Fusobacterium nucleatum-based cancer vaccines
Patent Information
- Application Number
- PCT/NL2026/050084
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2026-03-26
- Publication Date
- 2026-10-01
Abstract
Description
[0001] FUSOBACTERIUM NUCLEATUM-BASED CANCER VACCINES
[0002] FIELD OF THE INVENTION
[0003]
[0001] The invention pertains to the field of cancer, in particular colorectal cancer. In particular, it relates to the field of immune system directed approaches, i.e. immunologic approaches, for prevention and / or treatment of Fusobacterium nucleatum associated diseases, in particular cancer. This invention pertains in general to a vaccine for mammals against a tumour. Particularly, the invention pertains to a vaccine comprising outer membrane vesicles obtained from Fusobacterium nucleatum for use in the prevention and / or treatment in mammals of a tumour, preferably selected from a gastro-intestinal tumour, an oral tumour, a breast tumour, a pancreas tumour, or a colorectal tumour.
[0004] BACKGROUND OF THE INVENTION
[0005]
[0001] The background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is relevant prior art.
[0006]
[0002] The human microbiome comprises a diverse set of bacteria, viruses, fungi, protozoa, and archaea that exist on and within the human body. The highest number and the largest variety of species of these bacteria, viruses, fungi, protozoa, and archaea are found in the gastro-intestinal tract. While the majority of microbes of the microbiome may not directly harm humans and are actually beneficial, some bacteria are negatively associated with human health. One of such bacteria is the species Fusobacterium nucleatum (F. nucleatum).
[0007]
[0003] F. nucleatum is an anaerobic, gram negative bacterial species that has been associated with oral infections, pre-term labour, appendicitis, and inflammatory bowel disease. The last decade F. nucleatum has gained significant attention for its association with certain cancers, including colorectal cancer, oral cell cancer, breast cancer or pancreas cancer (Castellarin, M, et al. Genome research 2012; 22.2: 299-306) and was found in higher abundance in colorectal tumour tissues compared to normal tissues. Enrichment of the tumour microenvironment of colorectal cancer withF. nucleatum has been associated with a poor prognosis (Mima K, Nishihara R, Qian ZR, et al. Gut 2016;65:1973-1980).
[0008]
[0004] It has been suggested that F. nucleatum has implications for various pathological processes associated with cancer, including tumorigenesis, metastasis, and resistance to therapy and that its pro-carcinogenic effects are primarily linked to its interactions with host cells and its ability to modulate the immune microenvironment. F. nucleatum is understood to be that of an active contributor to tumour biology rather than a simple bystander. It has been found that F. nucleatum influences several key aspects of cancer progression. Through the interaction of its surface proteins with host cell receptors, it can directly activate oncogenic signalling pathways that promote tumour cell proliferation. Furthermore, F. nucleatum is known to alter the tumour microenvironment by inducing inflammatory cytokine production, which in turn promotes angiogenesis and metastatic conditioning. It is also believed to contribute to immune suppression by engaging inhibitory immune receptors and recruiting immunosuppressive cell populations. Finally, evidence suggests that F. nucleatum can induce chemoresistance by activating specific signalling pathways, thereby protecting them from therapies that target proliferating cells.
[0009]
[0005] A recent patent application (WQ2022 / 029024A1) describes a vaccine composed of a truncated Fusobacterium nucleatum adhesin A (FadA) protein and methods of treating or preventing cancer using such protein.
[0010]
[0006] To date there remains an unmet medical need to provide remedies for the prevention and treatment of pathologies involving F. nucleatum, in particular cancers.
[0011]
[0007] To overcome any one of the aforementioned limitations it is an object of the invention to provide for a vaccine that is able to prevent and / or treat pathologies involving F. nucleatum, in particular cancers.
[0012] SUMMARY OF THE INVENTION
[0013]
[0008] Surprisingly, the inventors found that by the invention as disclosed herein at least such object was met. As embodied and described herein, the present invention relates to the surprising finding that the outer membrane vesicles of F. nucleatum are promising as medicament and particularly as medicament in the prevention and / or treatment of a cancer.
[0009] It was found that the main advantage of using an OMV is that multiple mechanisms through which F. nucleatum induces and enhances F. nucleatum associated diseases and / or disorders, such as cancer, preferably such as colorectal cancer, can be targeted. The OMVs contain many surface antigens in their native conformation. As such, multiple antigens will be included, which can result in a highly efficacious medicament, preferably an immunogenic composition such as a vaccine.
[0014]
[0010] Without being bound by theory the inventors found that this medicament, preferably in the form of an immunogenic composition such as a vaccine, i.e., a medicament that targets F. nucleatum and comprises the outer membrane vesicles of F. nucleatum, can induce a F. nucleatum specific immune response that results in, for example, one or more of less colonization of F. nucleatum in the tumour and / or tumourmicroenvironment, increased sensitivity of the tumour to therapy, including chemotherapy, reduced growth of the tumour, and / or improved survival for mammals having the cancer. Further, the medicament was found to be safe.
