Oncolytic virus combination therapy with microbiome-targeting compositions

Abstract

The present invention is directed to an oncolytic vector together with one or more antibiotics, one or more a probiotics or other microbiome-targeting compositions, and / or one or more fecal transplant for use in the treatment of cancer. The invention is also directed to a pharmaceutical composition comprising an oncolytic vector with one or more of the following: an antibiotic, a probiotic, a microbiome-targeting composition and a fecal transplant. The invention is also directed to a kit which comprises a first container, a second container, and a package insert, wherein the first container comprises at least one dose of a pharmaceutical composition containing an oncolytic vector, the second container containing one of the following: an antibiotic, a probiotic, a microbiome-targeting composition or a fecal transplant, and the package insert comprises instructions for treating a subject having cancer using the compositions of the first and second containers, wherein said probiotic, microbiome-targeting composition or fecal transplant comprises or promotes a bacterial species which improves the effect of the treatment with an oncolytic vector in a subject when said bacterial species is present in the gut microbiome of said subject, and wherein said antibiotic is effective against a bacterial species which decreases the effect of the treatment with an oncolytic vector in a subject when said bacterial species is present in the gut microbiome of said subject.

Description

[0001] ONCOLYTIC VIRUS COMBINATION THERAPY WITH MICROBIOME-TARGETING COMPOSITIONS

[0002] FIELD

[0003] The present invention relates generally to virology, immunology and medicine. In certain aspects, the invention relates to therapy with oncolytic viruses, particularly oncolytic adenoviruses, in combination with antibiotics, probiotics, other microbiometargeting compositions and / or fecal transplantation for the treatment of cancer.

[0004] BACKGROUND

[0005] With cancer still being one of the leading causes of death worldwide, there is a high need for new cancer therapies. In spite of the emergence of paradigm shifting cancer therapy approaches, such as immunotherapies, only a fraction of patients derive benefit from it.

[0006] Oncolytic viruses are a promising cancer immunotherapy in which viruses lyse tumor cells (without harming healthy cells) and activate the immune system towards the tumor, via inherent and / or engineered properties. After years of development, oncolytic viruses are currently starting to be used as cancer therapeutics. Although there have been some discoveries relating to the mechanisms of action and factors that influence the efficacy of the viruses, there is still a need to identify pathways that determine the overall response to virotherapy. In 30 clinical trials, oncolytic viruses have demonstrated a favorable safety profile and promising efficacy. However, there is still room for improvement in the responses, especially in patients with a significant metastasis burden. Further characterization of pathways related to the activity of oncolytic viruses could reveal potential targets for improving the efficacy of virotherapy.

[0007] The gut microbiome has been proposed to affect a wide spectrum of diseases and treatments. Particularly, recent studies have shown that the microbiome plays an important role in several cancer immunotherapies, including aPD-L1 and aCTLA-4 immune checkpoint inhibitor therapies [1 , 2],

[0008] In the present invention, we utilize insights of the interaction of the microbiome and oncolytic virus therapy for improving therapeutic efficacy.

[0009] WO201 4170389 relates to oncolytic adenoviral vectors alone or together with therapeutic compositions for therapeutic uses and therapeutic methods for cancer.

[0010] WO201 6146894 discloses oncolytic adenoviral vectors coding for bi-specific antibodies. WO2023057697 discloses oncolytic virus vectors coding for interleukin-7 (IL-7) polypeptide.

[0011] SUMMARY

[0012] The invention is defined by the features of the independent claims. Some specific embodiments are defined in the dependent claims.

[0013] This disclosure provides a mean to improve cancer therapy response by enhancing immune activation with the administration of immunogenic agents, specifically an oncolytic virus, together with antibiotics, probiotics, other microbiome-targeting compositions and / or fecal transplantation. Oncolytic viruses are able to selectively replicate in and lyse cancer cells, and oncolytic viruses can be armed with transgenes to enhance therapeutic efficacy.

[0014] The present invention is based on the discovery that the efficacy of an oncolytic virus therapy associates with the baseline microbiome of the treated patient. In particular, the present data shows that there are significant differences between the baseline microbiome of oncolytic adenovirus-treated patients who experienced progressive disease (PD) and stable disease (SD).

