embodiments, the Clostridium bacterium is of a species selected from Clostridium septicum and Clostridium novyi. In some embodiments, the Clostridium bacterium is of the species Clostridium septicum.

[0028] In some embodiments, the modified microorganism is as defined in section (A) of the present disclosure.

[0029] The present disclosure also provides a Clostridium septicum bacterium for use in a method of medical treatment or prophylaxis.

[0030] The present disclosure also provides a Clostridium bacterium, Clostridium spore, or composition according to the present disclosure, for use in a method of medical treatment or prophylaxis.

[0031] The present disclosure also provides a Clostridium bacterium, Clostridium spore, or composition according to the present disclosure, for use in a method of treating or preventing a cancer.

[0032] The present disclosure also provides use of a Clostridium bacterium, Clostridium spore, or composition according to the present disclosure, in the manufacture of a medicament for treating or preventing a cancer. The present disclosure also provides a method of treating or preventing a cancer, comprising administering to a subject a therapeutically- or prophylactically-effective amount of a Clostridium bacterium, Clostridium spore, or composition according to the present disclosure.

[0033] In some embodiments, the cancer is selected from: a solid tumour, breast cancer, breast carcinoma, ductal carcinoma, gastric cancer, gastric carcinoma, gastric adenocarcinoma, colorectal cancer, colorectal carcinoma, colorectal adenocarcinoma, head and neck cancer, squamous cell carcinoma of the head and neck, lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung carcinoma, ovarian cancer, ovarian carcinoma, ovarian serous adenocarcinoma, renal cancer, renal cell carcinoma, renal clear cell carcinoma, renal cell adenocarcinoma, renal papillary cell carcinoma, pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, cervical cancer, cervical squamous cell carcinoma, skin cancer, melanoma, esophageal cancer, esophageal adenocarcinoma, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, uterine cancer, uterine corpus endometrial carcinoma, thyroid cancer, thyroid carcinoma, pheochromocytoma, paraganglioma, bladder cancer, bladder urothelial carcinoma, prostate cancer, prostate adenocarcinoma, sarcoma and thymoma.

[0034] In some embodiments, the cancer is characterised by hypoxia.

[0035] In some embodiments, the cancer comprises cells expressing a given antigen, and wherein the modified microorganism, or the Clostridium bacterium, is modified to express the given antigen.

[0036] In some embodiments, the method further comprises an additional therapeutic or prophylactic intervention selected from chemotherapy, immunotherapy, radiotherapy, surgery, and / or hormone therapy.

[0037] In some embodiments, the method comprises administering a chemotherapeutic agent.

[0038] In some embodiments, the method comprises administering an immunomodulatory agent.

[0039] In some embodiments, the method comprises administering an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is capable of inhibiting signalling mediated by PD-1 , CTLA-4, LAG-3, TIM-3, TIGIT or BTLA. In some embodiments, the immune checkpoint inhibitor is an anti- PD-1 antibody or fragment thereof, or anti-PD-L1 antibody or fragment thereof.

[0040] In some embodiments, the method comprises administering an immunomodulatory cytokine.

[0041] Also provided herein is a method of treating cancer in a subject, comprising:

[0042] (i) administering a modified microorganism, a spore derived from a modified microorganism, or a composition comprising a modified microorganism or spore derived from a modified microorganism to the subject, wherein the modified microorganism has been modified to express an antigen identified as being expressed in the cancer to be treated.

[0043] Also provided herein is a method of treating cancer in a subject, comprising:

[0044] (i) identifying an antigen expressed in the cancer to be treated; and

[0045] (ii) administering a modified microorganism, a spore derived from a modified microorganism, or a composition comprising a modified microorganism or spore derived from a modified microorganism to the subject, wherein the modified microorganism has been modified to express the antigen.

[0046] Also provided herein is a method of treating cancer in a subject, comprising:

[0047] (i) identifying an antigen expressed in the cancer to be treated;

[0048] (ii) producing a microorganism modified to express the antigen; and optionally,

[0049] (iii) administering the modified microorganism, a spore derived from the modified microorganism, or a composition comprising the modified microorganism or spore derived from the modified microorganism to the subject.

[0050] Description

[0051] The present invention relates to modified microorganisms, and their use in immunotherapy. In particular, the present disclosure pertains to modified Clostridia, and their use in the treatment of cancer.

[0052] A. Modified microorganisms of the disclosure

[0053] Aspects and embodiments of the present disclosure relate to modified microorganisms.

[0054] It will be appreciated that the microorganisms disclosed herein may relate to microorganisms that have been modified according to various aspects of the invention (e.g. genetically modified microorganisms). That is, microorganisms described herein may be microorganisms that have undergone genetic modification to exhibit various properties / attributes / features described herein.

[0055] Microorganisms include prokaryotic and eukaryotic cells. For example, the prokaryotic cell may be a bacteria or archaea, and the eukaryotic microorganism may be a fungi, protist, or microscopic animal or microscopic plant organism. In some embodiments, the modified microorganism is a bacterium. In some embodiments, the modified bacterium is Salmonella, Listeria or Clostridium.

[0056] In some embodiments, the modified microorganism is anaerobic. Anaerobic microorganisms may be obligate anaerobes or facultative anaerobes. Obligate anaerobes are organisms that can grow and survive only in the absence of oxygen. In aerobic conditions obligate anaerobes tend to die, e.g. they are killed by normal atmospheric concentrations of oxygen ( / .e. 20.95% O2). Facultative anaerobes are organisms that usually grow in aerobic conditions but have mechanisms (e.g. fermentation) that allow their growth and survival even under anaerobic conditions. Anaerobic microorganisms are able to colonise and / or grow in oxygen deficient / hypoxic environments, e.g. an oxygen deficient / hypoxic tumour microenvironment. In some embodiments, the modified microorganism is an obligate anaerobe. In some embodiments, the modified microorganism is a facultative anaerobe.

[0057] Microorganisms may grow selectively within a specific environment. For example, a microorganism may exhibit preferential growth within specific host tissues or microenvironments. In some embodiments, the modified microorganism exhibits preferential growth in hypoxic regions / areas / tissues. In some embodiments, the modified microorganism exhibits preferential growth in tumours e.g. in tumours comprising a hypoxic microenvironment and / or hypoxic cells. In some embodiments, the modified microorganism may be unable to survive in normoxic regions / areas / tissues.

[0058] One of the most common coping mechanisms for bacteria is forming spores to protect themselves against ecological degrading agents. Bacterial spores are the most dormant form of bacteria since they exhibit minimal metabolism and respiration, as well as reduced enzyme production. Typically, grampositive bacteria are best known for producing intracellular spores called endospores as a survival mechanism. Endospores are highly refractile and thick-walled structures formed inside the bacterial cells. It is most common for Bacillus species and Clostridium species to create endospores.

[0059] Endospores germinate back into vegetative cells ( / .e. an active bacterial cell that undergoes metabolism) when surrounding environmental conditions favour bacterial growth and reproduction. Several stimulants revert bacterial cells to their active vegetative cells, such as suitable oxygen levels, optimal close-to-body temperature, and diffusion of nutrients and water through bacterial cell walls through alteration of their surface tension.

[0060] The process of spore formation is a multistep process. It starts from replication of the bacterial DNA, followed by the formation of the forespore, which is, by definition, pinching of the cellular plasma membrane between the replicated chromosome. Then, a cortex forms between the inner and outer membrane by extending the second cellular membrane to enclose the forespore with calcium and dipicolinic acid. Finally, the external spore coat surrounds the endospore before its release.

[0061] In some embodiments, the modified microorganism is a spore-forming microorganism (e.g. an endospore-forming microorganism). In some embodiments, the modified microorganism is capable of forming a spore (e.g. an endospore). In some embodiments, the modified microorganism is capable of sporulation.

[0062] Sporulation is the process in which a spore (or spores) is formed from a vegetative cell. Spores may be derived from the modified microorganisms described herein ( / .e. spores formed following sporulation of the microorganisms described herein).

[0063] The present disclosure also provides a spore (e.g. an endospore) derived from a modified microorganism described herein. Germination is the process by which a spore is converted into a vegetative cell. It will be appreciated that a cell originated from a given spore ( / .e. a cell produced by germination of a given spore) will exhibit the properties of the microorganism from which the given spore was derived. In some embodiments, a spore is capable of germinating in hypoxic conditions. In some embodiments, a spore is only capable of germinating in hypoxic conditions.

[0064] In some embodiments, the modified microorganism is a Clostridium bacterium. A Clostridium bacteria may alternatively be described as a bacterium of the genus Clostridium.

[0065] Clostridium is a genus of anaerobic, gram-positive bacteria. Species of Clostridium inhabit soils and the intestinal tract of animals, including humans.

[0066] Species of Clostridium are obligate anaerobes and capable of producing endospores. They generally stain gram-positive, but are often described as gram-variable, because they show an increasing number of gram-negative cells as the culture ages. The normal, reproducing cells of Clostridium (i.e. the vegetative form) are rod-shaped. Clostridium endospores have a distinct bowling pin or bottle shape, distinguishing them from other bacterial endospores.

[0067] Clostridia are reviewed in Johnson EA, Clostridia, Encyclopaedia of Microbiology (Third Edition) 2009, pages 87-93, which is hereby incorporated by reference in its entirety.

[0068] In some embodiments, the Clostridium bacterium is of a species selected from: C. (Clostridium) aceticum, C. acetireducens, C. acetobutylicum, C. acidisoli, C. aciditolerans, C. aestuarii, C. akagii, C. algidicarnis, C. algifaecis, C. algoriphilum, C. amazonense, C. aminophilum, C. ammoniilyticum, C. amylolyticum, C. arbusti, C. arcticum, C. argentinense, C. aurantibutyricum, C. autoethanogenum, C. baratii, C. beihaiense, C. beijerinckii, C. diolis, C. bornimense, C. botulinum, C. bovifaecis, C. bowmanii, C. budayi, C. butyricum, C. cadaveris, C. caldaquaticum, C. carboxidivorans, C. carnis, C. cavendishii, C. celatum, C. cellulofermentans, C. cellulosi, C. cellulovorans, C. chartatabidum, C. chauvoei, C. chromiireducens, C. chrysemydis, C. cochlearium, C. colicanis, C. colinum, C. collagenovorans, C. combesii, C. composti, C. cylindrosporum, C. disporicum, C. drakei, C. estertheticum, C. facile, C. fallax, C. felsineum, C. fermenticellae, C. fessum, C. fimetarium, C. formicaceticum, C. frigidicarnis, C. frigoris, C. fungisolvens, C. ganghwense, C. gasigenes, C. gelidum, C. grantii, C. guangxiense, C. haemolyticum, C. herbivorans, C. homopropionicum, C. hominis, C. huakuii, C. hydrogeniformans, C. hylemonae, C. indicum, C. innocuum, C. intestinale, C. isatidis, C. jeddahense, C. kluyveri, C. kogasense, C. lacusfryxellense, C. lentum, C. leptum, C. liquoris, C. Ijungdahlii, C. lundense, C. luticellarii, C. magnum, C. malenominatum, C. manihotivorum, C. methoxybenzovorans, C. methylpentosum, C. mobile, C. moniliforme, C. muellerianum, C. neonatale, C. neuense, C. nexile, C. nitritogenes, C. nitrophenolicum, C. novyi, C. oceanicum, C. oryzae, C. pabulibutyricum, C. paradoxum, C. paraputrificum, C. pascui, C. pasteurianum, C. peptidivorans, C. perfringens, C. phytofermentans, C. piliforme, C. polyendosporum, C. polynesiense, C. polysaccharolyticum, C. porci, C. prolinivorans, C. psychrophilum, C. punense, C. puniceum, C. putrefaciens, C. putrificum, C. quinii, C. ragsdalei, C. saccharobutylicum, C. saccharoperbutylacetonicum, C. sardiniense, C. sartagoforme, C. saudiense, C. scatologenes, C. schirmacherense, C. scindens, C. senegalense, C. septicum, C. simiarum, C. sporogenes, C. sporosphaeroides, C. subterminale, C. sulfidigenes, C. swellfunianum, C. symbiosum, C. tagluense, C. tarantellae, C. tepidiprofundi, C. tepidum, C. tertium, C. tetani, C. tetanomorphum, C. thailandense, C. thermarum, C. thermoalcaliphilum, C. thermobutyricum, C. thermopalmarium, C. thermopapyrolyticum, C. thermosuccinogenes, C. thiosulfatireducens, C. tyrobutyricum, C. uliginosum, C. vincentii, C. viride, C. vitabionis, C. vulturis, C. weizhouense, and C. zeae.

[0069] In some embodiments, the Clostridium bacterium is of the species Clostridium septicum. In some embodiments, the Clostridium bacterium is of the species Clostridium novyi.

[0070] Microorganisms may be defined by strain. A strain is a genetic variant or subtype within a species of a microorganism. Strains can be identified from those available in public collections such as American Type Culture Collection (ATCC), National Collections of Industrial, Food and Marine Bacteria (NCIMB), or National Collection of Type Cultures (NCTC), or from depositary Institutions that are International Depositary Authority Under the Budapest Treaty.

[0071] In some embodiments, the Clostridium septicum strain is ATCC 11424.

[0072] In some embodiments, the Clostridium septicum strain is ‘CS11 ’. As used herein, ‘CS11 ’ refers to the C. septicum strain ATCC 11424 in which the csa gene has been disrupted. The Clostridium septicum strain ‘CS11 ’ is described herein at e.g. Example 1 “Generation of CS11” and Example 2.

[0073] In some embodiments the strain is C. novyi-NT. C. novyi-NT is an attenuated strain in which the lethal a- toxin gene has been deleted. C. novyi-NT is described in Dang LH et al, PNAS 2001 , 98(26):15155- 15160, which is hereby incorporated by reference in its entirety.

[0074] In some embodiments, the modified microorganism is non-pathogenic.

[0075] As used herein, a ‘non-pathogenic’ microorganism refers to a microorganism that does not cause or produce disease. The microorganism may be naturally non-pathogenic ( / .e. the wildtype organism is non- pathogenic) or may have been modified to be non-pathogenic. For example, C. beijerinckii is a naturally non-pathogenic microorganism, while C. novy / -NT has been modified to be non-pathogenic.

[0076] The modified microorganisms of the present disclosure may be modified to attenuate toxicity. A microorganism with attenuated toxicity may also be referred to as an attenuated strain.

[0077] A microorganism with attenuated toxicity may exhibit a reduced level of toxicity. In some embodiments, a modified microorganism may exhibit a reduced level of toxicity compared to an appropriate reference microorganism (e.g. a wildtype microorganism of the same species or strain). The toxicity of a given microorganism may be assessed in a suitable in vitro or in vivo assay. For example, cell viability may be assessed in vitro following exposure to a test microorganism. For example, the microorganism may be administered to an in vivo animal model and weight and / or survival of the animal model may be assessed. The toxicity of a given microorganism may be assessed essentially as described herein e.g. in Example 1 “Assessing the cytotoxicity of CS11”.

[0078] In some embodiments, the modified microorganism of the present disclosure exhibits a level of toxicity that is less than 1 times, e.g. <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times, <0.05 times, or <0.01 times the level of toxicity observed in the unmodified / wildtype microorganism.

[0079] The modified microorganisms of the present disclosure may be modified to reduce or prevent expression of a gene encoding a toxin. In some embodiments, the modified microorganism is modified to reduce / prevent expression of a gene encoding a lethal toxin.

[0080] As used herein, a ‘toxin’ refers to an agent (e.g. a protein) produced by the microorganism that enhances / induces cellular toxicity in a host organism.

[0081] In some embodiments, the modified microorganism of the present disclosure exhibits expression of a gene encoding a toxin that is less than 1 times, e.g. <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times, <0.05 times, or <0.01 times the level of expression observed in an appropriate reference microorganism (e.g. the unmodified / wildtype microorganism).

[0082] Bacterial toxins are reviewed e.g. in Henkel JS et al, EXS 2010, 100:1-29, which is hereby incorporated by reference in its entirety. Bacterial toxins include: exotoxins, endotoxins, heat-stable enterotoxins, e.g. STa toxins and STb toxins; pore-forming toxins (PFT), e.g. a-PFTs (e.g. colicins) and p-PFTs (e.g. cholesterol-dependent cytolysins); superantigens; and superantigen-like toxins.

[0083] The toxins of Clostridia are reviewed in Popoff MR & Bouvet P, Future Microbiol. 2009, 4(8):1021-62, and Moore RJ & Lacey JA, Microbiology Spectrum 2019, 7(3) which are hereby incorporated by reference in their entireties. Clostridia] toxins include alpha toxin, beta toxin, gamma toxin, delta toxin, epsilon toxin, iota toxin, enterotoxin, TpeL toxin, phospholipase C toxin, Botulinum neurotoxin, Toxins A and B, Perfringens enterotoxin, and Perfringiolysin O.

[0084] Clostridium septicum produces four toxins: alpha toxin, beta toxin, gamma toxin and delta toxin. In some embodiments, the modified microorganism (e.g. a Clostridium septicum bacterium) is modified to reduce / prevent expression of a gene or genes encoding alpha toxin, beta toxin, gamma toxin and / or delta toxin. In some embodiments, the gene encodes alpha toxin. In some embodiments, the gene is csa.

[0085] The alpha toxin of Clostridium septicum causes traumatic or non-traumatic gas gangrene and necrotizing enterocolitis in humans. Clostridium septicum alpha toxin in the protein identified by UniProt Q208M1 . This toxin is the only lethal virulence factor of C. septicum and is necessary for the pathogenesis of this bacterium. The protein is encoded by the csa gene and secreted via the type II secretion pathway. The alpha toxin of Clostridium septicum and the csa gene are described e.g. in Ballard J et al, Infection and Immunity 1995, 63(1):340-344.