[0015]
[0011] Therefore, in a first aspect the invention provides for an outer membrane vesicle from a F. nucleatum, for use as a medicament.
[0016]
[0012] In further aspects methods for the prevention or treatment of a mammal, of a cancer, of F. nucleatum in a tumour and / or tumour microenvironment, methods for preventing tumorigenesis, tumour growth, metastasis, and / or resistance to cancer therapy and methods for the raising of an immune response are provided.
[0017]
[0013] In addition, the inventors found that the reduction of the expression of FomA protein in F. nucleatum, preferably wherein F. nucleatum is substantially devoid of expression of functional FomA protein, has the benefit of having a significantly increased OMV production.
[0018]
[0014] In addition, the inventors have found that reduced expression of, or absence of, functional FomA protein in the OMVs (as the consequence of the reduction of, or absence of, expression of FomA in F. nucleatum from which the OMVs are obtained) provides OMVs that are less capable of inducing F. nucleatum autoaggregation and / or of less capable of inducing adhesion of F. nucleatum to host cells, including cancer cells (e.g. after OMV fusion with such host cell). In another embodiment, the OMVs thus obtained may be less capable of biofilm formation involving F. nucleatum and / or may be less capable of inducing F. nucleatum autoaggregation and / or less capable of inducing adhesion of F. nucleatum to host cells, including cancer cells. / PD
[0015] Accordingly, and without being bound by theory, the reduced expression of FomA in F. nucleatum from which the OMVs are obtained results in an immunogenic composition that is more safe and that is having a desirable safety profile.
[0019]
[0016] Accordingly, in an aspect there is provided for F. nucleatum and / or outer membrane vesicles obtained from such F. nucleatum, wherein the F. nucleatum is a F. nucleatum that is substantially devoid of expression of functional FomA protein, preferably wherein the F. nucleatum does not express FomA protein. Accordingly, in a final aspect there is provided for F. nucleatum outer membrane vesicles obtained from F. nucleatum, wherein the OMVs are substantially devoid of expression of functional FomA protein, preferably wherein the OMVs do not express (comprise) FomA protein.
[0020] DEFINITIONS
[0021]
[0017] A portion of this disclosure contains material that is subject to copyright protection (such as, but not limited to, diagrams, device photographs, or any other aspects of this submission for which copyright protection is or may be available in any jurisdiction.). The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent Office patent file or records, but otherwise reserves all copyright rights whatsoever.
[0022]
[0018] Various terms relating to the methods, compositions, uses and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein.
[0023]
[0019] For purposes of the present invention, the following terms are defined below.
[0024]
[0020] As used herein, the singular form terms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like. For example, a method for culturing a cell includes the culturing of a plurality of cells (e.g., 10's, 100's, 1000's, 10's of thousands, 100's of thousands, millions, or more).
[0021] As used herein, “comprising" or “to comprise” is construed as being inclusive and open ended, and not exclusive. Specifically, the term and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps, or components. It also encompasses the more limiting “to consist of.”
[0025]
[0022] As used herein, “comprising” when placed before the recitation of steps in a method means that the method encompasses one or more steps that are additional to those expressly recited, and that the additional one or more steps may be performed before, between, and / or after the recited steps, unless the content clearly dictates otherwise. For example, a method comprising steps a, b, and c encompasses a method of steps a, b, x, and c, a method of steps a, b, c, and x, as well as a method of steps x, a, b, and c. Furthermore, the term “comprising” when placed before the recitation of steps in a method does not (although it may) require sequential performance of the listed steps, unless the content clearly dictates otherwise. For example, a method comprising steps a, b, and c encompasses, for example, a method of performing steps in the order of steps a, c, and b, the order of steps c, b, and a, and the order of steps c, a, and b, etc.
[0026]
[0023] As used herein the term “outer membrane vesicle” also abbreviated herein as “OMV”, refers to a substantially spherical, microstructural vesicle of the outer membrane. OMVs are formed by a sort of budding process, resulting in vesicles with the outer membrane located on their surface-exposed side. OMVs are commonly produced by Gram-negative bacteria. Generally, OMVs have a diameter between about 20 to about 250 nanometers (nm). Within the context of the current invention the OMVs are OMVs derived from F. nucleatum by such budding process. Within the context of the current invention, OMVs do not include versicle or membrane preparations obtained from F. nucleatum by physical or chemical disruption of F. nucleatum.