[0015] The present inventors thus propose that manipulation of the patient microbiome with antibiotics, probiotics, other microbiome-targeting compositions and / or fecal transplantation before and / or after the onset of an oncolytic virus therapy will improve efficacy of the treatment. Antibiotics and bacteriophages can be used for eradicating bacterial species which decrease the effect of the treatment with an oncolytic vector in a subject while probiotics, other microbiome-targeting compositions and fecal transplants can be used to increase the number of bacterial species which improve the effect of the treatment.

[0016] Accordingly, in several embodiments, the present application provides a combination therapy for use in the treatment and / or prevention of cancer and / or the establishment of metastases in a mammal and / or for use in initiating, enhancing or prolonging an anti-tumor response in a mammal comprising administering to the mammal an effective amount of (a) an oncolytic vector with (b) one or more antibiotics (c) one or more probiotics or other microbiome-targeting compositions, and / or (d) one or more fecal transplants.

[0017] In an embodiment, the gut, urine and / or tumor microbiome of the patient is / are mapped before, on or after the onset of the treatment with the oncolytic vector of the present disclosure. Furthermore, the present invention relates to the use of oncolytic adenoviral vectors in said combination treatment, particularly those with an adenovirus serotype 5 (Ad5) backbone or an adenovirus serotype 3 (Ad3) backbone encoding a cytokine as a transgene, such as TNFalpha, IL-2, CD40L, and / or IL-7, or encoding a bispecific monoclonal antibody as a transgene. Such adenoviral vector preferably comprises

[0018] - a 5 / 3 chimeric fiber knob,

[0019] - E2F1 promoter for tumor specific expression of E1A,

[0020] - a 24 bp deletion (D24) in the Rb binding constant region 2 of adenoviral E1 ,

[0021] - a nucleic acid sequence deletion of viral gp19k and 6.7k reading frames, and

[0022] - a nucleic acid sequence encoding at least one cytokine or a bispecific monoclonal antibody as transgene in the place of the deleted gp19k / 6.7K '\n the E3 region resulting in replication-associated control of transgene expression under the viral E3 promoter.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figures illustrate the present invention in accordance with at least some embodiments of the present invention.

[0025] Figure 1. Microbiome depletion results in reduced tumor growth control by and oncolytic adenovirus encoding TNFa and IL-2 (TILT-123) in vivo, a) Treatment schedule of the in vivo experiment where the hamster microbiome was depleted using an antibiotic cocktail and tumors were treated every 3 days with either PBS or with TILT-123 therapy, b) Tumor growth curves shown in mm3. Statistical significance determined using paired t-test and presented as **p-value < 0.01 .

[0026] Figure 2. Differences between the microbiome of oncolytic adenovirus encoding TNFa and IL-2 (TILT-123)-treated patients, who have experienced progressive disease (PD) and Stable disease (SD) at taxonomic and functional level, a) Significantly higher abundance of the genus Alistipes in the baseline microbiome of TILT-123-treated patients who experienced SD compared to those who experienced PD. b) Significantly higher abundance of the species Eggerthella lenta in the baseline microbiome of TILT- 123-treated patients who experienced PD compared to those who experienced SD. Statistical significance determined using Wilcoxon t-test and presented as *p-value < 0.05. c) Higher abundance of the species Eggerthella lenta at the baseline microbiome results in different functional profiles between TILT-123-treated patients who experienced PD and SD. Figure 3. Differences in the gut microbiome of oncolytic adenovirus encoding TNFa and IL-2 (TILT-123)-treated patients at taxonomic and functional level. A) Significantly higher abundance of the genus Alistipes in the baseline microbiome of the Disease Control (DC) patients. B) Increase butyrate kinase pathway activity in DC patients. C) Significantly higher abundance of the Eggerthella lenta species in the baseline microbiome of No Disease Control (No_DC) patients. D) Increases arginine deiminase pathway activity in No_DC patients. Statistical significance determined using Wilcoxon test and presented as *p-value < 0.05. E) Animal experiment showing increased tumor growth control for TILT-123 therapy, administrated with Alistipes shahii probiotics (dark group). Statistical significance determined using the Kenward-Roger Linear Mixed model test and presented as *p-value < 0.05, Upvalue < 0.01 , ***p-value < 0.001 , ****p-value < 0.0001.