[0086] The modified microorganisms of the present disclosure may be modified to express a heterologous protein. In some embodiments, the modified microorganism expresses a heterologous protein.

[0087] In some embodiments, the microorganism may be modified to express a plurality of heterologous proteins ( / .e. two or more heterologous proteins). In some embodiments the modified microorganism expresses a plurality of heterologous proteins ( / .e. two or more heterologous proteins).

[0088] As used herein, a ‘heterologous protein’ refers to a protein that is heterologous ( / .e. derived from a different organism) to the microorganism in which it is expressed.

[0089] In some embodiments, the heterologous protein is an antigen.

[0090] In some embodiments, the heterologous protein is a tumour-associated antigen. In some embodiments, the heterologous protein is a tumour neoantigen, comprises an immunodominant epitope and / or comprises a mimotope.

[0091] The antigen may be a tumour-associated antigen. In some embodiments the antigen is an antigen whose expression is associated with the development, progression and / or severity of symptoms of a cancer / tumour. The tumour-associated antigen may be associated with the cause or pathology of the cancer / tumour, or may be expressed abnormally as a consequence of the cancer / tumour. In some embodiments, the antigen is an antigen whose expression is upregulated (e.g. at the RNA and / or protein level) by cells of a cancer / tumour, e.g. as compared to the level of expression of by comparable non- cancerous cells (e.g. non-cancerous cells derived from the same tissue / cell type).

[0092] In some embodiments, the tumour-associated antigen may be preferentially expressed by cancerous cells, and not expressed by comparable non-cancerous cells (e.g. non-cancerous cells derived from the same tissue / cell type). A tumour-associated antigen which is preferentially expressed by cancerous cells, and not expressed by comparable non-cancerous cells, may also be referred to as a tumour-specific antigen. In some embodiments, the tumour-associated antigen may be the product of a mutated oncogene or mutated tumour suppressor gene. In some embodiments, the tumour-associated antigen may be the product of an overexpressed cellular protein, a tumour antigen produced by an oncogenic virus, an oncofetal antigen, or a cell surface glycolipid or glycoprotein.

[0093] Tumour / cancer cell antigens are reviewed by Zarour HM, DeLeo A, Finn OJ, et al. Categories of Tumour Antigens. In: Kufe DW, Pollock RE, Weichselbaum RR, et al., editors. Holland-Frei Cancer Medicine. 6th edition. Hamilton (ON): BC Decker; 2003. Cancer cell antigens include oncofetal antigens: CEA, Immature laminin receptor, TAG-72; oncoviral antigens such as HPV E6 and E7; overexpressed proteins: BING-4, calcium-activated chloride channel 2, cyclin-B1 , 9D7, Ep-CAM, EphA3, HER2 / neu, telomerase, mesothelin, SAP-1 , surviving; cancer-testis antigens: BAGE, CAGE, GAGE, MAGE, SAGE, XAGE, CT9, CT10, NY-ESO-1 , PRAME, SSX-2; lineage restricted antigens: MARTI , Gp100, tyrosinase, TRP-1 / 2, MC1R, prostate specific antigen; mutated antigens: p-catenin, BRCA1 / 2, CDK4, CML66, Fibronectin, MART-2, p53, Ras, TGF-pRII; post-translationally altered antigens: MUC1 , idiotypic antigens: Ig, TCR. Other cancer cell antigens include heat-shock protein 70 (HSP70), heat-shock protein 90 (HSP90), glucose-regulated protein 78 (GRP78), vimentin, nucleolin, feto-acinar pancreatic protein (FAPP), alkaline phosphatase placental-like 2 (ALPPL-2), siglec-5, stress-induced phosphoprotein 1 (STIP1), protein tyrosine kinase 7 (PTK7), and cyclophilin B.

[0094] In some embodiments, the heterologous protein is selected from: an oncofetal antigen, an oncoviral antigen, an overexpressed protein, a cancer-testis antigen, a lineage restricted antigen, a mutated antigen, a post-translationally altered antigen, and an idiotypic antigen. In some embodiments, the heterologous protein is selected from: CEA, immature laminin receptor, TAG-72, HPV E6, HPV E7, BING- 4, calcium-activated chloride channel 2, cyclin-B1 , 9D7, Ep-CAM, EphA3, HER2 / neu, telomerase, mesothelin, SAP-1 , BAGE, CAGE, GAGE, MAGE, SAGE, XAGE, CT9, CT10, NY-ESO-1 , PRAME, SSX- 2, MARTI , Gp100, tyrosinase, TRP-1 / 2, MC1 R, prostate specific antigen, p-catenin, BRCA1 / 2, CDK4, CML66, fibronectin, MART-2, p53, Ras, TGF-pRII, MUC1 , heat-shock protein 70 (HSP70), heat-shock protein 90 (HSP90), glucose-regulated protein 78 (GRP78), vimentin, nucleolin, feto-acinar pancreatic protein (FAPP), alkaline phosphatase placental-like 2 (ALPPL-2), siglec-5, stress-induced phosphoprotein 1 (STIP1), protein tyrosine kinase 7 (PTK7), and cyclophilin B.

[0095] In some embodiments, the heterologous protein is a tumour neoantigen.

[0096] As used herein, a ‘neoantigen’ is an antigen produced by tumour or cancer cells, formed due to mutations that arise in self-proteins in the cells. Neoantigens are absent from normal / non-cancerous / healthy tissues. Neoantigens may be generated by tumour cells as a result of various tumour-specific alterations, such as genomic mutation, dysregulated RNA splicing, disordered post-translational modification, and integrated viral open reading frames. Neoantigens are reviewed in Xie N et al, Signal Transduction and Targeted Therapy 2023, 8(9), which is hereby incorporated by reference in its entirety.

[0097] In some embodiments, the heterologous protein comprises an immunodominant epitope. An epitope or antigenic determinant is a group of amino acids or other chemical groups exposed on the surface of a molecule (e.g. a protein) which can generate an antigenic response.

[0098] As used herein, an ‘immunodominant epitope’ refers to an epitope that is highly targeted by the immune system. Epitopes from a given antigen may be of variable immunogenicity. That is, distinct epitopes from the same antigen may illicit different strengths of immune response. The immune effector cell population that is reactive against a given antigen / tumour / cancer cell / pathogen may be dominated by cells that recognise only certain epitopes. Such epitopes are immunodominant epitopes.

[0099] In some embodiments, the heterologous protein comprises gp70423-43i (AH1).

[0100] In some embodiments, the heterologous protein comprises a mimotope.

[0101] As used herein, a ‘mimotope’ is a peptide that mimics the structure of an epitope. Mimotopes can be developed using e.g. phage display technology.

[0102] In some embodiments, the heterologous protein comprises A5 mimotope.

[0103] In some embodiments, the heterologous protein (e.g. the tumour-associated antigen) is secreted.

[0104] The heterologous protein may comprise a secretory signal. A secretory signal is an amino acid sequence which directs secretion of a protein. The secretory signal may be cleaved from the mature protein once export of the polypeptide chain is initiated / completed. A secretory signal may be at the N-terminus or C- terminus of the heterologous protein. Any suitable secretory signal may be used. The secretory signal may be a secretory signal present in endogenous secreted proteins of the modified microorganism, e.g. a csa secretory signal in C. septicum or a pic secretory signal in C. novyi. In some embodiments, the secretory signal is a csa secretory signal. In some embodiments, the secretory signal is a pic secretory signal.

[0105] B. Particular exemplary embodiments of the modified microorganisms

[0106] In some aspects of the invention, the modified microorganism is a Clostridium bacterium.

[0107] In some embodiments, the Clostridium bacterium is modified to express a heterologous protein and / or modified to reduce or prevent expression of a gene encoding a toxin.

[0108] In some embodiments, the heterologous protein is an antigen. In some embodiments, the antigen is a tumour-associated antigen. In some embodiments, the tumour-associated antigen is a tumour neoantigen, comprises an immunodominant epitope and / or comprises a mimotope.

[0109] In some embodiments, the gene encoding a toxin encodes an alpha toxin. In some embodiments, the gene is csa. In some embodiments, the Clostridium bacterium is of a species selected from Clostridium septicum and Clostridium novyi. In some embodiments the bacterium is of the species Clostridium septicum.

[0110] In some aspects of the invention, the modified microorganism is a Clostridium septicum bacterium.

[0111] In some embodiments, the Clostridium septicum bacterium is modified to express a heterologous protein and / or modified to reduce or prevent expression of a gene encoding a toxin.

[0112] In some embodiments, the heterologous protein is an antigen. In some embodiments, the antigen is a tumour-associated antigen. In some embodiments, the tumour-associated antigen is a tumour neoantigen, comprises an immunodominant epitope and / or comprises a mimotope.

[0113] In some embodiments, the gene encoding a toxin encodes an alpha toxin. In some embodiments, the gene is csa.

[0114] In some aspects of the invention, the microorganism is a Clostridium bacterium of the strain “CS11”. In some embodiments, the Clostridium bacterium of the strain “CS11” is modified to express at least one heterologous protein.

[0115] In some embodiments, the heterologous protein is an antigen. In some embodiments, the antigen is a tumour-associated antigen. In some embodiments, the tumour-associated antigen is a tumour neoantigen, comprises an immunodominant epitope and / or comprises a mimotope.

[0116] The present disclosure also provides a spore (e.g. an endospore) derived from a modified microorganism described hereinabove.

[0117] C. Producing the organisms of the disclosure

[0118] The present disclosure also provides a method for producing a modified microorganism (e.g. a modified Clostridium bacterium) according to the present disclosure.

[0119] In some embodiments, the method for producing a modified microorganism (e.g. a modified Clostridium bacterium) comprises one or more of the following steps: purify ing / isolating the microorganism; modifying (e.g. genetically-modifying) the microorganism; introducing exogenous nucleic acid / vector into the microorganism; culturing the microorganism / modified microorganism; purify ing / isolating the modified microorganism; preparing spores derived from the modified microorganism; and purify ing / isolating the spores. The methods for producing a modified microorganism according to the present disclosure may utilise any suitable microorganism.

[0120] The microorganism may be a bacterium. The microorganism may be a Salmonella, Listeria or Clostridium bacterium. The microorganism may be anaerobic (e.g. an obligate anaerobe). The microorganism may exhibit preferential growth in hypoxic regions. The microorganism may exhibit preferential growth in tumours e.g. in tumours comprising a hypoxic microenvironment and / or hypoxic cells. The microorganism may be a spore-forming microorganism (e.g. an endospore-forming microorganism). A spore derived from the microorganism may be capable of germinating in hypoxic conditions or may only be capable of germinating in hypoxic conditions.

[0121] The microorganism may be a Clostridium bacterium. The Clostridium bacterium may be of a species selected from C. (Clostridium) aceticum, C. acetireducens, C. acetobutylicum, C. acidisoli, C. aciditolerans, C. aestuarii, C. akagii, C. algidicarnis, C. algifaecis, C. algoriphilum, C. amazonense, C. aminophilum, C. ammoniilyticum, C. amylolyticum, C. arbusti, C. arcticum, C. argentinense, C. aurantibutyricum, C. autoethanogenum, C. baratii, C. beihaiense, C. beijerinckii, C. diolis, C. bornimense, C. botulinum, C. bovifaecis, C. bowmanii, C. budayi, C. butyricum, C. cadaveris, C. caldaquaticum, C. carboxidivorans, C. carnis, C. cavendishii, C. celatum, C. cellulofermentans, C. cellulosi, C. cellulovorans, C. chartatabidum, C. chauvoei, C. chromiireducens, C. chrysemydis, C. cochlearium, C. colicanis, C. colinum, C. collagenovorans, C. combesii, C. composti, C. cylindrosporum, C. disporicum, C. drakei, C. estertheticum, C. facile, C. fallax, C. felsineum, C. fermenticellae, C. fessum, C. fimetarium, C. formicaceticum, C. frigidicarnis, C. frigoris, C. fungisolvens, C. ganghwense, C. gasigenes, C. gelidum, C. grantii, C. guangxiense, C. haemolyticum, C. herbivorans, C. homopropionicum, C. horn inis, C. huakuii, C. hydrogeniformans, C. hylemonae, C. indicum, C. innocuum, C. intestinale, C. isatidis, C. jeddahense, C. kluyveri, C. kogasense, C. lacusfryxellense, C. lentum, C. leptum, C. liquoris, C. Ijungdahlii, C. lundense, C. luticellarii, C. magnum, C. malenominatum, C. manihotivorum, C. methoxybenzovorans, C. methylpentosum, C. mobile, C. moniliforme, C. muellerianum, C. neonatale, C. neuense, C. nexile, C. nitritogenes, C. nitrophenolicum, C. novyi, C. oceanicum, C. oryzae, C. pabulibutyricum, C. paradoxum, C. paraputrificum, C. pascui, C. pasteurianum, C. peptidivorans, C. perfringens, C. phytofermentans, C. piliforme, C. polyendosporum, C. polynesiense, C. polysaccharolyticum, C. porci, C. prolinivorans, C. psychrophilum, C. punense, C. puniceum, C. putrefaciens, C. putrificum, C. quinii, C. ragsdalei, C. saccharobutylicum, C. saccharoperbutylacetonicum, C. sardiniense, C. sartagoforme, C. saudiense, C. scatologenes, C. schirmacherense, C. scindens, C. senegalense, C. septicum, C. simiarum, C. sporogenes, C. sporosphaeroides, C. subterminale, C. sulfidigenes, C. swellfunianum, C. symbiosum, C. tagluense, C. tarantellae, C. tepidiprofundi, C. tepidum, C. tertium, C. tetani, C. tetanomorphum, C. thailandense, C. thermarum, C. thermoalcaliphilum, C. thermobutyricum, C. thermopalmarium, C. thermopapyrolyticum, C. thermos uccinogenes, C. thiosulfatireducens, C. tyrobutyricum, C. uliginosum, C. vincentii, C. viride, C. vitabionis, C. vulturis, C. weizhouense, and C. zeae. The microorganism may be from the species Clostridium septicum. The microorganism may be from the species Clostridium novyi. The microorganism may be of Clostridium septicum strain ATCC 11424, Clostridium septicum strain CS11 , or C. novy / -NT. The microorganism may be isolated. The method may comprise a step of purifying or isolating the microorganism. That is, the method may comprise a step of purifying or isolating the microorganism prior to a step of modifying the microorganism. The method may comprise a step of purifying or isolating the modified microorganism. That is, the method may comprise a step of purifying or isolating the modified microorganism after a step of modifying the microorganism.

[0122] The method may comprise a step of modifying the microorganism.

[0123] The method may comprise introducing a nucleic acid or vector into a microorganism. In some embodiments, introducing a nucleic acid or vector according to the present disclosure into a microorganism comprises transformation, transfection, electroporation, transduction (e.g. bacteriophage transduction) or conjugation. In some embodiments, introducing a nucleic acid or vector according to the present disclosure into a microorganism comprises conjugation.

[0124] In some embodiments, the nucleic acid / vector is integrated into the genome of the microorganism. In some embodiments, the nucleic acid / vector is not integrated into the genome of the microorganism.

[0125] Methods of introducing nucleic acid / vectors into microorganisms are well known to the skilled person and are reviewed in e.g. Liu D et al, Engineering Microbiology 2022, 10004, which is hereby incorporated by reference in its entirety.

[0126] The method may comprise modifying a microorganism to express a heterologous protein. In some embodiments, the microorganism is modified to express a heterologous protein. In some embodiments, the nucleic acid / vector introduced into the microorganism encodes a heterologous protein. The heterologous protein may be any heterologous protein described herein.

[0127] The method may comprise modifying a microorganism to express a plurality of heterologous proteins. In some embodiments, the microorganism is modified to express a plurality of heterologous proteins. In some embodiments, the nucleic acid / vector introduced into the microorganism encodes a plurality of heterologous proteins. In some embodiments, a plurality of nucleic acids / vectors encoding one or more heterologous proteins are introduced into the microorganism. The heterologous protein may be any heterologous protein described herein.

[0128] A nucleotide sequence (e.g. a nucleic acid sequence encoding a heterologous protein) may be contained in a vector, e.g. an expression vector. A “vector” as used herein is a nucleic acid molecule used as a vehicle to transfer exogenous nucleic acid into a cell. The vector may be a vector for expression of the nucleic acid in the cell. Such vectors may include a promoter sequence operably linked to the nucleotide sequence encoding the sequence to be expressed. In some embodiments, the promoter is a csa promoter. In some embodiments, the promoter is a flic promoter. A vector may also include a termination codon and expression enhancers. Any suitable vectors, promoters, enhancers and termination codons known in the art may be used to express a peptide or polypeptide from a vector according to the present disclosure.

[0129] The term “operably linked” may include the situation where a selected nucleic acid sequence and regulatory nucleic acid sequence (e.g. promoter and / or enhancer) are covalently linked in such a way as to place the expression of nucleic acid sequence under the influence or control of the regulatory sequence (thereby forming an expression cassette). Thus, a regulatory sequence is operably linked to the selected nucleic acid sequence if the regulatory sequence is capable of effecting transcription of the nucleic acid sequence. The resulting transcripts) may then be translated into a desired pe ptid e (s) / po ly pe pti de (s) .

[0130] The microorganism may be modified to reduce / prevent / knockout expression of an endogenous gene. In some embodiments, the microorganism is modified to reduce or prevent expression of a gene encoding a toxin. Altering / disrupting the nucleotide sequence to inhibit / prevent gene or protein expression from a gene may be referred to as gene ‘knockout’.

[0131] Nucleotide sequences may be disrupted e.g. by homologous recombination or by target nucleic acid modification using site-specific nucleases (SSNs, also referred to herein as ‘gene editing systems’). Genome editing in bacteria is reviewed e.g. in Arroyo-Olarte RD et al, Microorganisms 2021 , 9:844, which is hereby incorporated by reference in its entirety. Methods of genome editing in bacteria include suicide plasmids, the Lambda Red system, the ClosTron method (e.g. TargeTron™ Gene Knockout System), and CRISPR-Cas 9 genome editing systems.