[0027]
[0024] As used herein, a “pharmaceutically acceptable carrier” is any compatible medium, viz. a medium that after administration to a mammal does not induce significant adverse reactions in the treated subject and that, for example allows for presenting the OMVs and the antigen presented therein to the immune system of the subject, and or to the cells of the subject, after administration of a composition according to the invention and comprising such carrier. Such a pharmaceuticallyacceptable carrier may for example be a liquid containing water and / or any other compatible solvent or a solid carrier such as commonly used to obtain freeze-dried vaccines (for example, based on sugars and / or proteins), optionally comprising additional immune-stimulating agents (also called adjuvants). Optionally other substances such as stabilisers, viscosity modifiers or other components are added depending on the intended use or required properties of the corresponding medicament, preferably vaccine.
[0028]
[0025] As used herein, “pre vention” for example when used in relation to a cancer, e.g., a tumour, is the same as arriving at protective immunity, i.e. aiding in preventing, ameliorating or curing said tumour, for example to prevent or reduce of the actual occurrence of the tumour or one or more clinical signs resulting from the tumour.
[0029]
[0026] As used herein, the term “tumour1’, refers to an abnormal mass of tissue that forms when cells, such as human cells, grow and divide more than they should or do not die when they should. Tumours may be malignant or benign. As meant herein, the term “tumour1’ includes primary tumours and secondary tumours (metastases). The tumour may be a refractory tumour, i.e. a tumour that is resistant to a medical treatment (resistant cancer).
[0030]
[0027] As regards the embodiments described in this specification, it is intended that each embodiment be read independently as well as in combination with another embodiment. For example, in case of an embodiment 1 reciting 3 alternatives A, B and C, an embodiment 2 reciting 3 alternatives D, E and F and an embodiment 3 reciting 3 alternatives G, H and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless the content clearly dictates otherwise or unless it is specifically mentioned otherwise.
[0031] DETAILED DESCRIPTION
[0032]
[0028] The invention is defined herein, and in particular in the accompanying claims. Subject-matter which is not encompassed by the scope of the claims does not form part of the present claimed invention.
[0029] It is contemplated that any method, use, or composition described herein can be implemented with respect to any other method, use or composition described herein. Embodiments discussed in the context of methods, use and / or compositions of the invention may be employed with respect to any other method, use or composition described herein. Thus, an embodiment pertaining to one method, use or composition may be applied to other methods, uses and compositions of the invention as well.
[0033]
[0030] In a first aspect, the invention provides for an outer membrane vesicle (OMV) from a Fusobacterium nucleatum, for use as a medicament. Preferably, the F. nucleatum is an isolated F. nucleatum. A skilled person is familiar with methods known in the art for obtaining and / or isolating F. nucleatum. Preferably the medicament is suitable for use in mammals, in particular humans.
[0034]
[0031] In aspects, the invention provides for an OMV from F. nucleatum for use as a medicament for raising an immune response in a mammal. It is herein contemplated that an immune response can be induced / raised as a result of the administration of an outer membrane vesicle of F. nucleatum, or a composition comprising the same, to a mammal.
[0035]
[0032] Methods for obtaining OMVs from F. nucleatum have been described in the art and may be performed in accordance with the herein described Example 1. However, other methods for obtaining OMVs from F. nucleatum are also envisioned and encompassed by the current invention.
[0036]
[0033] In preferred embodiments, the mammal is a human.
[0037]
[0034] In embodiments there is provided for an OMV for use according to the invention, wherein the OMV is for use as a medicament in the prevention and / or treatment of a tumour in a mammal. It is contemplated that the OMV is capable of inducing an immune response that can prevent and / or treat tumours. In some preferred embodiments, the tumour is one or more selected from a gastro-intestinal tumour, an oral tumour, a breast tumour, a pancreas tumour, or a colorectal tumour. In more preferred embodiments, the tumour is a colorectal tumour. In further embodiments, the OMV for use according to the invention for the prevention and / or treatment of a mammal suspected of having, or diagnosed with, Lynch syndrome. In yet further embodiments, the OMV for use according to the invention is for raising an immune response in a mammal suspected of having, or diagnosed with, Lynch syndrome.Lynch syndrome is a genetic condition that increases the risk of several types of cancer, particularly colorectal and endometrial cancers.
[0038]
[0035] It is further contemplated that the invention as described and embodied herein is suitable for use in the prevention and / or treatment of diseases or disorders associated with F. nucleatum. Preferably, the invention as described and embodied herein is further suitable for use in the prevention and / or treatment of oral infections, dental infections, periodontitis, gingivitis, cardiovascular diseases, irritable bowel disease, inflammatory bowel disease, pre-term labour and / or appendicitis.
[0039]
[0036] In embodiments, the OMV for use according to the invention is for in the prevention and / or treatment of a tumour wherein the tumour is characterized by the presence of F. nucleatum in the tumour and / or tumour microenvironment, preferably the colorectal tumour and / or microenvironment of the colorectal tumour. It is contemplated that F. nucleatum can be present in the tumour, e.g., between tumorous cells and / or around the tumour, e.g., in the tumour microenvironment.