[0027] Figure 4. Differences in the baseline urobiome of TILT-123 treated patients. A) Relative abundance differences at genus level. B) Relative abundance differences in Prevotella species between DC and No_DC patients. Statistical significance determined using Wilcoxon test and presented as *p-value < 0.05.

[0028] Figure 5. Differences in the baseline oncobiome of TILT-123 treated patients, in injected and uninjected lesions. A) Highest abundant bacteria at genus levels in No_DC patients. B) Highest abundant bacteria at genus levels in DC patients. C) Alistipes and Prevotella in partial responder patient 20103.

[0029] EMBODIMENTS

[0030] In several embodiments, a combination therapy for use in the treatment of cancer and / or the establishment of metastases in a mammal is provided comprising administering to the mammal (i) an oncolytic vector with (ii) one or more antibiotics with (iii) one or more probiotics or other microbiome-targeting compositions and / or (iv) one or more fecal transplants. In preferred embodiments, said oncolytic vector is administered simultaneously or sequentially with the one or more antibiotics, one or more probiotics or one or more fecal microbiome transplants. In an embodiment, said oncolytic vector is administered sequentially with the antibiotic(s), the probiotic(s) or other microbiome-targeting compositions and / or fecal transplant(s) within 24 or 48 hours, or within 3-21 days in any order.

[0031] Oncolytic Virus

[0032] Examples of the oncolytic viral vector of the present disclosure include an adenovirus, herpes virus, lentivirus, vaccinia virus, Reo virus, maraba virus, Newcastle disease virus, sendai virus, pox virus, poliovirus, myxoma virus, and retrovirus [5, 6]. The term “oncolytic vector” may refer herein either to the viral particle capable of transferring a nucleic acid into a cell and lysing the cell or to the transferred nucleic acid itself. In some embodiments, the oncolytic virus of the combination is an oncolytic adenovirus.

[0033] As used herein “an oncolytic adenoviral vector” refers to an adenoviral vector capable of infecting and killing cancer cells by selective replication in tumor versus normal cells. Examples of cytokine expressing oncolytic adenoviral vectors are disclosed in WO2014170389, WO2016146894, and WO2023057697 such as oncolytic adenoviral vectors encoding cytokines TNFalpha, IL-2, CD40L and / or IL-7, or bi-specific monoclonal antibody as transgene(s) that can be used in this invention. Nucleic acid sequences of said oncolytic adenoviral vectors are disclosed, e.g., in WO2014170389.

[0034] The vectors may be modified in any way known in the art, e.g., by deleting, inserting, mutating or modifying any viral areas. The vectors are made tumor specific with regard to replication. For example, the adenoviral vector may comprise modifications in E1 , E3 and / or E4 such as insertion of tumor specific promoters (e.g., to drive E1 ), deletions of areas (e.g., the constant region 2 of E1 as used in “D24”, E3 / gp19k, E3 / 6.7k) and insertion of transgenes. Furthermore, fiber knob areas of the vector can be modified. In one embodiment of the invention the adenoviral vector is Ad5 / 3 comprising an Ad5 nucleic acid backbone and Ad3 fiber knob or Ad5 / 3 chimeric fiber knob.

[0035] As used herein, expression “adenovirus serotype 5 (Ad5) nucleic acid backbone” refers to the genome of Ad5.

[0036] “Ad5 / 3 vector” refers to a chimeric vector having parts of both Ad5 and Ad3 vectors. In a specific embodiment of the invention, the capsid modification of the vector is Ad5 / 3 chimerism. As used herein, “Ad5 / 3 chimeric fiber knob” refers to a chimerism, wherein the knob part of the fiber is from Ad serotype 3, and the rest of the fiber is from Ad serotype 5. Specifically, in one embodiment, the construct has the fiber knob from Ad3 while the remainder of the genome is from Ad5.