[0132] Modification by homologous recombination may involve the exchange of nucleic acid sequences through crossover events guided by homologous sequences. The homologous sequences flank all or part of the nucleotide sequence to be disrupted. Recombination may be catalysed by a recombinase. For example, in particular aspects and embodiments, the present disclosure contemplates disruption of a nucleotide sequence by homologous recombination between loxP sequences, catalysed by a Cre recombinase.

[0133] Homologous recombination-based methods include the use of suicide plasmids and the Lambda Red system, e.g. as reviewed e.g. in Arroyo-Olarte RD et al, Microorganisms 2021 , 9:844, which is hereby incorporated by reference in its entirety.

[0134] Gene editing using SSNs is reviewed e.g. in Eid and Mahfouz, Exp Mol Med. 2016, 48(10): e265, which is hereby incorporated by reference in its entirety. Enzymes capable of creating site-specific double strand breaks (DSBs) can be engineered to introduce DSBs to target nucleic acid sequences of interest. DSBs may be repaired by error-prone non-homologous end-joining (NHEJ), in which the two ends of the break are rejoined, often with insertion or deletion of nucleotides. Alternatively, DSBs may be repaired by highly homology-directed repair (HDR), in which a DNA template with ends homologous to the break site is supplied and introduced at the site of the DSB. SSNs capable of being engineered to generate target nucleic acid sequence-specific DSBs include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced palindromic repeats / CRISPR- associated-9 (CRISPR / Cas9) systems.

[0135] ZFN systems are reviewed e.g. in Umov et al, Nat Rev Genet. 2010, 11 (9):636-46, which is hereby incorporated by reference in its entirety. ZFNs comprise a programmable Zinc Finger DNA-binding domain and a DNA-cleaving domain (e.g. a Fok\ endonuclease domain). The DNA-binding domain may be identified by screening a Zinc Finger array capable of binding to the target nucleic acid sequence. TALEN systems are reviewed e.g. in Mahfouz et al., Plant Biotechnol J. 2014 12(8): 1006-14, which is hereby incorporated by reference in its entirety. TALENs comprise a programmable DNA-binding TALE domain and a DNA-cleaving domain (e.g. a Fok\ endonuclease domain). TALEs comprise repeat domains consisting of repeats of 33-39 amino acids, which are identical except for two residues at positions 12 and 13 of each repeat which are repeat variable di-residues (RVDs). Each RVD determines binding of the repeat to a nucleotide in the target DNA sequence according to the following relationship: “HD” binds to C, “Nl” binds to A, “NG” binds to T and “NN” or “NK” binds to G (Moscou and Bogdanove, Science 2009, 326(5959):1501). CRISPR / Cas9 and related systems e.g. CRISPR / Cpf1 , CRISPR / C2c1 , CRISPR / C2c2 and CRISPR / C2c3 are reviewed e.g. in Nakade et al, Bioengineered 2017 , 8(3):265-273, which is hereby incorporated by reference in its entirety. These systems comprise an endonuclease (e.g. Cas9, Cpf1 etc.) and the single-guide RNA (sgRNA) molecule. The sgRNA can be engineered to target endonuclease activity to nucleic acid sequences of interest.

[0136] The “ClosTron” system allows the directed construction of stable mutants in Clostridium species using a bacterial group II intron. These are broad host-range elements whose target specificity is determined largely by base-pairing between intron RNA and target site DNA. Such introns can therefore be rationally re-targeted by altering the sequence of an intron RNA-encoding plasmid. The ClosTron system (like most group II intron approaches) uses an element derived from the broad host range LI.LtrB intron of Lactococcus lactis. The ClosTron method is reviewed, e.g. in Heap JT et al, J Microbiol Methods. 2007, 70(3):452-64, and Heap et al, Methods Mol Biol. 2010, 646:165-82, which are hereby incorporated by reference in their entirety.

[0137] The method may comprise a step of culturing the microorganism. The method may comprise a step of culturing the modified microorganism. Methods for culturing / expanding microorganisms are well known in the art. Microorganisms / modified microorganisms of the present disclosure may be cultured in media / conditions conducive to survival / growth of the microorganism / modified microorganism. For example, Clostridia may be cultured essentially as described here in Example 1 “Bacterial strains and growth conditions”.

[0138] The method may comprise a step of preparing spores derived from the modified microorganism. The method may comprise a sporulation step. Methods for preparing spores are well known in the art. The method may comprise a step of culturing the microorganism in a sporulation medium to induce sporulation of the microorganism. Sporulation media are commercially available.

[0139] The method may further comprise a step of purifying the spores by e.g. density centrifugation and / or purification by heat, lysozyme or sonication.

[0140] D. Functional properties of modified microorganisms of the disclosure

[0141] The modified microorganisms described herein may be characterised by reference to certain functional properties. In some embodiments, the modified microorganisms (e.g. modified Clostridia) described herein may possess one or more of the following properties: does not induce / increase killing of cells (e.g. non-cancerous / healthy cells); does not induce death / reduce survival of a host organism; inhibits tumour growth and / or reduces tumour size / volume; increases survival of subjects having cancer; increases resistance to tumour rechallenge; and increases / induces anti-tumour activity of effector T cells (e.g. CD8+ T cells).

[0142] It will be appreciated that a given modified microorganism may display more than one of the properties recited in the preceding paragraph. A given modified microorganism may be evaluated for the properties in the preceding paragraph using suitable assays. The assays may be e.g., in vitro assays, e.g. cellbased assays. Alternatively, the assays may be e.g., in vivo assays, i.e. performed in non-human animals.

[0143] Where assays are cell-based assays, they may comprise contacting cells with a given modified microorganism in order to determine whether the modified microorganism displays one or more of the recited properties.

[0144] The microorganisms described herein may exhibit reduced toxicity / lethality to cells (e.g. non-cancerous cells) and / or a host organism.

[0145] In some embodiments, the modified microorganism does not induce / increase killing of cells (e.g. non- cancerous cells. In some embodiments, the modified microorganism does not reduce viability of cells (i.e. non-cancerous / healthy cells). In some embodiments, the modified microorganism does not induce death in a host organism. In some embodiments, the modified microorganism does not reduce survival in a host organism. In some embodiments, the modified microorganism is not lethal to a host organism.

[0146] The level of killing / viability of cells can be determined by measuring the number / proportion of viable and / or non-viable cells following exposure to different treatment conditions (e.g. following exposure to a given microorganism / modified microorganism). The level of lethality / death / survival in a host organism can be determined by measuring the survival of a host organism following exposure to different treatment conditions (e.g. following exposure to a given microorganism / modified microorganism).

[0147] In some embodiments, administration of a modified microorganism according to the present disclosure may cause one or more of: inhibition of the development / progression of the cancer, a delay to / prevention of onset of the cancer, a reduction in / delay to / prevention of tumour growth, a reduction in / delay to / prevention of tissue invasion, a reduction in / delay to / prevention of metastasis, a reduction in the severity of the symptoms of the cancer, a reduction in the number of cancer cells, a reduction in tumour size / volume, and / or an increase in survival (e.g. progression free survival or overall survival), e.g. as determined in an appropriate model.

[0148] Microorganisms of the present disclosure may be analysed for the properties described in the preceding paragraph in appropriate assays. Such assays include e.g. in vivo models, e.g. performed essentially as described in Example 1 “Subcutaneous tumour models and in vivo studies” and Examples 3-5 herein.

[0149] In some embodiments, the modified microorganism of the present disclosure is capable of reducing / inhibiting tumour growth to less than 1 times, e.g. <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times, <0.05 times, or <0.01 times the tumour growth observed in the absence of treatment with the modified microorganism (or following treatment with an appropriate control), in a given assay.

[0150] In some embodiments, the modified microorganism of the present disclosure is capable of increasing survival of subjects having a cancer (e.g. in an in vivo model) to more than 1 times, e.g. one of >1.01 times, >1.02 times, >1.03 times, >1 .04 times, >1 .05 times, >1.1 times, >1 .2 times, >1 .3 times, >1.4 times, >1 .5 times, >1.6 times, >1 .7 times, >1 .8 times, >1 .9 times, >2 times, >3 times, >4 times, >5 times, >6 times, >7 times, >8 times, >9 times or >10 times the level of survival observed in the absence of treatment with the modified microorganism (or following treatment with an appropriate control), in a given assay.

[0151] In some embodiments, administration of a modified microorganism according to the present disclosure increases resistance to tumour rechallenge. In some embodiments, administration of a modified microorganism according to the present disclosure induces long-term immunity.

[0152] Resistance to tumour rechallenge / long-term immunity may be determined by appropriate assays. Such assays include e.g. in vivo models. Such assays may comprise a subsequent exposure in an in vivo model to a cancer cell / cancer / tumour following a given time interval after a first exposure.

[0153] A modified microorganism may be determined to increase resistance to tumour rechallenge if a tumour is rejected following secondary / subsequent exposure. A modified microorganism may be determined to increase resistance to tumour rechallenge if there is reduced / inhibited tumour growth following secondary / subsequent exposure compared to an appropriate control (e.g. absence of treatment with the modified microorganism or treatment with an appropriate control microorganism).

[0154] In some embodiments, the modified microorganism of the present disclosure is capable of inducing / increasing the activity (e.g. anti-tumour activity) of CD8+ T cells (e.g. antigen specific CD8+ T cells).

[0155] In some embodiments, the modified microorganism of the present disclosure is capable of inducing / increasing the activity (e.g. anti-tumour activity) of CD8+ T cells (e.g. antigen specific CD8+ T cells) to more than 1 times, e.g. one of >1.01 times, >1 .02 times, >1.03 times, >1 .04 times, >1 .05 times, >1 .1 times, >1.2 times, >1 .3 times, >1 .4 times, >1 .5 times, >1 .6 times, >1 .7 times, >1 .8 times, >1 .9 times, >2 times, >3 times, >4 times, >5 times, >6 times, >7 times, >8 times, >9 times or >10 times the level of activity observed in the absence of treatment with the modified microorganism (or following treatment with an appropriate control), in a given assay.

[0156] E. Compositions

[0157] The present disclosure provides a composition comprising a microorganism or spore according to the present disclosure.

[0158] The microorganisms or spores described herein may be formulated as pharmaceutical compositions or medicaments for clinical use and may comprise a pharmaceutically-acceptable carrier, diluent, excipient or adjuvant. Thus, the present disclosure provides a pharmaceutical composition / medicament comprising a microorganism or spore described herein.

[0159] The pharmaceutical compositions / medicaments of the present disclosure may comprise one or more pharmaceutically-acceptable carriers (e.g. liposomes, micelles, microspheres, nanoparticles), diluents / excipients (e.g. starch, cellulose, a cellulose derivative, a polyol, dextrose, maltodextrin, magnesium stearate), adjuvants, fillers, buffers, preservatives (e.g. vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium citrate, methyl paraben, propyl paraben), anti-oxidants (e.g. vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium), lubricants (e.g. magnesium stearate, talc, silica, stearic acid, vegetable stearin), binders (e.g. sucrose, lactose, starch, cellulose, gelatin, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), xylitol, sorbitol, mannitol), stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents or colouring agents (e.g. titanium oxide).

[0160] The term ‘pharmaceutically-acceptable’ as used herein pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g. a human subject) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, adjuvant, filler, buffer, preservative, antioxidant, lubricant, binder, stabiliser, solubiliser, surfactant, masking agent, colouring agent, flavouring agent or sweetening agent of a composition according to the present disclosure must also be ‘acceptable’ in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, binders, stabilisers, solubilisers, surfactants, masking agents, colouring agents, flavouring agents or sweetening agents can be found in standard pharmaceutical texts, for example, Remington’s ‘The Science and Practice of Pharmacy’ (Ed. A. Adejare), 23rdEdition (2020), Academic Press.

[0161] Pharmaceutical compositions and medicaments of the present disclosure may be formulated for topical, parenteral, systemic, intracavitary, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoural, subcutaneous, intradermal, intrathecal, oral or transdermal routes of administration. In some embodiments, a pharmaceutical composition / medicament may be formulated for administration by injection or infusion, or administration by ingestion.

[0162] Medicaments and pharmaceutical compositions may be formulated in fluid, including gel, form. Fluid formulations may be formulated for administration by injection or infusion (e.g. via catheter) to a selected region of the human or animal body.

[0163] In some embodiments, the pharmaceutical compositions / medicament is formulated for injection or infusion, e.g. into a blood vessel, tissue / organ of interest, or a tumour.

[0164] The present disclosure also provides methods for the production of pharmaceutically useful compositions, such methods of production may comprise one or more steps selected from: producing a modified microorganism (e.g. a modified Clostridium bacterium) / spore described herein; isolating / purifying a modified microorganism (e.g. a modified Clostridium bacterium) / spore described herein; and / or mixing a modified microorganism (e.g. a modified microorganism) / spore described herein with a pharmaceutically-acceptable carrier, adjuvant, excipient or diluent.

[0165] For example, a further aspect of the present disclosure relates to a method of formulating or producing a medicament or pharmaceutical composition for use in the treatment of a disease / condition (e.g. a disease / condition described herein, e.g. a cancer), the method comprising formulating a pharmaceutical composition or medicament by mixing a microorganism / spore described herein with a pharmaceutically- acceptable carrier, adjuvant, excipient or diluent.

[0166] F. Therapeutic and prophylactic applications

[0167] Aspects and embodiments of the present disclosure relate to therapeutic and prophylactic intervention.

[0168] The modified microorganisms, spores and compositions described herein find use in therapeutic and prophylactic intervention for disease, e.g. cancers. The present disclosure provides a modified microorganism, spore or composition described herein for use in a method of medical treatment or prophylaxis.

[0169] Also provided is a modified microorganism, spore or composition described herein for use in a method of treating or preventing a cancer. Also provided is the use of a modified microorganism, spore or composition described herein in the manufacture of a medicament for treating or preventing a cancer. Also provided is a method of treating or preventing a cancer in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of a modified microorganism, spore or composition described herein.

[0170] The methods may be effective to reduce the development or progression of a cancer, alleviation of the symptoms of a cancer or reduction in the pathology of a cancer. The methods may be effective to prevent progression of the cancer, e.g. to prevent worsening of, or to slow the rate of development of, the cancer. In some embodiments, the methods may lead to an improvement in the cancer, e.g. a reduction in the symptoms of the cancer or reduction in some other correlate of the severity / activity of the cancer. In some embodiments, the methods may prevent development of the cancer to a later stage (e.g. a chronic stage or metastasis).

[0171] As used herein, a ‘cancer’ may be or comprise any unwanted cell proliferation (or any disease manifesting itself by unwanted cell proliferation), neoplasm or tumour. The cancer may be benign or malignant. The cancer may be primary or secondary (metastatic). A neoplasm or tumour may be any abnormal growth or proliferation of cells and may be located in any tissue. The cancer may be of tissues / cells derived from e.g. the adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain) cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g. renal epithelia), gallbladder, biliary tract, oesophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal glad, larynx, liver, lung, lymph, lymph node, lymphoblast, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissues, spleen, stomach, testis, thymus, thyroid gland, tongue, tonsil, trachea, uterus, vulva, or white blood cells.

[0172] Tumours to be treated may be nervous or non-nervous system tumours. Nervous system tumours may originate either in the central or peripheral nervous system, e.g. glioma, medulloblastoma, meningioma, neurofibroma, ependymoma, Schwannoma, neurofibrosarcoma, astrocytoma and oligodendroglioma. Non-nervous system cancers / tumours may originate in any other non-nervous tissue; examples include melanoma, mesothelioma, lymphoma, myeloma, leukemia, Non-Hodgkin’s lymphoma (NHL), Hodgkin’s lymphoma, chronic myelogenous leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma (CTCL), chronic lymphocytic leukemia (CLL), hepatoma, epidermoid carcinoma, prostate carcinoma, breast cancer, lung cancer, colon cancer, ovarian cancer, endometrial cancer, pancreatic cancer, thymic carcinoma, NSCLC, hematologic cancer and sarcoma. In some embodiments, the cancer is selected from: a solid tumour, breast cancer, breast carcinoma, ductal carcinoma, gastric cancer, gastric carcinoma, gastric adenocarcinoma, colorectal cancer, colorectal carcinoma, colorectal adenocarcinoma, head and neck cancer, squamous cell carcinoma of the head and neck, lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung carcinoma, ovarian cancer, ovarian carcinoma, ovarian serous adenocarcinoma, renal cancer, renal cell carcinoma, renal clear cell carcinoma, renal cell adenocarcinoma, renal papillary cell carcinoma, pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, cervical cancer, cervical squamous cell carcinoma, skin cancer, melanoma, esophageal cancer, esophageal adenocarcinoma, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, uterine cancer, uterine corpus endometrial carcinoma, thyroid cancer, thyroid carcinoma, pheochromocytoma, paraganglioma, bladder cancer, bladder urothelial carcinoma, prostate cancer, prostate adenocarcinoma, sarcoma and thymoma.

[0173] In some embodiments the cancer is a solid tumour. In some embodiments the cancer is colorectal cancer, breast cancer, or lung cancer.

[0174] In some embodiments, the cancer is associated with hypoxia.

[0175] Tumour cells that are situated close to the blood vessels are well oxygenated. As the distance from the blood vessels lengthens, less oxygen diffuses out to the tumour core, resulting in an interim layer of hypoxic viable cells and finally necrotic cells. This corresponds to a gradient of increasing chemotherapy and radiation resistance.