[0040]
[0037] In embodiments the OMV for use as a medicament according to the invention comprises that the medicament is in the form of an immunogenic composition, preferably a vaccine, preferably wherein the vaccine is a cancer vaccine. It is preferred that the immunogenic composition or vaccine comprises a pharmaceutically acceptable carrier.
[0041]
[0038] In preferred embodiments a multitude, e.g., 10.000s, 100.000s, 1.000.000s or even more, of outer membrane vesicles are present in any one of the afore-described forms of the medicament.
[0042]
[0039] In embodiments the OMV for use according to the invention comprises that F. nucleatum is one or more selected from F. nucleatum spp. animalis, F. nucleatum spp. fusiforme, F. nucleatum spp. nucleatum, F. nucleatum spp. polymorphum and F. nucleatum spp. Vincentii. In preferred embodiments, F. nucleatum is F. nucleatum spp. Animalis. Even more preferred embodiments comprise that F. nucleatum is the F. nucleatum spp. animalis clade C2. It is particularly envisioned by the inventors that the OMVs from F. nucleatum spp. animalis ATCC 51191 are particularly beneficial for at least one or more of the uses described herein. In other embodiments, the OMVs for use according to the invention comprises that F. nucleatum is a F. nucleatum from the Fusobacterium nucleatum group as described by Sivertsen (doi: https: / / doi.org / 10.1101 / 2025.03.20.644344).
[0040] In embodiments, the OMV for use according to the invention comprises that F. nucleatum is F. nucleatum with reduced expression of functional FomA protein, preferably wherein the F. nucleatum does not express FomA protein. In a preferred embodiment, the F. nucleatum has been genetically modified to reduce expression of functional FomA protein therein, preferably such that the F. nucleatum does not express FomA protein. FomA is a F. nucleatum Envelope Protein. FomA is one of the major outer membrane proteins of F. nucleatum and shares structural and functional similarities with the OmpA family proteins found in other Gram-negative bacteria. The natural function of FomA has been reported to be involved in adhesion of F. nucleatum to cells. Accordingly, FomA plays a role in bacterial (co)-aggregation and biofilm formation. As a major outer membrane protein, FomA is therefore contemplated by the inventors to be a primary mediator of the direct interactions with host cells. Reduced expression of FomA in a F. nucleatum comprises a reduced expression of the native protein FomA as compared to the native F. nucleatum. Reduced expression of FomA is in embodiments provided for by mutation or deletion of the FomA gene in the F. nucleatum. Preferably the F. nucleatum does not express FomA.
[0043]
[0041] It was surprisingly found that reduced expression of the FomA protein in F. nucleatum may significantly increase OMV production. In other words, the inventors found that F. nucleatum having a (point) mutation (e.g., substitution, insertion or deletion) in an endogenous FomA gene or having a deletion of the FomA gene may provide for a so-called high-blebbing phenotype, meaning that more OMVs are produced compared to a control comprising an unmutated, functional, FomA gene, e.g., a wildtype FomA gene. This provides the benefit of a higher yield of OMVs that are also substantially devoid, or fully absent, of the FomA protein implicated in tumourpromoting and immunosuppressive interactions. Further, reduced expression, or absence, of functional FomA protein in the OMVs provides OMVs that are less capable of inducing F. nucleatum autoaggregation and / or of less capable of inducing adhesion of F. nucleatum to host cells, including cancer cells (e.g. after OMV fusion with such host cell). In other embodiment, the OMVs thus obtained may be less capable of biofilm formation involving F. nucleatum. Accordingly, and without being bound by theory, the reduced expression of FomA in F. nucleatum from which the OMVs are obtained results in an immunogenic composition, preferably a vaccine composition, having a desirable safety profile, e.g., a vaccine that is safe to use and / or has animproved safety profile compared to a vaccine comprising functional FomA proteins. The resulting immunogenic composition, or vaccine composition, comprising OMVs wherein FomA is reduced or absent, is therefore contemplated to be more effective as compared to OMVs that do comprise FomA, as the host immune system can mount a response against other relevant F. nucleatum antigens without the potential confounding immunosuppressive and pro-tumorigenic effects mediated by the FomA protein.
[0044]
[0042] Mutation and / or a deletion of the FomA gene can be obtained by using routine gene engineering methods known and described in the art. One preferred method is SacB-based counterselection.
[0045]
[0043] In preferred embodiments, the FomA protein is not expressed by F. nucleatum from which an OMV according to the invention is obtained.
[0046]
[0044] In embodiments, the OMV for use according to the invention comprises one or more of the following:
[0047] wherein the OMV is a purified OMV;
[0048] wherein the OMV is a non-modified OMV obtained from F. nucleatum’, wherein the OMV is obtained with a method that does not involve the use of detergents and / or sonification; and / or
[0049] wherein the OMV is substantially devoid of FomA protein.