[0037] One approach for generation of a tumor specific oncolytic adenovirus is engineering a 24 base pair deletion (D24) affecting the constant region 2 (CR2) of E1 . In wild type adenovirus CR2 is responsible for binding the cellular Rb tumor suppressor / cell cycle regulator protein for induction of the synthesis (S) phase i.e. DNA synthesis or replication phase. The interaction between Rb and E1A requires eight amino acids (121 to 127) of the E1A protein conserved region, which are deleted in the present invention. The vector of the present invention comprises a deletion of nucleotides corresponding to amino acids 122-129 of the vector according to Heise C. et al. (2000, Nature Med 6, 1134-1139). Viruses with the D24 are known to have a reduced ability to overcome the G1-S checkpoint and replicate efficiently only in cells where this interaction is not necessary, e.g. in tumor cells defective in the Rb- p16 pathway, which includes most if not all human tumors.

[0038] It is also possible to replace E1 A endogenous viral promoter for example by a tumor specific promoter. In a specific embodiment of the invention hTERT promoter is utilized in the place of E1A endogenous viral promoter.

[0039] In a specific embodiment, the E1 B 19K gene, generally known to support replication of adenoviral vectors, has a disabling deletion dE1 B 19K in the present vectors. Deletion of E1 B 19K is known to sensitize cancer cells to TNFalpha and thus it promotes apoptosis.

[0040] The E3 region is nonessential for viral replication in vitro, but the E3 proteins have an important role in the regulation of host immune response, i.e. in the inhibition of both innate and specific immune responses. The gp19k / 6.7K deletion in E3 refers to a deletion of 965 base pairs from the adenoviral E3A region. In a resulting adenoviral construct, both gp19k and 6.7K genes are deleted (Kanerva A et al. 2005, Gene Therapy 12, 87-94). The gp19k gene product is known to bind and sequester major histocompatibility complex I (MHC1 , known as HLA1 in humans) molecules in the endoplasmic reticulum, and to prevent the recognition of infected cells by cytotoxic T-lymphocytes. Since many tumors are deficient in HLA1 / MHC1 , deletion of gp 19k increases tumor selectivity of viruses (virus is cleared faster than wild type virus from normal cells but there is no difference in tumor cells). 6.7K proteins are expressed on cellular surfaces and they take part in downregulating TNF-related apoptosis inducing ligand (TRAIL) receptor 2.

[0041] Both of these deletions provide an advantage. To regain expression of HLA / MHC for presentation of tumor epitopes, e.g., to the adoptively transferred T cells, expression of the gp19k protein is counterproductive and in fact, the upregulation of HLA / MHC requires deletion of gp19k. With regard to 6.7k, since an embodiment of our invention is production of TNFalpha from the virus, and one of its anti-tumor activities is a direct anti-tumor proapoptotic effect (on both transduced and nontransduced bystander cells), the presence of 6.7k is counterproductive.

[0042] In one embodiment of the invention, the cytokine transgene or transgenes are placed into a gp19k / 6.7k deleted E3 region, under the E3 promoter. This restricts transgene expression to tumor cells that allow replication of the virus and subsequent activation of the E3 promoter. E3 promoter may be any exogenous (e.g., CMV or E2F promoter) or endogenous promoter known in the art, specifically the endogenous E3 promoter. Although the E3 promoter is chiefly activated by replication, some expression occurs when E1 is expressed. As the selectivity of D24 type viruses occurs post E1 expression (when E1 is unable to bind Rb), these viruses do express E1 also in transduced normal cells. Thus, it is of critical importance to regulate also E1 expression to restrict E3 promoter mediated transgene expression to tumor cells.

[0043] In another embodiment of the invention E3 gp19k / 6.7k is kept in the vector but one or many other E3 areas have been deleted (e.g., E3 9-kDa, E3 10.2 kDa, E3 15.2 kDa and / or E3 15.3 kDa).

[0044] In a specific embodiment of the invention, the oncolytic adenoviral vector is based on an adenovirus serotype 5 (Ad5) nucleic acid backbone comprising a 5 / 3 chimeric fiber knob, and comprising the following: E2F1 promoter for tumor specific expression of E1 A, a 24 bp deletion (D24) in the Rb binding constant region 2 of adenoviral E1 , a nucleic acid sequence deletion of viral gp19k and 6.7k reading frames, with a transgene insertion into the deleted region, resulting in replication- associated control of transgene expression under the viral E3 promoter, and a nucleic acid sequence encoding at least one cytokine transgene in the place of the deleted adenoviral genes gp19k / 6.7K '\r\ the E3 region. In one embodiment of the invention, the adenoviral vector is based on a human adenovirus.