[0176] One of the characteristic hallmarks of solid tumours is hypoxia. Hypoxia is a state in which oxygen is not available in sufficient amounts at the tissue level to maintain adequate homeostasis. The rapidly proliferating tumour mass outgrows the vasculature support of the tumour microenvironment, thus resulting in a gradient of aerobic, hypoxic and necrotic tumour cells corresponding to the distance from the blood supply. Increasing hypoxia in the tumour is also correlated to increasing resistance of chemotherapeutic drugs and radiation. Besides reduced supply of chemotherapeutic drugs to the tumour core, hypoxia induces upregulation of several pathways involving hypoxia-inducible factor 1-alpha (HIF- 1a). These pathways can confer resistance to chemotherapeutic drugs through a myriad of ways, mainly by overexpression of drug efflux, dysregulation of apoptosis, induction of autophagy, inhibition of DNA damage, downregulation of mitochondrial activity and regulation of p53 activities. Radiation therapy primarily works by generating reactive oxygen species to form free radicals, creating oxidative stress and ultimately cell death, but since there is low oxygen content in the hypoxic region of the tumour mass, the free radicals produced do little to affect the cells. In the hypoxic regions of the tumours, upregulation of HIF-1a confers drug resistance through a variety of methods. In addition, the acidic pH of the tumour microenvironment leads to various phenomena which contribute to increased drug resistance.

[0177] All types of solid tumours, especially malignant solid tumours, are subject to hypoxia, often exhibiting oxygenation levels measurably lower than their tissue of origin. Recurring tumours often exhibit a higher hypoxic fraction than primary tumours. The pathophysiology of tumour hypoxia and methods for assessing tumour hypoxia are reviewed in Walsh JC et al, Antioxid Redox Signal. 2014, 21 (10):1516- 1554, which is hereby incorporated by reference in its entirety.

[0178] In some embodiments, hypoxia is a hallmark of the cancer. In some embodiments, the cancer: is associated with hypoxia; is characterised by hypoxia; is associated with tumour hypoxia; is characterised by tumour hypoxia; comprises a hypoxic tumour microenvironment; comprises a region / area of hypoxia; comprises hypoxic cells; has deficient oxygenation. In some embodiments, the cancer / tumour exhibits decreased oxygenation levels, e.g. compared to a reference tissue (e.g. the tissue of origin of the tumour).

[0179] In some embodiments, the cancer is characterised by expression of an antigen, e.g. a tumour-associated antigen.

[0180] In some embodiments, the cancer to be treated / prevented comprises cells expressing an antigen, e.g. a tumour-associated antigen. In some embodiments, the cancer to be treated / prevented is a cancer which is positive for an antigen, e.g. a tumour-associated antigen. In some embodiments, the cancer comprises cells that overexpress an antigen, e.g. a tumour-associated antigen. Overexpression can be determined by detection of a level of expression which is greater than the level of expression by equivalent non- cancerous cells / non-tumour tissue.

[0181] In some embodiments, the cancer is a cancer in which an antigen, e.g. a tumour-associated antigen is pathologically-implicated. That is, in some embodiments the cancer is a cancer which is caused or exacerbated by the expression of an antigen, a cancer for which expression of an antigen is a risk factor and / or a cancer for which expression of an antigen is positively associated with onset, development, progression, severity or metastasis of the cancer. The cancer may be characterised by expression of an antigen, e.g. a tumour-associated antigen, e.g. the cancer may comprise cells (e.g. cells of tumour tissue) expressing the given antigen. Such cancers may be referred to as being positive for an antigen. A cancer which is ‘positive’ for an antigen may be a cancer comprising cells expressing the antigen (e.g. at the cell surface). A cancer which is ‘positive’ for an antigen may overexpress the antigen.

[0182] It will be appreciated that modified microorganisms of the present disclosure that are modified to express a given antigen (e.g. a given tumour-associated antigen) find particular utility in the treatment / prevention of a cancer comprising cells expressing the given antigen.

[0183] Without being bound by theory, the modified microorganisms stimulate anti-tumour responses of the host immune system. Modified microorganisms expressing an antigen are able to recruit and activate immune cells (e.g. cytotoxic CD8+ T cells) specific to the expressed antigen. Therefore, where the modified microorganism expresses the same antigen as is expressed by a cancer / tumour, the modified microorganism effects the recruitment and activation of anti-tumour immune cells. In essence, the modified microorganisms function as live immunologic adjuvants. Also provided is a modified microorganism, spore or composition described herein for use in a method of treating or preventing a cancer, wherein the cancer to be treated / prevented comprises cells expressing a given antigen, and wherein the microorganism is modified to express the given antigen. Also provided is the use of a modified microorganism, spore or composition described herein in the manufacture of a medicament for treating or preventing a cancer, wherein the cancer to be treated / prevented comprises cells expressing a given antigen, and wherein the microorganism is modified to express the given antigen. Also provided is a method of treating or preventing a cancer in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of a modified microorganism, spore or composition described herein, wherein the cancer to be treated / prevented comprises cells expressing a given antigen, and wherein the microorganism is modified to express the given antigen.

[0184] In some embodiments, the cancer to be treated / prevented comprises cells expressing the given antigen. In some embodiments, the cancer to be treated / prevented is a cancer which is positive for the given antigen. In some embodiments, the cancer comprises cells that overexpress the given antigen. Overexpression can be determined by detection of a level of expression which is greater than the level of expression by equivalent non-cancerous cells / non-tumour tissue.

[0185] It will be appreciated that a cancer described in the above paragraphs may express a plurality of antigens, e.g. tumour-associated antigens. In some embodiments, the cancer is characterised by expression of a plurality of antigens, e.g. tumour-associated antigens. In some embodiments, the cancer to be treated / prevented comprises cells expressing (and / or overexpressing) a plurality of antigens, e.g. tumour-associated antigens.

[0186] Modified microorganisms of the present disclosure that are modified to express a given plurality of antigens (e.g. given tumour-associated antigens) find particular utility in the treatment / prevention of a cancer comprising cells expressing the given plurality of antigens. It will be appreciated that a plurality of modified microorganisms, each of which express one or more antigens, may be used in the treatment / prevention of a cancer comprising cells expressing a plurality of antigens. By way of explanation, an individual modified microorganism within the plurality of modified microorganisms may not express each of the plurality of antigens. Instead, a modified microorganism expressing a given antigen(s) may be used in combination with other modified microorganism(s) expressing a different antigen(s). Provided herein is a heterologous combination of modified microorganisms as described herein for use in a method of treating or preventing a cancer.

[0187] Expression may be determined by any suitable means. Expression may be gene expression or protein expression. Gene expression can be determined e.g. by detection of mRNA encoding the given antigen, for example by quantitative real-time PCR (qRT-PCR). Protein expression can be determined e.g. by antibody-based methods, for example by western blot, immunohistochemistry, immunocytochemistry, flow cytometry, or ELISA. In some embodiments the cancer is a cancer in which the given antigen is pathologically-implicated. That is, in some embodiments the cancer is a cancer which is caused or exacerbated by the expression of the given antigen, a cancer for which expression of the given antigen is a risk factor and / or a cancer for which expression of the given antigen is positively associated with onset, development, progression, severity and / or metastasis of the cancer. The cancer may be characterised by expression of the given antigen, e.g. the cancer may comprise cells (e.g. cells of tumour tissue) expressing the given antigen. Such cancers may be referred to as being positive for the given antigen. A cancer which is ‘positive’ for the given antigen may be a cancer comprising cells expressing the given antigen (e.g. at the cell surface). A cancer which is ‘positive’ for the given antigen may overexpress the given antigen.

[0188] Identification of tumour antigens is reviewed, e.g. in Leko V and Rosenberg SA, Cancer Cell 2020, 38(4): 454-472, which is hereby incorporated by reference in its entirety.

[0189] In some embodiments, the antigen is a neoantigen. Neoantigens can be determined as described in Xie N et al, Signal Transduction and Targeted Therapy 2023, 8(9), which is hereby incorporated by reference in its entirety.

[0190] A cancer may be analysed to determi ne / identify an antigen(s) which it expresses / overexpresses. In some embodiments, the method comprises a step of determining / identifying an antigen expressed in the cancer / tumour to be treated.

[0191] A modified microorganism may be produced which is modified to express an antigen, wherein the antigen is expressed in the cancer to be treated. In some embodiments, the method comprises a step of producing a microorganism modified to express an antigen, wherein the antigen is expressed in the cancer to be treated.

[0192] In some embodiments, the method of treating cancer in a subject comprises:

[0193] (i) administering a modified microorganism, a spore derived from a modified microorganism, or a composition comprising a modified microorganism or spore derived from a modified microorganism according to the present disclosure, to the subject, wherein the modified microorganism has been modified to express an antigen (e.g. a tumour associated antigen) identified as being expressed in the cancer to be treated.

[0194] In some embodiments, the method of treating cancer in a subject comprises:

[0195] (i) identifying an antigen (e.g. a tumour associated antigen) expressed in the cancer to be treated; and

[0196] (ii) administering a modified microorganism, a spore derived from a modified microorganism, or a composition comprising a modified microorganism or spore derived from a modified microorganism according to the present disclosure, to the subject, wherein the modified microorganism has been modified to express the antigen. In some embodiments, the method of treating cancer in a subject comprises:

[0197] (i) identifying an antigen (e.g. a tumour associated antigen) expressed in the cancer to be treated;

[0198] (ii) producing a microorganism modified to express the antigen; and optionally,

[0199] (iii) administering the modified microorganism, a spore derived from the modified microorganism, or a composition comprising the modified microorganism or spore derived from the modified microorganism according to the present disclosure, to the subject.

[0200] It will be appreciated that a plurality of antigens may be / may have been identified. The microorganism may be / may have been modified to express said plurality of antigens. It will also be appreciated that the plurality of antigens may be expressed across a plurality of microorganisms (e.g. each individual microorganism may not express each of the plurality of antigens, instead, a microorganism may be / may have been modified to express a given antigen(s) and may be used in combination with other microorganism(s) which may be / may have been modified to express a different antigen(s)).

[0201] Also provided herein is a modified microorganism, a spore derived from a modified microorganism, or a composition comprising a modified microorganism or spore derived from a modified microorganism, for use in a method of debulking a tumour. Also provided herein is the use of a modified microorganism, a spore derived from a modified microorganism, or a composition comprising a modified microorganism or spore derived from a modified microorganism in the manufacture of a medicament for debulking a tumour. Also provided herein is a method of debulking a tumour comprising administering to a subject a therapeutically- or prophylactically-effective amount of a modified microorganism, a spore derived from a modified microorganism, or a composition comprising a modified microorganism or spore derived from a modified microorganism. The tumour may be a tumour as described hereinabove.

[0202] The modified microorganisms, spores and compositions described herein find use in combination therapies.

[0203] In some embodiments, the modified microorganisms, spores and compositions of the present disclosure are used in a method of treatment further comprising an additional therapeutic or prophylactic intervention.

[0204] In some embodiments, the methods comprise additional therapeutic or prophylactic intervention, e.g. for the treatment / prevention of a cancer. In some embodiments, the therapeutic or prophylactic intervention is selected from chemotherapy, immunotherapy, radiotherapy, surgery and / or hormone therapy.

[0205] Simultaneous administration refers to administration of the modified microorganism, spore or composition and therapeutic agent together, for example as a pharmaceutical composition containing both agents (combined preparation), or immediately after each other and optionally via the same route of administration, e.g. to the same artery, vein or other blood vessel. Sequential administration refers to administration of one of the modified microorganism / spore / composition or therapeutic agent followed after a given time interval by separate administration of the other agent. It is not required that the two agents are administered by the same route, although this is the case in some embodiments. The time interval may be any time interval.

[0206] Chemotherapy and radiotherapy respectively refer to treatment of a cancer with a drug or with ionising radiation (e.g. radiotherapy using X-rays or y-rays). The drug may be a chemical entity, e.g. small molecule pharmaceutical, antibiotic, DNA intercalator, protein inhibitor (e.g. kinase inhibitor), or a biological agent, e.g. antibody, antibody fragment, aptamer, nucleic acid (e.g. DNA, RNA), peptide, polypeptide, or protein. The drug may be formulated as a pharmaceutical composition or medicament. The formulation may comprise one or more drugs (e.g. one or more active agents) together with one or more pharmaceutically acceptable diluents, excipients or carriers.

[0207] A treatment may involve administration of more than one drug. A drug may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. For example, the chemotherapy may be a co-therapy involving administration of two drugs, one or more of which may be intended to treat the cancer.

[0208] The chemotherapy may be administered by one or more routes of administration, e.g. parenteral, intravenous injection, oral, subcutaneous, intradermal or intratumoural.

[0209] The chemotherapy may be administered according to a treatment regime. The treatment regime may be a pre-determined timetable, plan, scheme or schedule of chemotherapy administration which may be prepared by a physician or medical practitioner and may be tailored to suit the patient requiring treatment. The treatment regime may indicate one or more of: the type of chemotherapy to administer to the patient; the dose of each drug or radiation; the time interval between administrations; the length of each treatment; the number and nature of any treatment holidays, if any etc. For a co-therapy a single treatment regime may be provided which indicates how each drug is to be administered.

[0210] In some embodiments, the method of treatment comprises administration of a chemotherapeutic agent.