[0050]
[0045] Purification of OMVs can be performed by using methods known in the art such as those described in Example 1. It is particularly preferred that an OMV for use according to the invention is not obtained by methods involving the use of detergents and / or sonification. It was found that such methods comprising detergent and / or sonication may alter the protein composition and properties of the resulting OMVs compared to spontaneously produced OMV.
[0051] a) In embodiments, the OMV for use according to the invention comprises that the route of administration of the OMV is intradermal, intramuscular, intranasal, oral, sublingual, or buccal. A skilled person is aware that dosing strongly depends on the route of administration. In an example, it is contemplated that a suitable dose for OMV for use as a medicament may be in the 25 pg - 1.5 mg range. Any one dose in said range is herein encompassed. In the case the OMV of the invention are administered as a vaccine, said vaccine can be administered once or more than once, for example in a prime-boost or prime-boost-boost regimen, wherein a first vaccine (prime) isadministered between about 1 - 12 months, preferably between about 1 - 6 months, more preferably about 1-3 months, for example with a 3 - 6 weeks interval, prior to a second vaccine (boost). It is contemplated and encompassed herein that further boost vaccines (third, fourth, fifth, etc.) can be administered to the mammal in need thereof, for example in a case wherein it is required to boost an immune response against F. nucleatum.
[0052]
[0046] In a second aspect there is provided for a method of treating a mammal, wherein the method comprises administering an OMV from Fusobacterium nucleatum to the mammal, for example according to a dosage regimen as disclosed herein (e.g. in the previous paragraph).
[0053]
[0047] In a further aspect there is provided for a method of raising an immune response in a mammal, wherein in the method comprises administering an OMV from F. nucleatum to the mammal in need thereof, preferably wherein the immune response is against F. nucleatum, preferably against F. nucleatum in the tumour and / or tumour microenvironment.
[0054]
[0048] In a further aspect there is provided for a method of preventing or treating cancer in a mammal, wherein the method comprises administering an OMV from F. nucleatum to the mammal.
[0055]
[0049] In a further aspect there is provided for a method of preventing or treating F. nucleatum in a mammal or in a tumour and / or tumour microenvironment comprised in the mammal, wherein the method comprises administering an OMV from F. nucleatum to the mammal, preferably wherein the mammal comprises a tumour characterized by the presence of F. nucleatum in the tumour and / or tumour microenvironment.
[0056]
[0050] In a further aspect there is provided for a method of preventing tumorigenesis and preventing and / or treating tumour growth, metastasis, and / or resistance to cancer therapy in a mammal, wherein the method comprises administering an outer membrane vesicle from F. nucleatum to the mammal.
[0057]
[0051] In embodiments of the above-described one or more methods, preferably all methods, provided herein, the OMV is as defined in the current disclosure and / or comprise that the OMV is in the form of an immunogenic composition, preferably a vaccine, preferably wherein the vaccine is a cancer vaccine.
[0058]
[0052] In a further aspect of the invention there is provided for a F. nucleatum and / or at least one, preferably a multitude of, OMVs obtained from such F. nucleatum,wherein the F. nucleatum is a F. nucleatum that is substantially devoid of expression of functional FomA protein. In preferred embodiments, F. nucleatum does not express FomA protein.
[0059]
[0053] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein), readily modify and / or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein.
[0060]
[0054] All references cited herein, including journal articles or abstracts, published or corresponding patent applications, patents, or any other references, are entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also entirely incorporated by references.
[0061]
[0055] It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.
[0062]
[0056] It will be understood that all details, embodiments, and preferences discussed with respect to one aspect of embodiment of the invention is likewise applicable to any other aspect or embodiment of the invention and that there is therefore not need to detail all such details, embodiments, and preferences for all aspect separately.
[0063]
[0057] Having now generally described the invention, the same will be more readily understood through reference to the following examples which is provided by way of illustration and is not intended to be limiting of the present invention. Further aspects and embodiments will be apparent to those skilled in the art.
[0064] EXAMPLES
[0065] These examples are non-limiting demonstrations of the current invention.Example 1: OMV vaccine production
[0066] One example for the production of a vaccine according to the invention is provided below and is divided in the following topics:
[0067] 1) Culturing F. nucleatum and OMV isolation,
[0068] 2) Formulation of F. nucleatum vaccine,
[0069] 1. Culturing F. nucleatum and OMV isolation and characterization
[0070] F. nucleatum is cultured using methods known in the art, for example, as described by Wang et al. (Wang et al, Nat Commun. 2024 Apr 30;15(1):3669). In short, F. nucleatum is cultured in anaerobic conditions at temperature of about 37 °C.