[0045] The exact functions of the Early Region (E3) proteins in adenovirus 3 are not known. Generally, in adenoviruses they do not seem to impair replication when deleted and they seem to affect anti-viral host response to adenoviruses. The E3 of the human adenovirus genome contains the highest level of genetic diversity among the six species (A-F) of adenoviruses found in humans. This diversity in genetic content is primarily located between the highly conserved E3-gp19K and E3-RIDa open reading frames (ORFs) where species-specific arrays of genes are encoded.

[0046] Cytotoxic T-cell mediated killing of viral-infected cells is modulated by E3-gp19K. This is accomplished by blocking transport of MHC class I to the plasma membrane, and inhibiting the TAP-MHC class I complex formation.

[0047] Thus, in one aspect of the invention the important molecule E3-gp19K is comprised in the adenoviral vector to make virus replication stealthier and enable more time for oncolysis and its beneficial effects. Also, retaining E3-gp19K can reduce induction of anti-adenovirus-cytotoxic T cells, resulting in more anti-tumor T cells.

[0048] Cytokines participate in immune response by acting through various mechanisms including recruitment of T cells towards the tumor. The nucleotide sequence encoding a cytokine transgene may be from any animal such as a human, ape, rat, mouse, hamster, dog or cat, but specifically it is encoded by a human sequence. The nucleotide sequence encoding the transgene may be modified in order to improve its effects, or unmodified i.e., of a wild type.

[0049] Particular embodiments of the present invention include viral vectors coding for at least one cytokine, preferably selected from a group consisting of interferon alpha, interferon beta, interferon gamma, complement C5a, TNFalpha, IL-2, CD40L, IL-7, IL- 12, IL-23, IL-15, IL-17, CCL1 , CCL11 , CCL12, CCL13, CCL14-1 , CCL14-2, CCL14- 3, CCL15-1 , CCL15-2, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21 , CCL22, CCL23-1 , CCL23-2, CCL24, CCL25-1 , CCL25-2, CCL26, CCL27, CCL28, CCL3, CCL3L1 , CCL4, CCL4L1 , CCL5, CCL6, CCL7, CCL8, CCL9, CCR10, CCR2, CCR5, CCR6, CCR7, CCR8, CCRL1 , CCRL2, CX3CL1 , CX3CR, CXCL1 , CXCL10, CXCL11 , CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCR1 , CXCR2, CXCR4, CXCR5, CXCR6, CXCR7 and XCL2. In a specific embodiment of the invention the cytokine is IL-2 or TNFalpha, preferably the viral vectors are coding for both cytokines. In one embodiment of the invention the viral vectors are coding for IL-2 and / or TNFalpha and a further cytokine.

[0050] Cytokine TNFalpha (tumor necrosis factor alpha) functions by attracting and activating the T cells and reducing tumor immunosuppression, while IL-2 (interleukin-2) induces the propagation of T-cells.

[0051] In a specific embodiment of the invention, said oncolytic adenoviral vector is i) an adenovirus serotype 5 (Ad5) vector comprising

[0052] - a 5 / 3 chimeric fiber knob,

[0053] - E2F1 promoter for tumor specific expression of E1A,

[0054] - a 24 bp deletion (D24) in the Rb binding constant region 2 of adenoviral E1 ,

[0055] - a nucleic acid sequence deletion of viral gp19k and 6.7k reading frames, and

[0056] - a nucleic acid sequence encoding at least one cytokine transgene, such as TNFalpha, IL-2, and / or IL-7 in the place of the deleted gp19k / 6.7K '\n the E3 region resulting in replication-associated control of transgene expression under the viral E3 promoter; or ii) an adenovirus serotype 3 (Ad3) vector comprising - a promoter for tumor specific expression of E1 A,

[0057] - a deletion in the E3 area affecting E3 9 kDa, E3 10.2 kDa, E3 15.2 kDa and E3 15.3 kDa, and

[0058] - an exogenous or tumor specific promoter for expression of at least one cytokine, such as CD40L, in the place of the deleted area of E3.