[0211] Chemotherapeutic drugs / agents may be selected from: Abemaciclib, Abiraterone Acetate, Abitrexate (Methotrexate), Abraxane (Paclitaxel Albumin-stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE- PC, AC, Acalabrutinib, AC-T, Adcetris (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, Adriamycin (Doxorubicin Hydrochloride), Afatinib Dimaleate, Afinitor (Everolimus), Akynzeo (Netupitant and Palonosetron Hydrochloride), Aldara (Imiquimod), Aldesleukin, Alecensa (Alectinib), Alectinib, Alemtuzumab, Alimta (Pemetrexed Disodium), Aliqopa (Copanlisib Hydrochloride), Alkeran for Injection (Melphalan Hydrochloride), Alkeran Tablets (Melphalan), Aloxi (Palonosetron Hydrochloride), Alunbrig (Brigatinib), Ambochlorin (Chlorambucil), Amboclorin (Chlorambucil), Amifostine, Aminolevulinic Acid, Anastrozole, Aprepitant, Aredia (Pamidronate Disodium), Arimidex (Anastrozole), Aromasin (Exemestane), Arranon (Nelarabine), Arsenic Trioxide, Arzerra (Ofatumumab), Asparaginase Erwinia chrysanthemi, Atezolizumab, Avastin (Bevacizumab), Avelumab, Axicabtagene Ciloleucel, Axitinib, Azacitidine, Bavencio (Avelumab), BEACOPP, Becenum (Carmustine), Beleodaq (Belinostat), Belinostat, Bendamustine Hydrochloride, BEP, Besponsa (Inotuzumab Ozogamicin) , Bevacizumab, Bexarotene, Bexxar (Tositumomab and Iodine I 131 Tositumomab), Bicalutamide, BiCNU (Carmustine), Bleomycin, Blinatumomab, Blincyto (Blinatumomab), Bortezomib, Bosulif (Bosutinib), Bosutinib, Brentuximab Vedotin, Brigatinib, BuMel, Busulfan, Busulfex (Busulfan), Cabazitaxel, Cabometyx (Cabozantinib-S- Malate), Cabozantinib-S-Malate, CAF, Calquence (Acalabrutinib), Campath (Alemtuzumab), Camptosar (Irinotecan Hydrochloride), Capecitabine, CAPOX, Carac (Fluorouracil-Topical), Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, Carmubris (Carmustine), Carmustine, Carmustine Implant, Casodex (Bicalutamide), CEM, Ceritinib, Cerubidine (Daunorubicin Hydrochloride), Cervarix (Recombinant HPV Bivalent Vaccine), Cetuximab, CEV, Chlorambucil, CHLORAMBUCIL-PREDNISONE, CHOP, Cisplatin, Cladribine, Clafen (Cyclophosphamide), Clofarabine, Clofarex (Clofarabine), Clolar (Clofarabine), CMF, Cobimetinib, Cometriq (Cabozantinib-S-Malate), Copanlisib Hydrochloride, COPDAC, COPP, COPP-ABV, Cosmegen (Dactinomycin), Cotellic (Cobimetinib), Crizotinib, CVP, Cyclophosphamide, Cyfos (Ifosfamide), Cyramza (Ramucirumab), Cytarabine, Cytarabine Liposome, Cytosar-U (Cytarabine), Cytoxan (Cyclophosphamide), Dabrafenib, Dacarbazine, Dacogen (Decitabine), Dactinomycin, Daratumumab, Darzalex (Daratumumab), Dasatinib, Daunorubicin Hydrochloride, Daunorubicin Hydrochloride and Cytarabine Liposome, Decitabine, Defibrotide Sodium, Defitelio (Defibrotide Sodium), Degarelix, Denileukin Diftitox , Denosumab, DepoCyt (Cytarabine Liposome), Dexamethasone, Dexrazoxane Hydrochloride, Dinutuximab, Docetaxel, Doxil (Doxorubicin Hydrochloride Liposome), Doxorubicin Hydrochloride, Doxorubicin Hydrochloride Liposome, Dox-SL (Doxorubicin Hydrochloride Liposome), DTIC-Dome (Dacarbazine), Durvalumab, Efudex (Fluorouracil-Topical), Elitek (Rasburicase), Ellence (Epirubicin Hydrochloride), Elotuzumab, Eloxatin (Oxaliplatin), Eltrombopag Olamine, Emend (Aprepitant), Empliciti (Elotuzumab), Enasidenib Mesylate, Enzalutamide, Epirubicin Hydrochloride, EPOCH, Erbitux (Cetuximab), Eribulin Mesylate, Erivedge (Vismodegib), Erlotinib Hydrochloride, Erwinaze (Asparaginase Erwinia chrysanthemi), Ethyol (Amifostine), Etopophos (Etoposide Phosphate), Etoposide, Etoposide Phosphate, Evacet (Doxorubicin Hydrochloride Liposome), Everolimus, Evista (Raloxifene Hydrochloride), Evomela (Melphalan Hydrochloride), Exemestane, 5-FU (Fluorouracil Injection), 5-FU (Fluorouracil-Topical), Fareston (Toremifene), Farydak (Panobinostat), Faslodex (Fulvestrant), FEC, Femara (Letrozole), Filgrastim, Fludara (Fludarabine Phosphate), Fludarabine Phosphate, Fluoroplex (Fluorouracil-Topical), Fluorouracil Injection, Fluorouracil— Topical, Flutamide, Folex (Methotrexate), Folex PFS (Methotrexate), FOLFIRI, FOLFIRI-BEVACIZUMAB, FOLFIRI-CETUXIMAB, FOLFIRINOX, FOLFOX, Folotyn (Pralatrexate), FU-LV, Fulvestrant, Gardasil (Recombinant HPV Quadrivalent Vaccine), Gardasil 9 (Recombinant HPV Nonavalent Vaccine), Gazyva (Obinutuzumab), Gefitinib, Gemcitabine Hydrochloride, GEMCITABINE-CISPLATIN, GEMCITABINEOXALIPLATIN, Gemtuzumab Ozogamicin, Gemzar (Gemcitabine Hydrochloride), Gilotrif (Afatinib Dimaleate), Gleevec (Imatinib Mesylate), Gliadel (Carmustine Implant), Gliadel wafer (Carmustine Implant), Glucarpidase, Goserelin Acetate, Halaven (Eribulin Mesylate), Hemangeol (Propranolol Hydrochloride), Herceptin (Trastuzumab), HPV Bivalent Vaccine, Recombinant, HPV Nonavalent Vaccine, Recombinant, HPV Quadrivalent Vaccine, Recombinant, Hycamtin (Topotecan Hydrochloride), Hydrea (Hydroxyurea), Hydroxyurea, Hyper-CVAD, Ibrance (Palbociclib), Ibritumomab Tiuxetan, Ibrutinib, ICE, Iclusig (Ponatinib Hydrochloride), Idamycin (Idarubicin Hydrochloride), Idarubicin Hydrochloride, Idelalisib, Idhifa (Enasidenib Mesylate), Ifex (Ifosfamide), Ifosfamide, Ifosfamidum (Ifosfamide), IL-2 (Aldesleukin), Imatinib Mesylate, Imbruvica (Ibrutinib), Imfinzi (Durvalumab), Imiquimod, Imlygic (Talimogene Laherparepvec), Inlyta (Axitinib), Inotuzumab Ozogamicin, Interferon Alfa-2b, Recombinant, lnterleukin-2 (Aldesleukin), Intron A (Recombinant Interferon Alfa-2b), Iodine I 131 Tositumomab and Tositumomab, Ipilimumab, Iressa (Gefitinib), Irinotecan Hydrochloride, Irinotecan Hydrochloride Liposome, Istodax (Romidepsin), Ixabepilone, Ixazomib Citrate, Ixempra (Ixabepilone), Jakafi (Ruxolitinib Phosphate), JEB, Jevtana (Cabazitaxel), Kadcyla (Ado-Trastuzumab Emtansine), Keoxifene (Raloxifene Hydrochloride), Kepivance (Palifermin), Keytruda (Pembrolizumab), Kisqali (Ribociclib), Kymriah (Tisagenlecleucel), Kyprolis (Carfilzomib), Lanreotide Acetate, Lapatinib Ditosylate, Lartruvo (Olaratumab), Lenalidomide, Lenvatinib Mesylate, Lenvima (Lenvatinib Mesylate), Letrozole, Leucovorin Calcium, Leukeran (Chlorambucil), Leuprolide Acetate, Leustatin (Cladribine), Levulan (Aminolevulinic Acid), Linfolizin (Chlorambucil), LipoDox (Doxorubicin Hydrochloride Liposome), Lomustine, Lonsurf (Trifluridine and Tipiracil Hydrochloride), Lupron (Leuprolide Acetate), Lupron Depot (Leuprolide Acetate), Lupron Depot-Ped (Leuprolide Acetate), Lynparza (Olaparib), Marqibo (Vincristine Sulfate Liposome), Matulane (Procarbazine Hydrochloride), Mechlorethamine Hydrochloride, Megestrol Acetate, Mekinist (Trametinib), Melphalan, Melphalan Hydrochloride, Mercaptopurine, Mesna, Mesnex (Mesna), Methazolastone (Temozolomide), Methotrexate, Methotrexate LPF (Methotrexate), Methylnaltrexone Bromide, Mexate (Methotrexate), Mexate-AQ (Methotrexate), Midostaurin, Mitomycin C, Mitoxantrone Hydrochloride, Mitozytrex (Mitomycin C), MOPP, Mozobil (Plerixafor), Mustargen (Mechlorethamine Hydrochloride), Mutamycin (Mitomycin C), Myleran (Busulfan), Mylosar (Azacitidine), Mylotarg (Gemtuzumab Ozogamicin), Nanoparticle Paclitaxel (Paclitaxel Albumin-stabilized Nanoparticle Formulation), Navelbine (Vinorelbine Tartrate), Necitumumab, Nelarabine, Neosar (Cyclophosphamide), Neratinib Maleate, Nerlynx (Neratinib Maleate), Netupitant and Palonosetron Hydrochloride, Neulasta (Pegfilgrastim), Neupogen (Filgrastim), Nexavar (Sorafenib Tosylate), Nilandron (Nilutamide), Nilotinib, Nilutamide, Ninlaro (Ixazomib Citrate), Niraparib Tosylate Monohydrate, Nivolumab, Nolvadex (Tamoxifen Citrate), Nplate (Romiplostim), Obinutuzumab, Odomzo (Sonidegib), OEPA, Ofatumumab, OFF, Olaparib, Olaratumab, Omacetaxine Mepesuccinate, Oncaspar (Pegaspargase), Ondansetron Hydrochloride, Onivyde (Irinotecan Hydrochloride Liposome), Ontak (Denileukin Diftitox), Opdivo (Nivolumab), OPPA, Osimertinib, Oxaliplatin, Paclitaxel, Paclitaxel Albumin-stabilized Nanoparticle Formulation, PAD, Palbociclib, Palifermin, Palonosetron Hydrochloride, Palonosetron Hydrochloride and Netupitant, Pamidronate Disodium, Panitumumab, Panobinostat, Paraplat (Carboplatin), Paraplatin (Carboplatin), Pazopanib Hydrochloride, PCV, PEB, Pegaspargase, Pegfilgrastim, Peginterferon Alfa-2b, PEG-lntron (Peginterferon Alfa-2b), Pembrolizumab, Pemetrexed Disodium, Perjeta (Pertuzumab), Pertuzumab, Platinol (Cisplatin), Platinol-AQ (Cisplatin), Plerixafor, Pomalidomide, Pomalyst (Pomalidomide), Ponatinib Hydrochloride, Portrazza (Necitumumab), Pralatrexate, Prednisone, Procarbazine Hydrochloride, Proleukin (Aldesleukin), Prolia (Denosumab), Promacta (Eltrombopag Olamine), Propranolol Hydrochloride, Provenge (Sipuleucel-T), Purinethol (Mercaptopurine), Purixan (Mercaptopurine), [No Entries], Radium 223 Dichloride, Raloxifene Hydrochloride, Ramucirumab, Rasburicase, R-CHOP, R-CVP, Recombinant Human Papillomavirus (HPV) Bivalent Vaccine, Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine, Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine, Recombinant Interferon Alfa-2b, Regorafenib, Relistor (Methylnaltrexone Bromide), R-EPOCH, Revlimid (Lenalidomide), Rheumatrex (Methotrexate), Ribociclib, R-ICE, Rituxan (Rituximab), Rituxan Hycela (Rituximab and Hyaluronidase Human), Rituximab, Rituximab and Hyaluronidase Human, Rolapitant Hydrochloride, Romidepsin, Romiplostim, Rubidomycin (Daunorubicin Hydrochloride), Rubraca (Rucaparib Camsylate), Rucaparib Camsylate, Ruxolitinib Phosphate, Rydapt (Midostaurin), Sclerosol Intrapleural Aerosol (Talc), Siltuximab, Sipuleucel-T, Somatuline Depot (Lanreotide Acetate), Sonidegib, Sorafenib Tosylate, Sprycel (Dasatinib), STANFORD V, Sterile Talc Powder (Talc), Steritalc (Talc), Stivarga (Rego rate nib), Sunitinib Malate, Sutent (Sunitinib Malate), Sylatron (Peginterferon Alfa-2b), Sylvant (Siltuximab), Synribo (Omacetaxine Mepesuccinate), Tabloid (Thioguanine), TAC, Tafinlar (Dabrafenib), Tagrisso (Osimertinib), Talc, Talimogene Laherparepvec, Tamoxifen Citrate, Tarabine PFS (Cytarabine), Tarceva (Erlotinib Hydrochloride), Targretin (Bexarotene), Tasigna (Nilotinib), Taxol (Paclitaxel), Taxotere (Docetaxel), Tecentriq (Atezolizumab), Temodar (Temozolomide), Temozolomide, Temsirolimus, Thalidomide, Thalomid (Thalidomide), Thioguanine, Thiotepa, Tisagenlecleucel, Tolak (Fluorouracil-Topical), Topotecan Hydrochloride, Toremifene, Torisel (Temsirolimus), Tositumomab and Iodine I 131 Tositumomab, Totect (Dexrazoxane Hydrochloride), TPF, Trabectedin, Trametinib, Trastuzumab, Treanda (Bendamustine Hydrochloride), Trifluridine and Tipiracil Hydrochloride, Trisenox (Arsenic Trioxide), Tykerb (Lapatinib Ditosylate), Unituxin (Dinutuximab), Uridine Triacetate, VAC, Valrubicin, Valstar (Valrubicin), Vandetanib, VAMP, Varubi (Rolapitant Hydrochloride), Vectibix (Panitumumab), VelP, Velban (Vinblastine Sulfate), Velcade (Bortezomib), Velsar (Vinblastine Sulfate), Vemurafenib, Venclexta (Venetoclax), Venetoclax, Verzenio (Abemaciclib), Viadur (Leuprolide Acetate), Vidaza (Azacitidine), Vinblastine Sulfate, Vincasar PFS (Vincristine Sulfate), Vincristine Sulfate, Vincristine Sulfate Liposome, Vinorelbine Tartrate, VIP, Vismodegib, Vistogard (Uridine Triacetate), Voraxaze (Glucarpidase), Vorinostat, Votrient (Pazopanib Hydrochloride), Vyxeos (Daunorubicin Hydrochloride and Cytarabine Liposome), Wellcovorin (Leucovorin Calcium), Xalkori (Crizotinib), Xeloda (Capecitabine), XELIRI, XELOX, Xgeva (Denosumab), Xofigo (Radium 223 Dichloride), Xtandi (Enzalutamide), Yervoy (Ipilimumab), Yescarta (Axicabtagene Ciloleucel), Yondelis (Trabectedin), Zaltrap (Ziv-Aflibercept), Zarxio (Filgrastim), Zejula (Niraparib Tosylate Monohydrate), Zelboraf (Vemurafenib), Zevalin (Ibritumomab Tiuxetan), Zinecard (Dexrazoxane Hydrochloride), Ziv-Aflibercept, Zofran (Ondansetron Hydrochloride), Zoladex (Goserelin Acetate), Zoledronic Acid, Zolinza (Vorinostat), Zometa (Zoledronic Acid), Zydelig (Idelalisib), Zykadia (Ceritinib) and Zytiga (Abiraterone Acetate).

[0212] In some embodiments, the method of treatment comprises administration of an immunomodulatory agent.

[0213] As used herein, an ‘immunomodulatory agent’ refers to an agent that modulates / modifies the immune system. An immunomodulatory agent may stimulate or supress the immune system. An immunomodulatory agent may also be referred to as an ‘immunomodulator’.

[0214] An immunomodulatory agent that stimulates / increases an immune response may also be called an immunostimulatory agent or an immunostimulator. As used herein, an ‘immunostimulatory agent’ or ‘immunostimulator’ is an immunomodulatory agent that stimulates / increases the immune system. In some embodiments, the method of treatment comprises administration of an immunostimulatory agent. Immunomodulatory / immunostimulatory agents may stimulate / increase an immune response by increasing the immune activity of effector cells. Immunomodulatory / immunostimulatory agents may stimulate / increase an immune response by suppressing / reducing immunosuppressive activity.

[0215] Immunomodulatory therapeutics are reviewed e.g. in Nash A et al, Advanced Drug Delivery Reviews 2021 , 176:113896, which is hereby incorporated by reference in its entirety.

[0216] In some embodiments, the immunomodulatory agent is an immune checkpoint inhibitor.

[0217] In some embodiments, the immune checkpoint inhibitor is an agent capable of inhibiting signalling mediated by an immune checkpoint molecule. In some embodiments, the immune checkpoint molecule is e.g. PD-1 , CTLA-4, LAG-3, TIM-3, TIGIT or BTLA.

[0218] Agents capable of inhibiting signalling mediated by immune checkpoint molecules are known in the art, and include e.g. antibodies capable of binding to immune checkpoint molecules or their ligands, and inhibiting signalling mediated by the immune checkpoint molecule. Other agents capable of inhibiting signalling mediated by an immune checkpoint molecule include agents capable of reducing gene / protein expression of the immune checkpoint molecule or a ligand for the immune checkpoint molecule (e.g. through inhibiting transcription of the gene(s) encoding the immune checkpoint molecule / ligand, inhibiting post-transcriptional processing of RNA encoding the immune checkpoint molecule / ligand, reducing stability of RNA encoding the immune checkpoint molecule / ligand, promoting degradation of RNA encoding the immune checkpoint molecule / ligand, inhibiting post-translational processing of the immune checkpoint molecule / ligand, reducing stability the immune checkpoint molecule / ligand, or promoting degradation of the immune checkpoint molecule / ligand), and small molecule inhibitors.

[0219] In some embodiments, the immune checkpoint inhibitor is an agent capable of inhibiting signalling mediated by PD-1. The agent capable of inhibiting signalling mediated by PD-1 may be a PD-1- or PD- L1 -targeted agent. The agent capable of inhibiting signalling mediated by PD-1 may e.g. be an antibody capable of binding to PD-1 or PD-L1 and inhibiting PD-1 -mediated signalling. In some embodiments, the agent is an anti-PD-1 antibody or fragment thereof (e.g. an antagonist anti-PD-1 antibody or fragment thereof). In some embodiments, the agent is an anti-PD-L1 antibody or fragment thereof (e.g. an antagonist anti-PD-L1 antibody or fragment thereof).

[0220] In some embodiments, the immune checkpoint inhibitor is an agent capable of inhibiting signalling mediated by CTLA-4. The agent capable of inhibiting signalling mediated by CTLA-4 may be a CTLA-4- targeted agent, or an agent targeted against a ligand for CTLA-4 such as CD80 or CD86. In some embodiments, the agent capable of inhibiting signalling mediated by CTLA-4 may e.g. be an antibody capable of binding to CTLA-4, CD80 or CD86 and inhibiting CTLA-4-mediated signalling.

[0221] Immune checkpoint inhibitors are reviewed in e.g. Darvin P et al., Experimental & Molecular Medicine, 2018, 50:165; de Miguel M & Calvo E, Cancer Cell, 2020, 38:326; and Marin-Acevedo JA et al., J Hematol Oncol. 2021 , 14:45, which are hereby incorporated by reference in their entirety. Immune checkpoint inhibitors include ipilimumab, tremelimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, cemiplimab, and dostarlimab.

[0222] In some embodiments, the immunomodulatory agent is an agent capable of promoting signalling mediated by a costimulatory receptor. In some embodiments, the costimulatory receptor is e.g. CD28, CD80, CD40L, CD86, 0X40, 4-1 BB, CD27 or ICOS.

[0223] Agents capable of promoting signalling mediated by costimulatory receptors are known in the art, and include e.g. agonist antibodies capable of binding to costimulatory receptors and triggering or increasing signalling mediated by the costimulatory receptor. Other agents capable of promoting signalling mediated by costimulatory receptors include agents capable of increasing gene / protein expression of the costimulatory receptor or a ligand for the costimulatory receptor (e.g. through promoting transcription of the gene(s) encoding the costimulatory receptor / ligand, promoting post-transcriptional processing of RNA encoding the costimulatory receptor / ligand, increasing stability of RNA encoding the costimulatory receptor / ligand, inhibiting degradation of RNA encoding the costimulatory receptor / ligand, promoting post- translational processing of the costimulatory receptor / ligand, increasing stability of the costimulatory receptor / ligand, or inhibiting degradation of the costimulatory receptor / ligand), and small molecule agonists.

[0224] In some embodiments, the immunomodulatory agent is an immunomodulatory cytokine.

[0225] As used herein, an ‘immunomodulatory cytokine’ refers to a cytokine that modulates / modifies the immune system. An immunomodulatory cytokine may stimulate or supress the immune system.

[0226] Examples of immunomodulatory cytokines which stimulate the immune system include IFN-a, IL-2, IL-15, IL-21 , IL-12, IL-18, and GM-CSF.

[0227] Cytokines and their use in the treatment of cancer are reviewed in Berraondo P et al, British Journal of Cancer 2019, 120:6-15, and Conlon KC et al, Journal of Interferon & Cytokine Research 2019, 39(1):6, which are hereby incorporated by reference in their entirety.

[0228] In some embodiments methods employing a combination of a microorganism / spore / composition of the present disclosure and an immunomodulatory agent (e.g. an agent capable of inhibiting signalling mediated by an immune checkpoint molecule (e.g. PD-1 and / or PD-L1)) provide an improved treatment effect as compared to the effect observed when either agent is used as a monotherapy. In some embodiments, the combination of a microorganism / spore / composition of the present disclosure and an immunomodulatory agent (e.g. an agent capable of inhibiting signalling mediated by an immune checkpoint molecule (e.g. PD-1 and / or PD-L1)) provide a synergistic ( / .e. super-additive) treatment effect. It will be appreciated that singular forms ‘a modified microorganism’ and ‘a spore’ include plural referents. Accordingly, it will be appreciated that the above embodiments may be performed with modified microorganisms, with spores derived from modified microorganisms, or with compositions comprising said microorganisms or spores, according to the present disclosure.