[0071] From the culture OMV may be isolated, for example, by using any one of the methods as reviewed by Klimentova et al. (Klimentova et al, Microbiological Research 2015, Volume 170, Pages 1-9) or combinations of these methods. The procedure usually consists in the following steps: cultivation of bacteria, removal of intact bacteria and OMV isolation from the culture filtrate by concentration and purification, for example by tangential flow filtration, high speed centrifugation and size exclusion chromatography or a combination of these steps. As a last step a buffer exchange and sterile filtration of the purified and concentrated OMVs is foreseen. OMVs are characterized (e.g. purity, identity, particle size and distribution, composition) by a variety of assays including DLS, PCR, protein concentration and MS.
[0072] 2. Formulation of the vaccine
[0073] To obtain a vaccine formulation according to the invention, the purified and concentrated OMV are diluted and mixed with an adjuvant, such as aluminiumhydroxide (AI(OH)3) and followed by a fill / finish step using methods known in the art.
[0074] Example 2: Efficacy & safety of the OMV vaccine
[0075] One example of a safety and efficacy test of a vaccine formulation according to the invention is provided below and is divided in the following topics:
[0076] 1 ) Determination of the immunogenicity of the OMV vaccine,
[0077] 2) The use of the OMV vaccine to prevent colorectal cancer,
[0078] 3) The use of the OMV vaccine as therapeutic treatment for colorectal cancer;1. Determination of the immunogenicity and efficacy of the OMV vaccine
[0079] To determine the immunogenicity and preventative and therapeutic efficacy of the OMV isolated and formulated in Example 1, OMV can be evaluated in assays well known to the skilled person in the art, for example in immunogenicity studies in animals, like mice, as described by Rudi et al (Rudi E et aL, Front Immunol. 2024 Nov 28;15:1506638), Singh Solanki et al (Solanki KS, et aL, Int ImmunopharmacoL 2021 Jan;90:107148.) and / or Grandi et al (Grandi A et al. Vaccines (Basel). 2023 Sep 29;11(10):1546). In short, mice are vaccinated with the OMV vaccine following a prime-boost or a prime-boost-boost regimen. Two weeks after the last vaccination, blood samples are obtained to determine humoral and cellular immune responses against the OMV or Fusobacterium nucleatum using ELISA, Elispot and / or flow cytometry. Also, mucosal samples are collected and analysed for mucosal immune responses.
[0080] To determine the efficacy of preventative and therapeutic vaccinations, colorectal tumour cells growing subcutaneously or orthotopically are inoculated with F. nucleatum. Chemotherapy is given, for example in the form of oxaliplatin. The effect of OMV vaccination on tumour growth will be determined using a digital caliper.
[0081] In another set of experiments the capacity of vaccination to prevent F. nucleatum-induced metastases is determined.
[0082] Safety of the vaccine is determined by measuring clinical abnormalities that could be attributed to the vaccine.
[0083] 2. The use of the vaccine to prevent colorectal cancer
[0084] In one exemplary envisioned clinical setting, the vaccine according to the invention will be administered intranasally following a prime-boost or a prime-boost-boost interval with approximately 3-6 weeks in between vaccinations. Optionally, more booster vaccinations are given approximately every 1 - 5 years.The vaccine will induce a specific immune response against F. nucleatum which prevents or reduces F. nucleatum infection, thereby also preventing the translocation of F. nucleatum to other sites in the human body avoiding F. nucleatum induced initiation and progression of cancer.
[0085] The vaccine is foreseen to be used to prevent colorectal cancer in persons at risk for the development of colorectal cancer, such as persons with Lynch syndrome or familial adenomatous polyposis. However, the vaccine is likely suitable for the general population.
[0086] 3. The use of the vaccine as therapeutic treatment for colorectal cancer
[0087] In another exemplary envisioned clinical setting, the vaccine according to the invention will be administered intradermally following a prime-boost or a prime-boost-boost interval with approximately 3-4 weeks in between vaccinations in patients diagnosed with colorectal cancer.
[0088] Frequent booster vaccinations are foreseen because of the suboptimal immune system of cancer patients in general. After vaccination patients will be treated with standard of care for both the primary tumour and metastases, including (a combination of) surgical removal, chemotherapy, radiotherapy, and immunotherapy.
[0089] Example 3: : Efficacy & safety of the OMV vaccine
[0090] One example for the production of a genetically modified vaccine according to the invention is provided below and is divided in the following topics:
[0091] 1 ) Knock out of specific target proteins in F. nucleatum.