[0059] In another specific embodiment of the invention, the oncolytic adenoviral vector comprises:

[0060] 1 ) E2F1 promoter for tumor specific expression of E1 A

[0061] 2) a 24 bp deletion (D24) in the Rb binding constant region 2 of adenoviral E1 ;

[0062] 3) a nucleic acid sequence deletion of viral gp19k and 6.7k reading frames; and

[0063] 4) a nucleic acid sequence encoding a bispecific monoclonal antibody comprising a single chain variable fragment (scFv) specific for a cell surface molecule and a scFv specific for a tumor antigen in the place of the deleted nucleic acid sequence as defined in point 3), wherein, preferably, the tumor antigen is EpCAMI or MUC 1 and the cell surface molecule is CD3.

[0064] A bispecific monoclonal antibody (BsMAb, BsAb) is an artificial protein that is composed of fragments of two different monoclonal antibodies and consequently is able to bind two different types of antigens. In other words, bispecific antibodies combine two or more antigen-recognizing elements into a single construct, which is able to bind to two or more targets. In one embodiment of the invention, the virus vector comprises an internal ribosomal entry site (IRES) or optionally a ribosome shunt site 2A between the two transgenes. Thus, IRES or a ribosome shunt site 2A may be between any cytokines, such as IL-2 and any other cytokine, preferably selected from the above listed cytokine group. As used herein “IRES” refers to a nucleotide sequence that enables initiation of the translation in the middle of a messenger RNA sequence in protein synthesis. IRES can be from any virus, but in one embodiment of the invention IRES is from encephalomyocarditis virus (EMCV). As used herein “a ribosome shunt site 2A” refers to a translation initiation site in which ribosomes physically bypass parts of the 5' untranslated region to reach the initiation codon. Both the IRES and the A2 enable viruses to produce two transgenes from one promoter (the E3 promoter).

[0065] Examples of detailed structures of the oncolytic adenoviral vector encoding TNFalpha and / or IL-2 as a transgene are disclosed in WO2014170389.

[0066] In summary, the key advantages of utilizing viral vectors comprising at least one cytokine transgene are: i) cytokines and virus perse cause a danger signal which recruits T cells and other immune cells to tumors, ii) cytokines induce T-cell proliferation both at the tumor and in local lymphoid organs, iii) cytokines and virus per se are able to induce T cells (natural, innate anti-tumor T cells) to propagate at the tumor, iv) cytokine and / or virus induce the upregulation of antigen-presenting molecules (HLA) on cancer cells, rendering them sensitive to recognition and killing by T cells, and v) cytokines and virus replication favorably alter tumor microenvironment by reducing immunosuppression and cellular anergy.

[0067] The viral vectors utilized in the present inventions may also comprise other modifications than described above. Any additional components or modifications may optionally be used but are not obligatory for the present invention.

[0068] Insertion of exogenous elements may enhance effects of vectors in target cells. The use of exogenous tissue or tumor-specific promoters is common in recombinant vectors and they can also be utilized in the present invention.

[0069] Antibiotics

[0070] “Antibiotics” are types of antimicrobial substances active against certain type of bacteria. In the present invention, a suitable antibiotic is preferably an antimicrobial substance to which Alistipes and / or Prevotella strains are resistant. As illustrated in the experimental part below, antibiotics can be useful to modulate the gut microbiome of a patient, through the use of antibiotics and / or probiotics, for increasing the anticancer effects of an oncolytic vector treatment. In particular, an antibiotic can be used for increasing the relative amount of strains of genus Alistipes and / or Prevotella as well as the relative amount of other advantageous bacteria in the gut microbiome. In another embodiment, a suitable antibiotic kills strains of species Eggerthella lenta.

[0071] Probiotics and other microbiome-targeting compositions

[0072] "Probiotics" are generally described as micro-organisms that have claimed health benefits when consumed. As used herein, the term “probiotic” can be used to describe, for example, probiotic bacteria and can include the bacteria described herein as well as other bacteria. Probiotic bacteria or spores thereof may be administered in the form of a liquid, a suspension, a dried (e.g., lyophilized) powder, a tablet, a capsule, or a suppository, and may be administered orally, nasogastrically, or rectally. Preferred probiotic bacterial strains in the present invention belong to genus Alistipes and / or Prevotella.