[0229] E. Kits

[0230] In some aspects of the present disclosure a kit of parts is provided. A kit according to the present disclosure may comprises components for performing a method described herein, in whole or in part.

[0231] The kit may have at least one container having a predetermined quantity of a microorganism, spore or composition described herein. The microorganism / spore may be lyophilised.

[0232] The kit may provide a microorganism, spore or composition described herein together with instructions for administration to a patient in order to treat a specified disease / condition (e.g. a disease / condition described herein, e.g. a cancer).

[0233] The kit may further comprise reagents, buffers and / or standards required for execution of a method according to the present disclosure. Kits according to the present disclosure may include instructions for use, e.g. in the form of an instruction booklet or leaflet. The instructions may include a protocol for performing any one or more of the methods described herein.

[0234] F. Subjects

[0235] The subject in accordance with aspects described herein may be any animal or human. The subject is preferably mammalian, more preferably human. The subject may be a non-human mammal, but is more preferably human. The subject may be male or female. The subject may be a patient. A subject may have been diagnosed with a disease or condition requiring treatment (e.g. a cancer, e.g. a cancer described herein), may be suspected of having such a disease / condition, or may be at risk of developing / contracting such a disease / condition.

[0236] In some embodiments, the subject to be treated according to a therapeutic or prophylactic method of the present disclosure herein is a subject having, or at risk of developing, a cancer, e.g. a cancer described herein. In embodiments according to the present disclosure, a subject may be selected for treatment according to the methods based on characterisation for certain markers of such disease / condition.

[0237] In some embodiments, a patient may be selected for treatment described herein based on the detection of a cancer expressing / overexpressing a given antigen, e.g. in a sample obtained from the subject (e.g. a biopsy, e.g. of a tumour). Where a patient is selected for treatment on the basis of having a cancer expressing a given antigen, the treatment may comprise administration of a microorganism modified to express the antigen. ***

[0238] The present disclosure includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

[0239] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0240] Aspects and embodiments of the present disclosure will now be illustrated, by way of example, with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0241] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word ‘comprise,’ and variations such as ‘comprises’ and ‘comprising,’ will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0242] As used herein, an amino acid sequence or a region of a polypeptide which ‘corresponds’ to a specified reference amino acid sequence or region of a polypeptide has at least 60% (e.g. one of >60%, >65%, >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to the amino acid sequence of the amino acid sequence / polypeptide / region. An amino acid sequence / region / position of a polypeptide / amino acid sequence which ‘corresponds’ to a specified reference amino acid sequence / region / position of a polypeptide / amino acid sequence can be identified by sequence alignment of the subject sequence to the reference sequence, e.g. using sequence alignment software such as ClustalOmega (Soding, J. 2005, Bioinformatics 21 , 951-960).

[0243] It must be noted that, as used in the specification and the appended claims, the singular forms ‘a,’ ‘an,’ and ‘the’ include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from ‘about’ one particular value, and / or to ‘about’ another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent ‘about,’ it will be understood that the particular value forms another embodiment.

[0244] Where a nucleic acid sequence is disclosed herein, the reverse complement thereof is also expressly contemplated.

[0245] Methods described herein may preferably be performed in vitro. The term ‘in vitro’ is intended to encompass procedures performed with cells in culture whereas the term ‘in vivo’ is intended to encompass procedures with / on intact multi-cellular organisms. Brief Description of the Figures

[0246] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures.

[0247] Figures 1A to 1F. CS11 spores are safe and exert anti-tumour activities. (1A) Southern blotting with group II intron-specific probes confirms intronic insertions into csa of CS10, CS11 and CS13. (1B) The cytotoxicity of CS-WT was heavily ablated in CS11 in vitro, as determined with calcein-AM staining and with t tests, and in vivo with the injection of CS11 and CS-WT spores into CT26-BALB / c mice. (1C) Intravenous injections of CS11 spores result in less than a 20% weight loss, indicating tolerable weight loss. (1D, 1 E) CS11 delays CT26 tumour growth, prolongs survival, and confers a 1 / 13 complete response rate. Ttests of relative tumour volumes were performed for each timepoint. Survival was calculated using the Mantel-Cox survival test. (1F) Tumour tissue was fixed, sectioned then gram-stained for the visualization of CS11. CS11 , marked by arrows, germinates and grows within tumours 24 hours after intravenous injection. All error bars represent SEM. * < 0.05, “ < 0.01 , “* < 0.001 .

[0248] Figure 2. Table showing CS11 and its derivative neoantigen-secreting strains effects on in vivo models. CS11 and its derivative neoantigen-secreting strains can induce complete and lasting clearances of solid tumours in BALB / c mice. Mice that experience complete tumour remission are unlikely to have recurrences of tumours and are able to prevent the establishment of tumours when rechallenged. The treatment of mice with tumour-matched-neoantigen-secreting strains of CS11 with aPD-L1 mAb increases the likelihood of complete tumour clearance, thus the induction of anti-tumour immune memory.

[0249] Figures 3A to 3C. CS11 causes hemorrhagic necrosis and fibrosis of CT26 tumours. (3A) Hematoxylin and eosin staining of CS11- and PBS-treated CT26 tumour sections revealed extensive hemorrhage, necrosis, fibrosis 24 hours after CS11 treatment. (3B) Weakly staining bacilli can be found in gram stained CT26 sections, particularly in fibrotic regions (3C) A microscopic view of the blackened tissue and unscathed pink tissue (Figure 8) following CS11 treatment.

[0250] Figures 4A to 4E. The anti-tumour effects of CS11 can be synergised by its secretion of the A5 mimotope and the co-administration of aPD-L1 mAb. (4A) Spore and ICI treatment regime. Grafted CT26 cells are given 7 days to establish a tumour before treatment begins. (4B, 4C) The administration of aPD- L1 mAb in combination with CS11 spores results in better tumour survival, and significantly better growth control on Day 4. (4D, 4E) CS11-sA5 with aPD-L1 mAb can cause drastic tumour shrinkage and confers a 8 / 10 survival rate. The intratumoural injection of A5 with the intravenous injection of CS11 spores did not engender the same synergistic effects achieved with CS11-sA5. Relative tumour volumes were compared by / tests performed at each time point, and survival rates were analyzed with the Mantel-Cox survival test. Asterisks in the table of (4D) and legend of (4E) indicate significant differences found between the CS11-sA5 and aPD-L1 mAb combination therapy group and the asterisked group. Error bars represent SEM. * < 0.05, “ < 0.01 , *“ < 0.001 . Figures 5A to 5B. Neoantigen-secreting strains of CS11 and C. novyi-NT can be produced. (5A) Schematics of the expression cassette carried by neoantigen-secreting strains of CS11 and C. novyi-NT. In CS11 strains, the promoter and secretion signal of the alpha-toxin gene, csa, are placed upstream of the neoantigen. In CN-sOVA, the sequence for amino acids 257 to 264 of ovalbumin is placed downstream of the pic secretory signal, under the transcriptional control of the fliC promoter. The neoantigens are fused to a FLAG tag, enabling the detection of secreted neoantigens. (5B) Measurement of neoantigens secreted by different Clostridium species using ELISA. Error bars represent the SEM of three biological replicates. Each dot represents the average of three technical replicates.

[0251] Figures 6A to 6E. CD8+ T cells are critical to complete tumour clearance after CS11 and aPD-L1 mAb treatment. (6A) The treatment schedules for mice receiving different immune depletion treatments and CS11-sA5 with aPD-L1 mAb. The effectiveness of schedules were verified in Figure 7. (6B, 6C) Both the abolishment of T cell egress into circulation early in treatment with FTY720 and the depletion of CD8+ T cells ablate the therapeutic effects of CS11-sA5 and aPD-L1 mAb and these two groups did not survive past Day 20. (6D) The transfer of splenocytes or purified CD8+ T cells from CT26-mice cured with CS11- sA5 and aPD-L1 mAb into naive mice prevents the establishment of tumours from grafted CT26 cells. All mice that received CD4+ T cells developed tumours. (6E) Treatment with CS11-sA5 instead of CS11 spores results in more AH1 -responsive immune cells as measured with ELISpot and intracellular cytokine staining. Error bars represent SEM. * < 0.05, “ < 0.01 , “* < 0.001 .

[0252] Figure 7A to 7C. T cell trafficking and depletion can be achieved with (A-B) FTY720 Depletion and (C) aCD4 mAb and aCD8 mAb depletion. (A) Treatment of mice with FTY720 results in significantly lower CD3+ splenocytes as determined by flow cytometry. Furthermore, the treatment of mice with aPD-L1 mAb and CS11-sA5 causes a higher level of CD3+ cells than treatment with CS11 . (B) Mice have significantly lower circulating CD3+ cells even 4 days after treatment with FTY720, ascertained by flow cytometry. (C) Treatment of mice with aCD4 mAb causes significantly decreased CD45+ CD4+ cells in tumour draining lymph nodes and the spleen measured by flow cytometry. Similar decreases in CD45+CD8+ cells were found in mice treated with aCD8 mAb. Each dot represents cells taken from one mouse, and error bars represent SEM. * < 0.05, “ < 0.01 , “* < 0.001 .

[0253] Figures 8A to 8B. T cells are necessary for the anti-tumour effects of CS11-sA5 and aPD-L1 mAb treatment. (8A) Representative CT26 tumour of a BALB / c mouse treated with CS11-sA5 and aPD-L1 mAb. The majority of the tumour turns black and shrivels up, leaving behind a pinkish rim of tissue (lighter grey in the image). The pinkish rim of tissue is eventually cleared, leaving behind a scab that heals. CS11 monotherapy, or treatments of aPD-L1 mAb with CS11 or CS11-sAH1 leave the pinkish rim unscathed, allowing it to grow until the mouse reaches the euthanasia end point of the experiment (images not shown). (8B) Representative CT26 tumour of a BALB / c mouse from the FTY720 D-10 group. The tumour blackens and shrivels up to a lesser extent than in Figure 8A, leaving behind a necrotic core and a more pronounced pink rim that rapidly grows. Evidently, the clearance of the pink rim is performed by T cells. Figure 9. Representative well images obtained with ELISpot analysis. 2,500,000 splenocytes were incubated with the respective stimulants or DMSO overnight before spot-forming units were developed and counted according to the supplied instructions.

[0254] Figures 10A and 10B. Intracellular cytokine staining gating strategy. 1 ,500,000 splenocytes were incubated with the respective stimulant or DMSO for 6 hours, then checked for the production of IFNy. (10A) The gating strategy for identifying AH 1 -responsive CD3+ CD8+ splenocytes. (10B) Positive gates for IFNy production were drawn using the DMSO setup.

[0255] Figures 11A to 11C. Preparation of Ovalbumin-Expressing Cell Lines. (11 A) Fluorescence microscopy of 4T1 and B16F10 cells that had integrated the gene expression cassette containing GFP and ovalbumin. (11 B) GFP+ cells were sorted with FACS, and flow cytometry was used to ascertain their purity. Positive gates for GFP were drawn. (11 C) The expression of ovalbumin was confirmed by RT- PCR. (11 D) AH1 is expressed by 4T1 and CT26.

[0256] Figure 12. 4T1-OVA and 4T1 mice succumb to disease before they can reach the euthanasia end point. Mice often develop tumours with volumes smaller than 1500 mm3but still succumb to disease, exemplified by the depicted mouse. Necropsies often reveal the presence of metastases, particularly in the liver or spleen.

[0257] Figures 13A to 13E. Synergistic therapeutic effects are observed when aPD-L1 mAb is combined with CS11 strains secreting the tumour-matched neoantigens in other cancer models. (13A) Co-administration of CS11-sA5 and CS11-sOVA with aPD-L1 mAb induce drastic 4T1-OVA tumour remissions. Ttests were performed between the dual-spore treatment group with aPD-L1 mAb and all the other groups for each time point. (13B) The administration of neoantigen-secreting CS11 strains with aPD-L1 mAb confers improved survival outcomes, as shown with the Mantel-Cox test performed for pairwise comparisons between the CS11-sOVA + CS11-sA5 and aPD-L1 mAb group and all other groups. (13C) Treatment schedule for C. novyi-NT and anti-PD-L1 mAb for OVA-B16F10. (13D) CN-sOVA delays OVA-B16F10 tumour growth. Paired t tests were only performed between the two groups that received 2 x 107 spores. Error bars represent SEM. (13E) CN-sOVA improves the survival of OVA-B16F10 mice in combination with anti-PD-L1 mAb. * < 0.05, ** < 0.01 , *** < 0.001 .

[0258] Examples

[0259] Example 1 : Materials and Methods

[0260] Bacterial strains and growth conditions. Clostridium species were all maintained in an anaerobic chamber at 37°C with a mixed gas atmosphere of 85% N2, 10% H2, and 5% CO2. CS-WT was grown in BD Bacto™ Brain Heart Infusion Broth (BD, cat. no. 237500, “BHI”) supplemented with 0.5 g / L L-cysteine (“BHIS”). C. novyi-NT was grown in BHI supplemented with 10% FBS.

[0261] Conjugation of CS-WT and C. novyi-NT. E. coli S17-1 Apir (Biomedal S.L) transformed with the desired plasmids were cultured aerobically at 37°C in 2 x YT broth supplemented with either 10 pg / mL chloramphenicol or 500 pg / mL erythromycin. Overnight cultures of E. coli S17-1 Apir served as conjugant donors for CS-WT or C.novy / -NT over a 6 hour mating period. After mating, polymyxin B at 60 U / mL was added to the media to select against the conjugant donors, and transconjugants were selected for using 10 pg / mL thiamphenicol or 2.5 pg / mL erythromycin.

[0262] Generation of CS11. \Ne used the TargeTron™ Gene Knockout System (Sigma-Aldrich) to disrupt the alpha-toxin of CS-WT by inserting the intron into its gene, csa. The insertion sites in csa were predicted and scored with an algorithm (Perutka et al., 2004). To ensure complete disruption of the alpha-toxin, we chose high scoring insertion sites near the N-terminus. Putative transconjugants were screened with PCR using a gene specific and universal intron primers, and the transconjugants were subcultured to shed their plasmids. Genomic DNA was isolated from each culture using the original protocol described by Pospiech and Neumann (1995), with a few modifications: RNase A and Proteinase K (Roche) treatment was only done following crude DNA extraction for 2 hours each at 37°C. gDNA was then purified by phenol / chloroform extraction and quantified with Picogreen (Life Technologies) using lambda DNA (NEB) as a standard. 0.5 pg of gDNA was digested with 10 U of Xbal, electrophoresed in a 0.8% agarose gel, then transferred to a nylon membrane (Amersham Hybond-N+) with a vacuum blotting system (GE, VacuGeneXL). A 378bp digoxigenin-labelled probe complementary to the inserted intron was synthesized with the DIG-High Prime DNA Labelling and Detection Starter Kit II (Roche, cat. no. 11585614910) and PCR DIG Labelling Mix (Roche, cat. no. 11585550910) before it was hybridized to the blot to confirm csa- specific insertion of the intron.

[0263] Generation of neoantigen-expressing Clostridium strains. Vectors for expressing neoantigens were prepared using NEB Hifi assembly (New England Biolabs, cat. no. E5520S). Vectors transferred into CS11 contain the csa promoter and secretory signal (Ballard et al., 1995) to drive the expression and secretion of the neoantigen. Vectors for C. novyi-NT instead have the fliC promoter and pic secretory signal to drive neoantigen expression and secretion. All vectors have the neoantigen fused to a FLAG tag (Figure 5A) to permit detection using ELISA.

[0264] Preparation of Clostridium spores. C. novyi-NT spores were prepared as previously described (Diaz et al., 2005). To obtain spores of CS-WT and its derivative strains, starter cultures were first prepared by inoculating 1 x 106of spores into 5 mL of BHIS containing the appropriate antibiotics. After growth under anaerobic conditions at 37°C, 2 mL of the overnight cultures were inoculated into 98 mL of BHIS with the necessary antibiotics, then allowed to grow for another 7 hours, after which the 100 mL culture was added into 900 mL of sporulation media prepared from 50 g / L DIFCO™ Cooked Meat Medium (BD, cat. no. 226730), 30 g / L Polypeptone™ Peptone (Gibco™, cat. no. 211910), 0.5 g / L L-cysteine, and 10% FBS (iDNA, cat. no. S1810). After five days, the spores were harvested and separated from vegetative cells by centrifugation in an 80% Percoll gradient, similar to that previously described by Diaz et al (2005) for C. novyi-NT spores.

[0265] ELISA for neoantigen detection. 1 x 109 spores of CS11 strains and C. novyi-NT strains were inoculated in 5 mL of BHIS or BHI / FBS respectively, and the cultures were incubated overnight at 37°C under anaerobic conditions with the appropriate antibiotics. 100 pL of the overnight cultures were then transferred into 1 mL of fresh media with antibiotics and the bacteria were allowed to grow for another 6 hours. The cultures were then collected and centrifuged at 4000 x g for 15 minutes before the supernatants were collected and analyzed with the FLAG-Tag (DYKDDDDK-Tag Protein) ELISA Kit (Fine Test, cat. no. EU2607) as per the supplied instructions. The OD450nm readings of supernatants from neoantigen-expressing strains were normalized against those obtained from CS11 or C. novyi-NT.