[0092] 1. Knock out of specific target proteins in F. nucleatum
[0093] In order to obtain F. nucleatum with reduced expression of protein FomA, the gene encoding protein FomA can be knocked out using methods well-known to the skilled person, for example, by using a method that basically consist of counterselection using the Bacillus subtilis sacB gene coding for levansucrase (see, for example Zhou Pet al. Microbiol Spectr. 2025 Jan 7;13(1):e0206624) that allows introducing markerless deletion in Gram-negative bacteria. In short, cloning of sacB in gram-negative bacterialeads to the death of the transformed bacteria when they are plated in the presence of sucrose and the counterselection system is based on two recombination events. In the first recombination event SacB and a resistance marker are inserted at the target region in the bacterial genome via homologous recombination to delete the gene of interest. In the second recombination step SacB will be removed and the SacB containing region will be replaced with the final desired genetic modification (i.e. a full gene deletion). Bacteria will be selected by culturing in sucrose-containing medium. Surviving bacteria have the desired knockout modification. The effect of gene deletion on blebbing formation can, for example, be measured by determining the number of OMV produced per bacteria in comparison to bacteria still containing the gene encoding FomA. Further, bacterial co-aggregation and biofilm formation can be measured.
[0094] CLAUSES:
[0095] 1. An outer membrane vesicle from a Fusobacterium nucleatum, for use as a medicament.
[0096] 2. An outer membrane vesicle from Fusobacterium nucleatum for use as a medicament for raising an immune response in a mammal.
[0097] 3. An outer membrane vesicle for use according to any one of the previous clauses in the prevention or treatment of a tumour in a mammal, preferably wherein the tumour is selected from a gastro-intestinal tumour, an oral tumour, a breast tumour, a pancreas tumour, or a colorectal tumour, more preferably wherein the tumour is a colorectal tumour.
[0098] 4. An outer membrane vesicle for use according to any one of the previous clauses, wherein the tumour is characterized by the presence of Fusobacterium nucleatum in the tumour and / or tumour microenvironment.
[0099] 5. An outer membrane vesicle for use as a medicament according to any one of the previous clauses, wherein the medicament is in the form of an immunogeniccomposition, preferably a vaccine, preferably wherein the vaccine is a cancer vaccine.
[0100] 6. An outer membrane vesicle for use as a medicament according to any one of the previous clauses, wherein Fusobacterium nucleatum is selected from Fusobacterium nucleatum spp. animalis, Fusobacterium nucleatum spp. fusiforme, Fusobacterium nucleatum spp. nucleatum, Fusobacterium nucleatum spp. polymorphum and Fusobacterium nucleatum spp. vincentii, preferably wherein the Fusobacterium nucleatum is Fusobacterium nucleatum spp. animalis, more preferably Fusobacterium nucleatum spp. animalis clade C2.
[0101] 7. An outer membrane vesicle for use as a medicament according to any one of the previous clauses, wherein the Fusobacterium nucleatum is a Fusobacterium nucleatum with reduced expression of functional FomA protein, preferably wherein the Fusobacterium nucleatum does not express FomA protein.
[0102] 8. An outer membrane vesicle for use as a medicament according to any one of the previous clauses
[0103] - wherein the outer membrane vesicle is a purified outer membrane vesicle,
[0104] - wherein the outer membrane vesicle is a non-modified outer membrane vesicle obtained from Fusobacterium nucleatum,
[0105] - wherein the outer membrane vesicle is obtained with a method that does not involve the use of detergents and / or sonification, and / or - wherein the outer membrane vesicle is substantially devoid of FomA protein.
[0106] 9. An outer membrane vesicle for use as a medicament according to any one of the previous clauses wherein administration of the outer membrane vesicle is intradermal, intramuscular, intranasal, oral, sublingual, or buccal.A method of treating a mammal, wherein the method comprises administering outer membrane vesicles from Fusobacterium nucleatum to the mammal.
[0107] A method of raising an immune response in a mammal, wherein in the method comprises administering outer membrane vesicles from Fusobacterium nucleatum to the mammal in need thereof, preferably wherein the immune response is against Fusobacterium nucleatum, preferably against Fusobacterium nucleatum in the tumour and / or tumour microenvironment.
[0108] A method of preventing or treating cancer in a mammal, wherein the method comprises administering outer membrane vesicles from Fusobacterium nucleatum to the mammal.
[0109] A method of preventing or treating Fusobacterium nucleatum in a mammal or in a tumour comprised in the mammal, wherein the method comprises administering an outer membrane vesicle from Fusobacterium nucleatum to the mammal, preferably wherein the mammal comprises a tumour characterized by the presence of Fusobacterium nucleatum in the tumour and / or tumour microenvironment.
[0110] A method of preventing tumorigenesis, tumour growth, metastasis, and / or resistance to cancer therapy in a mammal, wherein the method comprises administering an outer membrane vesicle from Fusobacterium nucleatum to the mammal.
[0111] The method according to any one of the previous clauses 10 - 14, wherein the outer membrane vesicles are a defined in any one of the previous clauses 1 -9 are in the form of an immunogenic composition, preferably a vaccine, preferably wherein the vaccine is a cancer vaccine.
[0112] A Fusobacterium nucleatum and / or outer membrane vesicles obtained from such Fusobacterium nucleatum, wherein the Fusobacterium nucleatum is a Fusobacterium nucleatum that is substantially devoid of expression offunctional FomA protein, preferably wherein the Fusobacterium nucleatum does not express FomA protein.
[0113] An outer membrane vesicle, an immunogenic composition, preferably a vaccine as defined in any one of the previous clauses 1 - 9 or 16.
Claims
CLAIMS1. An outer membrane vesicle from a Fusobacterium nucleatum, for use as a medicament, wherein the Fusobacterium nucleatum is a Fusobacterium nucleatum with reduced expression of functional FomA protein.
2. An outer membrane vesicle from Fusobacterium nucleatum for use as a medicament for raising an immune response in a mammal.
3. An outer membrane vesicle for use according to any one of the previous claims in the prevention or treatment of a tumour in a mammal, preferably wherein the tumour is selected from a gastro-intestinal tumour, an oral tumour, a breast tumour, a pancreas tumour, or a colorectal tumour, more preferably wherein the tumour is a colorectal tumour.
4. An outer membrane vesicle for use according to any one of the previous claims, wherein the tumour is characterized by the presence of Fusobacterium nucleatum in the tumour and / or tumour microenvironment.
5. An outer membrane vesicle for use as a medicament according to any one of the previous claims, wherein the medicament is in the form of an immunogenic composition, preferably a vaccine, preferably wherein the vaccine is a cancer vaccine.
6. An outer membrane vesicle for use as a medicament according to any one of the previous claims, wherein Fusobacterium nucleatum is selected from Fusobacterium nucleatum spp. animalis, Fusobacterium nucleatum spp. fusiforme, Fusobacterium nucleatum spp. nucleatum, Fusobacterium nucleatum spp. polymorphum and Fusobacterium nucleatum spp. vincentii, preferably wherein the Fusobacterium nucleatum is Fusobacterium nucleatum spp. animalis, more preferably Fusobacterium nucleatum spp. animalis clade C2.
7. An outer membrane vesicle for use as a medicament according to any one of the previous claims wherein the Fusobacterium nucleatum does not express FomA protein.
8. An outer membrane vesicle for use as a medicament according to any one of the previous claims- wherein the outer membrane vesicle is a purified outer membrane vesicle,- wherein the outer membrane vesicle is a non-modified outer membrane vesicle obtained from Fusobacterium nucleatum,- wherein the outer membrane vesicle is obtained with a method that does not involve the use of detergents and / or sonification, and / or - wherein the outer membrane vesicle is substantially devoid of FomA protein.
9. An outer membrane vesicle for use as a medicament according to any one of the previous claims wherein administration of the outer membrane vesicle is intradermal, intramuscular, intranasal, oral, sublingual, or buccal.
10. A method of treating a mammal, wherein the method comprises administering outer membrane vesicles from Fusobacterium nucleatum to the mammal.
11. A method of raising an immune response in a mammal, wherein in the method comprises administering outer membrane vesicles from Fusobacterium nucleatum to the mammal in need thereof, preferably wherein the immune response is against Fusobacterium nucleatum, preferably against Fusobacterium nucleatum in the tumour and / or tumour microenvironment.
12. A method of preventing or treating cancer in a mammal, wherein the method comprises administering outer membrane vesicles from Fusobacterium nucleatum to the mammal.
13. A method of preventing or treating Fusobacterium nucleatum in a mammal or in a tumour comprised in the mammal, wherein the method comprises administering an outer membrane vesicle from Fusobacterium nucleatum to the mammal, preferably wherein the mammal comprises a tumour characterized by the presence of Fusobacterium nucleatum in the tumour and / or tumour microenvironment.
14. A method of preventing tumorigenesis, tumour growth, metastasis, and / or resistance to cancer therapy in a mammal, wherein the method comprises administering an outer membrane vesicle from Fusobacterium nucleatum to the mammal.
15. The method according to any one of the previous claims 10 - 14, wherein the outer membrane vesicles are a defined in any one of the previous claims 1 - 9 are in the form of an immunogenic composition, preferably a vaccine, preferably wherein the vaccine is a cancer vaccine.
16. A Fusobacterium nucleatum and / or outer membrane vesicles obtained from such Fusobacterium nucleatum, wherein the Fusobacterium nucleatum is a Fusobacterium nucleatum that is substantially devoid of expression of functional FomA protein, preferably wherein the Fusobacterium nucleatum does not express FomA protein.
17. An outer membrane vesicle, an immunogenic composition, preferably a vaccine as defined in any one of the previous claims 1 - 9 or 16.
18. An outer membrane vesicle for use according to any of previous claims 1-9, wherein the mammal is suspected of having, or is diagnosed with, Lynch syndrome.