[0266] Mammalian cell culture. CT26 cells (ATCC CRL-2638), 4T1 cells (ATCC CRL-2539), and B16F10 cells (ATCC CRL-6475) were purchased from the American Type Culture Collection and were maintained in McCoy's 5A Medium (Gibco™, cat. no. 16600-082), RPMI-1640 Medium (Gibco™, cat. no. 21870-076), and DMEM (Gibco™, cat. no. 10313-021) respectively, all of which contained 10% HyClone Characterized FBS (Cytiva, cat. no. SH30071 .03HI). All cell lines were maintained at 37°C under 5% CO2, and were regularly checked for Mycoplasma contamination with the MycoStrip™ (Invivogen, cat. no. rep-mysnc-100). Cells were not passaged past the ninth passage.

[0267] Assessing the cytotoxicity of CS11 in vitro. 1 x 104CT26 cells were seeded into 96-well plates and allowed to grow overnight. 1 x 105CS1 1 or CS-WT spores were added to each culture, then further incubated for 48 hours. Cells were then washed twice with PBS to remove vegetative CS11 or CS-WT and dead CT26 cells before the addition of calcein-AM (Invitrogen™, cat. no. C1430) to a final concentration of 2 pM. The cultures were incubated for 30 minutes at 37°C before their fluorescence was measured. Live cells are distinguished by the enzymatic production of intensively fluorescent calcein (Ex / Em 494 / 517 nm) from non-fluorescent cell-permeant calcein-AM through intracellular esterease activity.

[0268] Generation of ovalbumin-expressing cell lines. The Sleeping Beauty transposon system (Geurts et al., 2003; Kowarz et al., 2015) was used to integrate the complete coding sequence of the oval gene from Gallus gallus (GenBank: MH360742.1) into the chromosomes of B16F10 and 4T1 cell lines. The bidirectional E1 Fa promoter drives the expression of ovalbumin and GFP as a reporter gene. B16F10 and 4T1 cell lines were transfected with plasmids encoding for the Sleeping Beauty transposase and ovalbumin, and the transfected cell lines were subjected to selection by treatment with 400 pg / mL and 200 pg / mL G418 for B16F10 and 4T1 respectively. To obtain pure populations of ovalbumin expressing cells, the GFP+transfectants were purified using FACS using the MoFlo™ XDP cell sorter and maintained with G418. The cell lines are referred to as B16F10-OVA and 4T1-OVA.

[0269] Animal Husbandry. Female BALB / c mice and C57 / BI6 were purchased from Jackson Laboratory (InVivos) and allowed to acclimatize for a week prior to any experiments. Animals were maintained under specific pathogen-free conditions in a climate-controlled holding room with a controlled 12 h dark / light cycle, an ambient temperature of 21 ± 2°C, and relative humidity ranging between 30% and 70%. Mice were used for experiments at 6-10 weeks of age. All animal experiments were approved by the Institutional Animal Care and Use Committee of Temasek Life Sciences Laboratory. Subcutaneous tumour models and in vivo studies. Mice that were to be treated with aPD-1 mAb (clone: RMP1-14, BioXcell, cat. no. BE0146) or aPD-L1 mAb (clone: 10F.9G2, BioXcell, cat. no. BE0101) and / or spores were first subcutaneously injected with 1 x 107of tumour cells using a 26-gauge needle into their shaved right flanks. Tumours were allowed to establish for 7 days, when they would be at least 150 mm3, with the tumour volume calculated by measuring the length and breadth of the tumour and with electronic calipers and then applying the formula length x width2x 0.5. The mice were then randomized into treatment groups to obtain similar average tumour volumes on Day 0. Seven 200 pg doses of aPD-1 or aPD-L1 mAb were administered every two to three days. Three days after the first dose of aPD-1 ZaPD-L1 mAb, 2 x 107of C. novyi-NT spores or 2 x 108of CS spores were delivered intravenously through the tail vein. For studies involving intratumoural injections of tumour antigens, 2 pg of antigen in 20 pL of 1x PBS was slowly injected into the largest sections of the tumour using a 31 G insulin syringe on days 1 and 3. Mice having tumours exceeding 1500 mm3or relative body weights below 80% were euthanized as per IACUC protocol. All mice were monitored until study day 60, or in some cases, day 90. For isotype controls, either 200 pg of InVivoMAb rat lgG2b isotype control (clone: LTF-2, BioXcell, cat. no. BE0090) or 200 pg of InVivoMAb rat lgG2a isotype control (clone: 2A3, BioXcell, cat. no. BE0089) were administered.

[0270] FTY720 treatment and in vivo depletion of immune cells. To prevent the egress of T cells into circulation, FTY720 was administered at 3 pg per gram of mouse every 3 to 4 days (Figure 7A, Figure 7B). Specific T cell populations were depleted by administering 250 pg of depleting antibody or isotype controls intraperitoneally beginning from on the day of tumour inoculation as indicated: CD4+ T cells with aCD4 mAb (clone: GK1.5, BioXCell, cat. no. BE0003-1), CD8+ T cells with aCD8p mAb (clone Lyt3.2, BioXCell, cat. no. BE0223). The isotype controls were InVivoMAb rat lgG2b isotype control (clone: LTF-2, BioXcell, cat. no. BE0090) or 200 pg InVivoMAb rat IgG 1 isotype control (clone HRPN, BioXCell, cat. no. BE0088). Cellular depletions of CD8+ T cells and CD4+ T cells were confirmed by flow cytometry of splenic tissues and tumour draining lymph nodes of CT26-tumour bearing mice (Figure 7C). All cells were incubated with Mouse TruStain FcX Anti-mouse CD16 / 32 antibody (Clone: 93, Biolegend, cat. no. 101319) prior to staining with the following antibodies: APC anti-mouse CD4 Antibody (Clone: RM4-5, Biolegend, cat. no. 100515), PE anti-mouse CD8a Antibody (Clone: 53-6.7, Biolegend, cat. no. 100707), FITC anti-mouse CD45.2 Antibody (Clone: 104, Biolegend, cat. no. 109805). Viable cells were stained with 7-AAD (Biolegend). All flow cytometry data were acquired with LSR Fortessa (BD Biosciences) and analyzed with DIVA software (BD Biosciences) and FlowJo™ v10.8 Software (BD Life Sciences).

[0271] Isolation of splenocytes. Mice were euthanized by CO2 asphyxiation and had their spleens aseptically removed and suspended in RPMI-1640 containing 10% FBS, 100 units / mL penicillin and 100 pg / mL streptomycin. The spleens were macerated using plungers removed from syringes, then passed through 70 pm pore cell strainer (SPL Life Sciences) and flushed twice with ice-cold PBS containing 2% FBS and 1 mM EDTA. The lysate was centrifuged at 500 x g for 5 minutes at 4°C. The pellet was then resuspended in 5 mL of eBioscience™ 1X RBC Lysis Buffer (eBioscience™, cat. no. 00-4333-57) for 5 minutes with gentle shaking to lyse erythrocytes. The lysate was diluted with 20 mL of ice-cold PBS containing 2% FBS and 1 mM EDTA, and was re-filtered with a 70 pm pore cell strainer, then centrifuged as before. The cell pellet was resuspended in RPMI-1640 with RPMI-1640 containing 10% FBS, 100 units / mL penicillin and 100 pg / mL streptomycin.

[0272] Adoptive transfer experiments. Splenocytes were isolated from CS11-sA5 + aPD-L1 cured mice, and CD4+ T cells and CD8+ T cells purified using the EasySep Mouse CD4+ T Cell Isolation Kit (STEMCELL Technologies, cat. no. 19852) and EasySep Mouse CD8+ T Cell Isolation Kit as per manufacturer instructions (STEMCELL Technologies, cat. no. 19853). Purities of 84% - 90% were obtained as ascertained by flow cytometry. 2 x 107crude splenocytes, 2 x 106purified CD4+ T cells or 2 x 104to 2 x 106CD8+ T cells were intravenously injected into naive BALB / c mice that were then challenged with 2 x 105CT26 cells on either the same day or a day later. Mice were then monitored for 30 days for tumour establishment. In all experiments, at least one naive mouse that did not receive any adoptive transfers was challenged with 2 x 105CT26 to ensure that the CT26 preparation is tumourigenic.

[0273] Visualization of CS11 within tumours. CT26-tumours on BALB / c mice were treated with CS11 . 24 hours after spore administration, the mice were euthanized and tumours were fixed in 4% paraformaldehyde for another 24 hours. The tumours were dehydrated in an ascending series of ethanol, cleared with xylene, and then embedded in paraffin wax. 5 pm thick sections were cut and placed onto glass slides. The slides were dewaxed in xylene and hydrated with a descending series of ethanol before being stained for Gram. The slides were stained in 0.5% crystal violet, Gram’s iodine and counterstained with 1% neutral red. After which, the slides were dehydrated through an ascending series of ethanol to xylene before being coverslipped and imaged.

[0274] Detection ofAH1-responsive splenocytes and CD8+ T cells. The ELISpot Plus: Mouse IFN-y (ALP) kit (MabTech, cat. no. 3321-4APW) was used to quantify the number of AH 1 -responsive splenocytes obtained from CT26-tumour bearing BALB / c mice that received different treatments. 2.5 x 106or 1 x 106splenocytes in RPMI-1640 containing 10% FBS, 100 units / mL penicillin and 100 pg / mL streptomycin were added into each well and stimulated with AH1 peptide (SPSYVYHQF; Genscript peptide synthesis service) at a final concentration of 5 pg / mL, or phytohemagglutinin(Selleck Chemicals, cat. no. S6990) at a final concentration of 5 pg / mL for 24 hours at 37°C under 5% CO2. 2.5 x 106splenocytes were incubated with DMSO as a no-stimulation control. Spot development was then performed as per the manufacturer’s instructions. Intracellular cytokine staining was used to quantify AH1 -responsive CD3+ CD8+ splenocytes. 1.5 x 106splenocytes were pulsed with 10 pg / mL of AH1 peptide for 2 hours at 37°C under 5% CO2, before BD GolgiPlug™ Protein Transport Inhibitor (BD Biosciences, cat. no. 555029) was added to the cells for a further 4 hour-incubation. For each sample, a stimulation positive control was prepared by incubating 1 .5 x 106splenocytes with 5 ng / mL of Phorbol 12-myristate13-acetate (PM A, Sigma Aldrich, cat. no. P8139) and 500 ng / mL of ionomycin (Santa Cruz Biotechnology, cat. no. sc- 3592). 1 .5 x 106splenocytes were incubated with only BD GolgiPlug™ and DMSO for 6 hours as a nostimulation control. The antigen-pulsed splenocytes were collected and resuspended in FACS Buffer (1x PBS containing 2% FBS and 2 mM EDTA). The cells were then blocked with 10 pg of Mouse TruStain FcX Anti-mouse CD16 / 32 antibody (Clone: 93, Biolegend, cat. no. 101319) for 10 mins on ice, then stained with 0.28 pg of APC-eFluor™ 780 CD3 Monoclonal Antibody (Clone: 17A2, eBioscience™, cat. no. 47-0032-82), 0.28 pg of Brilliant Violet 785™ anti-mouse CD8a Antibody (Clone: 53-6.7, Biolegend, cat. no. 100749), and 0.5 pL of Fixable Viability Dye eFluor™ 506 (Invitrogen™) in 100 pL volumes for 20 min. The cells were then washed once with 200 pL of FACS buffer before fixation with 100 pL of Fixation / Permeabilization solution (BD Biosciences, cat. no. 51-2090KZ) for 20 mins on ice, followed by two washes with 200 pL of 1x BD Perm / Wash™ Buffer (BD Biosciences, cat. no. 51-2091 KZ). The splenocytes were then stained with 0.138 pg PE-eFluor™ 610 anti-mouse IFNy Antibody (eBioscience™, Clone: XMG1 .2) for 30 mins on ice. The cells were again washed twice with 200 pL 1x BD Perm / Wash™ Buffer before they were resuspended in 500 pL of FACS Buffer for flow cytometric analysis using the BD LSRFortessa™ Cell Analyzer. The gating strategy is shown in Figure 10A.

[0275] Data analysis. The generation of figures and statistical analysis was performed on Graphpad Prism 8 for Windows (Graphpad software). In experiments comparing two sets of values, an unpaired / test was performed with Welch correction. Mouse survival data was represented with Kaplan-Meier survival curves and Mantel Cox test were used to compute statistical significance.

[0276] Example 2: Generation of CS11 , a non-lethal strain of C. septicum

[0277] Wildtype C. septicum infections are highly lethal if untreated with antibiotics, and this lethality is attributed to its aerolysin-like pore-forming alpha-toxin (14,15). Genetic inactivation of the csa gene which codes for alpha-toxin has been shown to fully abrogate C. septicum’s virulence (16). Hence, group II intron insertional mutagenesis (17) was used to genetically inactivate csa in Clostridium septicum ATCC 11424 (“CS-WT”). Three csa knockout clones (CS10, CS11 and CS13) were identified by Southern Blotting using an intron-specific probe, and CS11 was chosen for downstream experiments as it had the highest band intensity (Figure 1 A). To verify that csa had been attenuated, spores of CS11 or CS-WT were inoculated into CT26 monolayer cell cultures for 48 hours after which CT26 cell viability was measured using calcein-AM. CS-WT killed practically all CT26 cells whereas 90% of CT26 cells were still viable after treatment with CS11 (Figure 1 B).

[0278] Next, BALB / c mice bearing subcutaneous CT26 allografts were intravenously treated with CS11 or CS- WT spores. All 10 mice treated with CS-WT spores died within 48 hours whereas all 13 mice treated with CS11 spores survived (Figure 1 B). CS11-treated mice lost 3.6% of their weight within the first 2 days but reverted to a weight gain trajectory similar to control mice after 2 days (Figure 1 C), showing that CS11 was well tolerated.

[0279] CS11 treated tumours exhibited blackening in the central region, followed by scabbing and hollowing of the area. This resulted in a temporary and modest decrease in tumour volume (Figure 1 D) after which the well-vascularized rim tissue expanded and drove an increase in tumour volume. These observations are similar to previous studies with C. novyi-NT (18-20). There was a minor but statistically significant survival benefit for CS11 which was driven by a single complete response in the CS11 treatment group (Figure 1 E, Figure 2). This mouse remained tumour-free past day 60, and resisted CT26 tumour rechallenge (Figure 2), suggesting that CS11 treatment may have resulted in long-term anti-CT26 immune memory. To verify that CS11 did indeed germinate within the CT26 tumours, CT26 tumours were resected from BALB / c mice 24 hours after the administration of CS11 or PBS, then fixed and visualized with H&E or Gram staining. The accumulation of Gram variable bacilli in the fibrotic and necrotic tumour regions was observed in CS11 -treated tumours (Figure 1 F, Figure 3B). Previous studies have reported similar Gram variability for C. septicum and other Clostridium (21 , 22). H&E staining revealed greater infiltration of polymorphonuclear cells, extensive hemorrhage, necrosis and fibrosis in CS11-treated tumours but not in the controls (Figure 3A).

[0280] Example 3: Neoantiqen-secretion enhances the anti-tumour effects of CS11

[0281] While CS11 was able to debulk the hypoxic core of tumours, the rim of well-oxygenated tumour tissue remained refractory to colonization. Previous studies have demonstrated that complementing hypoxia- targeted therapy with a further therapy that attacks well-oxygenated tissue (e.g. chemotherapy, radiotherapy) can enhance the efficacy of treatment (23-25). Chemo- and radiotherapy often have immunosuppressive effects which severely handicap the anti-tumour immune response. Since treatment with CS11 alone produced a single complete response which was resistant to tumour rechallenge (Figure 1 E), it was investigated if this modest complete response rate could be enhanced without the need for chemo- or radio-therapy.

[0282] It was hypothesised that CS11 engendered a low-level anti-tumour immune response, and that immune checkpoint blockade might make such a response more pronounced. Since CT26 tumours express PD- L1 (26,27) and respond to PD-1 blockade (28), combinations of CS11 and aPD-1 mAb or aPD-L1 mAb were tested on CT26-tumour bearing BALB / c mice (Figure 4A). Only 1 out of 5 mice treated with CS11 and aPD-1 mAb experienced complete and lasting tumour clearance, while a slight improvement of 3 out of 9 complete and lasting tumour clearances were obtained with CS11 and aPD-L1 mAb combination therapy (Figure 2). As with the CS11 monotherapy, all mice that remained tumour free past day 60 were able to resist CT26 rechallenge, showing that CS11 -induced complete responses were correlated with anti-tumour immune memory. Despite the non-significant differences in survival (Figure 4B), the combination with aPD-L1 mAb resulted in significantly lower relative tumour volumes (Figure 4C). Since aPD-L1 mAb produced a more efficacious result, downstream experiments were focused on aPD-L1 mAb.

[0283] The expression of neoantigens by anti-tumour bacteria is also known to improve therapeutic responses through the induction of anti-tumour effector T cells (29). Strains of CS11 were generated that express and secrete the immunodominant neoantigen in CT26, AH1 (30) (CS11-sAH1), or the more immunogenic A5 mimotope (CS11-sA5) (31) (Figure 5A, Figure 5B), and were administered with aPD-L1 mAb or its isotype control in CT26-tumour bearing BALB / c mice (Figure 4D, Figure 4E).

[0284] The best response was elicited by CS11-sA5 and aPD-L1 , which saw the greatest tumour regression at day 15 and 80% survival at day 60. All surviving mice on day 60 remained tumour free and resisted CT26 tumour rechallenge (Figure 2). Injecting A5 intratumourally instead of secretion by CS11 greatly worsened the result. This may have been due to oxygen introduced by the injection reducing the hypoxic niche for CS11 . The persistence of expression by CS11 may also have advantages over the periodic injections of tumour antigens. The worst response in contrast was aPD-L1 alone, which saw no tumour regressions and no mice surviving beyond day 36. The other experimental arms did not show improvement beyond CS11 + aPD-L1 in the previous experiment.

[0285] Overall, the results showed a synergistic effect for A5 secretion and aPD-L1 in enhancing the baseline anti-tumour immune response observed with just CS11 alone. This highlights that the right neoantigen delivered by CS11 in combination with immune checkpoint inhibition can produce a high complete response rate.

[0286] Example 4: CD8+ T cells mediate CS11-induced anti-tumour immunity

[0287] Prior studies have demonstrated that Clostridium-mduced anti-tumour activity is mediated by CD8+ T cells (32). Experiments were performed to test whether lymphocytes were necessary for efficacy of C. septicum. Mice were treated with antibodies to deplete either their CD4+ or CD8+ T cells before treatment with CS11-sA5 + aPD-L1 (Figure 6A). Additionally, other mice were treated with the FTY720 on days -10, 1 or 7 relative to CS11 spore administration. FTY720 is an immunomodulator which attenuates lymphocytic egress from secondary lymphoid tissues and the thymus (Figure 7A, Figure 7B). Hence, any decrease in efficacy would indicate that lymphocyte trafficking from lymph nodes to blood might be important for efficacy.

[0288] The results were sharply contrasting (Figure 6B, Figure 6C, Figure 2). While depleting CD8+ T cells substantially destroyed efficacy, CD4+ T cell-depletion had practically no effect at all, hence establishing that CD8+ T cells are important for CS11 ’s anti-tumour effect. Further, FTY720 substantially negated efficacy when administered 10 days before CS11-sA5 administration, but had no effect on efficacy when administered either 1 or 7 days after CS11-sA5 administration. This implies that T cells which have infiltrated the tumour prior to CS11-sA5 colonization are crucial for tumour clearance. In the worst performing groups (CD8+ depletion and FTY720 at day -10), no mice survived past 18 days (Figure 6C, Figure 2). In contrast, complete response rates between 80% and 100% were observed in all other groups. Cured mice resisted CT26 rechallenge 100% of the time except when CD4+ T cells were depleted (2 / 5) or FTY720 was administered from day 1 , with 1 / 8 mice experiencing recurrence and only 4 / 7 of the survivors being able to resist rechallenge (Figure 3C, Figure 2). This observation points to a role for CD4+ T cells in engendering long-term immune memory, even if they were not as crucial as CD8+ T cells for the initial tumour debulking and complete response.

[0289] Visually, tumours treated with CS11-sA5 and aPD-L1 would form a scab which fell off after a week. In most mice, the remaining wound resolved and healed without any evidence of remaining tumour tissue (Figure 8A). In all mice treated additionally with FTY720 from day -10 however, there was residual tumour rim tissue a week after CS11-sA5 treatment which quickly expanded to reconstitute the tumour mass (Figure 8B). Halting lymphocyte migration before spore treatment impairs the clearance of well- oxygenated tumour rim tissue. Investigating further, splenocytes, CD4+ T cells, or CD8+ T cells from mice cured after treatment with CS11-sA5 and aPD-L1 mAb were adoptively transferred into naive mice which were then challenged with tumour cells (Figure 6D). All mice which received CD8+ T cells, and all mice but one which received splenocytes resisted tumour challenge. In contrast, all mice that received CD4+ T cells failed tumour rechallenge and formed solid tumours. This further supports our finding that CD8+ T cells, not CD4+ T cells, are crucial for the initial tumour debulking following CS11-sA5 spore treatment.

[0290] A5 is a more immunogenic mimotope of AH1 , the immunodominant antigen in CT26. Experiments were performed to test whether the secretion of A5 by CS11 led to superior activation of AH1 -responsive CD8+ T cells. Splenocytes were obtained from CT26-tumour bearing BALB / c mice 14 days after they were treated with aPD-L1 mAb and CS11 or CS11-sA5. The proportion of AH 1 -responsive splenocytes was then quantified with ELISpot, while the proportion of AH1 -responsive CD8+ T cells was quantified with intracellular cytokine staining (Figure 6E, Figure 9, Figure 10A, Figure 10B). Mice that were treated with A5-secreting CS11 had approximately 3 times more spot-forming units (p = 0.0009) and AH 1 -responsive CD8+ T cells (p = 0.0384). This higher proportion of activation reasonably explains the superior efficacy of CS11-sA5 compared to CS11.

[0291] Example 5: Generalization to other tumour models and another Clostridium

[0292] Neoantigen secretion by CS11 was investigated in a different tumour model. An OVA-expressing version of the 4T1 breast cancer cell line was engineered, 4T1-OVA (Figure 11A, Figure 11B, Figure 11C). A strain of CS11 which secretes OVA257-264, the immunodominant CD8+ T-cell epitope for OVA (33) was also created, CS11-sOVA (Figure 5A, Figure 5B). The 4T1-OVA cell line also incidentally expresses AH1 , hence allowing further validation of CS11-A5 in this model (34) (Figure 11 D). The propensity of 4T1 to metastasize and its resistance to checkpoint blockade make this cell line a challenging model (35).

[0293] 4T1-OVA tumour-bearing BALB / c mice were treated either with aPD-L1 alone, or with aPD-L1 in combination with various CS11 strains. The same treatment schedule was used as before (Figure 4A). All mice which received only aPD-L1 increased in tumour volume and died from metastatic disease (Figure 12) by day 9 prior to reaching the tumour volume endpoint (Figure 13A, Figure 13B). Despite the speed of metastatic spread, the combination of aPD-L1 with any of the CS11 strains produced significantly improved tumour reduction and survival outcomes (Figure 13A, Figure 13B). The best response was observed with CS11-sA5 + CS11-sOVA + aPD-L1 where 7 out of 9 mice were cured and disease free on day 60. All 7 cured mice were resistant to tumour rechallenge. The complete response rate for the other CS11 strains varied widely. CS11 produced a complete response rate of 1 / 8 while the antigen-secreting versions generally did better: CS11-sAH1 (2 / 8), CS11-sA5 (4 / 9), CS11-sOVA (5 / 9). All these cured mice were disease free on day 60 and resisted tumour challenge. In summary, two CS11-secreted antigens are more effective than one, and one is far more effective than none.

[0294] A strain of C. novyi-NT was created that expresses OVA257-264 under the transcriptional control of the C. novyi-NT flagellin gene promoter and C. perfrigens ferrodoxin gene terminator, CN-sOVA. Secretion was facilitated by adding the C. novyi-NT phospholipase C gene secretion signal (Figure 5A, Figure 5B). Since C. novyi-NT is known to colonize B16 tumours, B16F10 was used (a subline of B16 with high lung metastatic ability). As with 4T1 , B16F10 was engineered to express OVA. Tumours were subcutaneously established in C57BL / 6 mice. Either CN or CN-sOVA were then administered in combination with aPD-L1 according to the schedule in Figure 13C. Compared to mice that received CN, mice that instead received CN-sOVA experienced a longer period of steady disease before regrowth (Figure 13D) and a correspondingly longer survival (Figure 13E). Although no complete responses were observed, this experiment shows using a different Clostridium, that secretion of a tumour-matched neoantigen enhances its therapeutic potential.

[0295] Summary

[0296] Chemotherapy and radiotherapy eradicate cancer cells based mostly on their direct cytotoxic effects. Yet, myelosuppression is also one of their most common side effects. Such collateral damage to the immune system negatively impacts the activation of anti-tumour immune cells, possibly lowering the chances of the immune system of clearing residual disease. Yet, current immune checkpoint inhibitors are used in combination with chemo-and radiotherapy because immunotherapy alone is insufficient for a robust clinical response.

[0297] Immune checkpoint inhibitors only release the brakes on existing anti-tumour responses. The data herein demonstrates that Clostridium is an ideal vehicle to stimulate these anti-tumour responses in the first place. Used in combination, neoantigen-secreting Clostridium and immune checkpoint inhibition is able to synergistically recruit and activate anti-tumour immune cells. Clostridium therapy also has the important advantage of being immunostimulatory instead of immunosuppressive. It is, in essence, a live immunologic adjuvant which can be delivered to the tumour with exquisite specificity due to its inability to survive in normoxic tissues.

[0298] This is the first study where Clostridium bacteria have been engineered to secrete neoantigens and tested for efficacy in animal models. Clostridium septicum has a low incidence of infections in parallel with the unusually high correlation between C. septicum and existing malignancies (many times occult). This association remains unexplained but biases C. septicum towards being a safer and more tumour-tropic agent.

[0299] The anti-tumour efficacy observed with neoantigen-secreting C. septicum and aPD-L1 was surprising. The data suggest that engineering Clostridium to secrete neoantigens may increase the efficacy radius of Clostridium without cytotoxic chemo-or radiotherapy.

[0300] The data shows CS11 to be a generalizable delivery platform for neoantigen delivery. Therefore, customized neoantigens and combinations thereof can be designed to match specific tumour neoantigen profiles and delivered via CS11 . If required, CS11 can be rapidly engineered to accommodate novel neoantigens in a patient-specific manner with minimal turnaround time. The boost in efficacy from neoantigen secretion also extends to C. novyi-NT, showing that our observations are applicable to other tumour-colonizing Clostridium. References

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Claims

Claims1. A Clostridium bacterium, wherein the bacterium is genetically modified.

2. The Clostridium bacterium according to claim 1 , wherein the bacterium is modified to express a heterologous protein.

3. The Clostridium bacterium according to claim 2, wherein the heterologous protein is an antigen.

4. The Clostridium bacterium according to claim 3, wherein the antigen is a tumour-associated antigen.

5. The Clostridium bacterium according to claim 3 or claim 4, wherein the antigen is a tumour neoantigen, comprises an immunodominant epitope and / or comprises a mimotope.

6. The Clostridium bacterium according to any one of claims 1 to 5, wherein the bacterium is modified to express a plurality of heterologous proteins.

7. The Clostridium bacterium according to any one of claims 1 to 6, wherein the bacterium is modified to reduce or prevent expression of a gene encoding a toxin.

8. The Clostridium bacterium according to claim 7, wherein the gene encodes an alpha toxin.

9. The Clostridium bacterium according to claim 7 or claim 8, wherein the gene is csa.

10. The Clostridium bacterium of any one of claims 1 to 9, wherein the bacterium is of a species selected from Clostridium septicum and Clostridium novyi.

11. The Clostridium bacterium of any one of claims 1 to 10, wherein the bacterium is of the species Clostridium septicum .

12. A method of producing the Clostridium bacterium of any one of claims 1 to 11 .

13. A Clostridium bacterium obtainable by the method of claim 12.

14. An isolated Clostridium bacterium according to any one of claims 1 to 11 or claim 13.

15. A population comprising the Clostridium bacterium according to any one of claims 1 to 11 or claims 13 to 14.

16. A Clostridium spore obtained from the Clostridium bacterium according to any one of claims 1 to 11 or claims 13 to 14.

17. A composition comprising the Clostridium bacterium according to any one of claims 1 to 11 or claims 13 to 14, or the Clostridium spores according to claim 16.

18. A modified microorganism, spore derived from a modified microorganism, or a composition comprising a modified microorganism or spore derived from a modified microorganism, for use in a method of treating or preventing a cancer, wherein the modified microorganism is modified to express a heterologous protein and / or to reduce or prevent expression of a gene encoding a toxin.

19. Use of a modified microorganism, spore derived from a modified microorganism, or a composition comprising a modified microorganism or spore derived from a modified microorganism, in the manufacture of a medicament for treating or preventing a cancer, wherein the modified microorganism is modified to express a heterologous protein and / or to reduce or prevent expression of a gene encoding a toxin.

20. A method of treating or preventing a cancer, comprising administering to a subject a therapeutically- or prophylactically-effective amount of a modified microorganism, spore derived from a modified microorganism, or a composition comprising a modified microorganism or spore derived from a modified microorganism, wherein the modified microorganism is modified to express a heterologous protein and / or to reduce or prevent expression of a gene encoding a toxin.21 . The modified microorganism, spore or composition, the use, or the method, according to any one of claims 18 to 20, wherein the heterologous protein is an antigen.

22. The modified microorganism, spore or composition, the use, or the method, according to claim 21 , wherein the antigen is a tumour-associated antigen.

23. The modified microorganism, spore or composition, the use, or the method, according to claim 21 or claim 22, wherein the antigen is a tumour neoantigen, comprises an immunodominant epitope and / or comprises a mimotope.

24. The modified microorganism, spore or composition, the use, or the method, according to any one of claims 18 to 23, wherein the modified microorganism is modified to express a plurality of heterologous proteins.

25. The modified microorganism, spore or composition, the use, or the method, according to any one of claims 18 to 24, wherein the gene encodes an alpha toxin.

26. The modified microorganism, spore or composition, the use, or the method, according to any one of claims 18 to 25, wherein the gene is csa.

27. The modified microorganism, spore or composition, the use, or the method, according to any one of claims 18 to 26, wherein the modified microorganism is a Clostridium bacterium.

28. The modified microorganism, spore or composition, the use, or the method, according to claim27, wherein the Clostridium bacterium is of a species selected from Clostridium septicum and Clostridium novyi.

29. The modified microorganism, spore or composition, the use, or the method, according to claim28, wherein the Clostridium bacterium is of the species Clostridium septicum.

30. The modified microorganism, spore or composition, the use, or the method, according to any one of claims 18 to 29, wherein the modified microorganism is as defined in section A or section B of the present disclosure.31 . A Clostridium septicum bacterium for use in a method of medical treatment or prophylaxis.

32. The Clostridium bacterium according to any one of claims 1 to 11 or claims 13 to 14, Clostridium spore according to claim 16, or a composition according to claim 17, for use in a method of medical treatment or prophylaxis.

33. The Clostridium bacterium according to any one of claims 1 to 11 or claims 13 to 14, Clostridium spore according to claim 16, or a composition according to claim 17, for use in a method of treating or preventing a cancer.

34. Use of the Clostridium bacterium according to any one of claims 1 to 11 or claims 13 to 14, Clostridium spore according to claim 16, or a composition according to claim 17, in the manufacture of a medicament for treating or preventing a cancer.

35. A method of treating or preventing a cancer, comprising administering to a subject a therapeutically- or prophylactically-effective amount of the Clostridium bacterium according to any one of claims 1 to 11 or claims 13 to 14, Clostridium spore according to claim 16, or a composition according to claim 17.

36. The modified microorganism, the Clostridium bacterium, spore or composition, the use, or the method, according to any one of claims 18 to 30 or claims 33 to 35, wherein the cancer is selected from: a solid tumour, breast cancer, breast carcinoma, ductal carcinoma, gastric cancer, gastric carcinoma, gastric adenocarcinoma, colorectal cancer, colorectal carcinoma, colorectal adenocarcinoma, head andneck cancer, squamous cell carcinoma of the head and neck, lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung carcinoma, ovarian cancer, ovarian carcinoma, ovarian serous adenocarcinoma, renal cancer, renal cell carcinoma, renal clear cell carcinoma, renal cell adenocarcinoma, renal papillary cell carcinoma, pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, cervical cancer, cervical squamous cell carcinoma, skin cancer, melanoma, esophageal cancer, esophageal adenocarcinoma, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, uterine cancer, uterine corpus endometrial carcinoma, thyroid cancer, thyroid carcinoma, pheochromocytoma, paraganglioma, bladder cancer, bladder urothelial carcinoma, prostate cancer, prostate adenocarcinoma, sarcoma and thymoma.

37. The modified microorganism, the Clostridium bacterium, spore or composition, the use, or the method according to any one of claims 18 to 30 or claims 33 to 36, wherein the cancer is characterised by hypoxia.

38. The modified microorganism, the Clostridium bacterium, spore or composition, the use, or the method according to any one of claims 18 to 30 or claims 33 to 37, wherein the cancer comprises cells expressing a given antigen, and wherein the modified microorganism, or the Clostridium bacterium, is modified to express the given antigen39. The modified microorganism, the Clostridium bacterium, spore or composition, the use, or the method according to any one of claims 18 to 30 or claims 33 to 38, further comprising an additional therapeutic or prophylactic intervention selected from chemotherapy, immunotherapy, radiotherapy, surgery, and / or hormone therapy.

40. The modified microorganism, the Clostridium bacterium, spore or composition, the use, or the method according to claim 39, wherein the method comprises administering a chemotherapeutic agent.41 . The modified microorganism, the Clostridium bacterium, spore or composition, the use, or the method according to claim 39 or claim 40, wherein the method comprises administering an immunomodulatory agent.

42. The modified microorganism, the Clostridium bacterium, spore or composition, the use, or the method according to any one of claims 39 to 41 , wherein the method comprises administering an immune checkpoint inhibitor.

43. The modified microorganism, the Clostridium bacterium, spore or composition, the use, or the method according to claim 42, wherein the immune checkpoint inhibitor is capable of inhibiting signalling mediated by PD-1 , CTLA-4, LAG-3, TIM-3, TIGIT or BTLA.

44. The modified microorganism, the Clostridium bacterium, spore or composition, the use, or the method according to claim 43, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody or fragment thereof, or anti-PD-L1 antibody or fragment thereof.

45. The modified microorganism, the Clostridium bacterium, spore or composition, the use, or the method according to any one of claims 39 to 44, wherein the method comprises administering an immunomodulatory cytokine.

46. A method of treating cancer in a subject, comprising:(i) administering a modified microorganism, a spore derived from a modified microorganism, or a composition comprising a modified microorganism or spore derived from a modified microorganism to the subject, wherein the modified microorganism has been modified to express an antigen identified as being expressed in the cancer to be treated.

47. A method of treating cancer in a subject, comprising:(i) identifying an antigen expressed in the cancer to be treated; and(ii) administering a modified microorganism, a spore derived from a modified microorganism, or a composition comprising a modified microorganism or spore derived from a modified microorganism to the subject, wherein the modified microorganism has been modified to express the antigen.

48. A method of treating cancer in a subject, comprising:(i) identifying an antigen expressed in the cancer to be treated;(ii) producing a microorganism modified to express the antigen; and optionally,(iii) administering the modified microorganism, a spore derived from the modified microorganism, or a composition comprising the modified microorganism or spore derived from the modified microorganism to the subject.

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