[0073] A "prebiotic" is a substance that promotes the growth, proliferation and / or survival of one or more bacteria or yeast. As used herein, the term “prebiotic” can be used to describe, for example, a nutritional supplement including plant fiber, or one or more of poorly-absorbed complex carbohydrates, oligosaccharides, inulin-type fructans or arabinoxylans. Preferred prebiotics for this invention include those substances capable of promoting the growth, proliferation and / or survival of bacterial strains belonging to the genus Alistipes and / or Prevotella.

[0074] A "postbiotic" is a substance derived from a probiotic organism. As used herein, the term “postbiotic” can be used to describe, for example, a protein expressed by one or more bacteria, a metabolic product of one or more bacteria, or media from a culture of one or more strains of bacteria. A “postbiotic” metabolic product can be, e.g., riboflavin (B2 vitamin), pantothenate (B5 vitamin) or thiamine (B1 vitamin). Preferred postbiotics for this invention include those substances that derive from the genus Alistipes and / or Prevotella.

[0075] According to the present disclosure, probiotics, prebiotics and postbiotics can be used to increase the number of bacterial species which improves the effectiveness of the treatment with an oncolytic vector in a subject when said bacterial species are present in the gut microbiome of said subject.

[0076] In the present disclosure, bacteriophages are considered as microbiome-targeting compositions, which target bacterial species decreasing the effect of the present treatment with an oncolytic vector. In a preferred embodiment, said microbiometargeting composition is a bacteriophage targeting Eggerthella lenta strains.

[0077] Fecal transplants

[0078] As used herein, the term “fecal transplant” refers to fecal bacteria isolated from a donor which fecal bacteria is subsequently transplanted into a recipient. In the art, fecal transplant is also referred to as fecal microbiome transplantation (FMT), stool transplant or bacteriotherapy. In a particular embodiment, the fecal transplant is fecal material (fecal filtrate) having reduced volume and / or fecal aroma relative to unprocessed fecal material. In a more particular embodiment, the fecal transplant is a fecal bacterial sample. In a preferred embodiment, the “fecal transplant” of the present disclosure is the process of transplantation of fecal bacteria isolated from a healthy individual or an individual having SD (stable disease) after oncolytic virus therapy into a recipient, which can be an individual who has been treated with an oncolytic virus and is suffering from a PD (progressive disease) or an individual who will be subsequently treated with an oncolytic virus for the first time. In another preferred embodiment, the fecal transplant or the donor for the fecal transplant is selected based on the presence of suitable bacteria in the gut microbiome of the donor, wherein said suitable bacteria improves the effect of the treatment with an oncolytic vector in a subject when present in the gut microbiome, said suitable bacteria preferably being bacterial species belonging to Alistipes and / or Prevotella genus. In another preferred embodiment, the fecal transplant is selected based on the absence of bacteria, such as Eggerthella lenta, decreasing the effect of the treatment with an oncolytic vector in a subject when present in the gut microbiome.

[0079] Cancer

[0080] The recombinant oncolytic vectors of the present invention are replication competent in tumor cells. In one embodiment of the invention, the vectors are replication competent in cells, which have defects in the Rb-pathway, specifically Rb-p16 pathway. These defective cells include all tumor cells in animals and humans. As used herein “defects in the Rb-pathway” refers to mutations and / or epigenetic changes in any genes or proteins of the pathway. Due to these defects, tumor cells overexpress E2F and thus, binding of Rb by E1 A CR2, that is normally needed for effective replication, is unnecessary. Further selectivity is mediated by the E2F promoter, which only activates in the presence of free E2F, as seen in Rb / p16 pathway defective cells. In the absence of free E2F, no transcription of E1A occurs and the virus does not replicate. Inclusion of the E2F promoter is important to prevent expression of E1A in normal tissues, which can cause toxicity both directly and indirectly through allowing transgene expression from the E3 promoter.

[0081] In another embodiment, the presence of a human telomerase (hTERT) promoter in the viral construct allows for tumor selective viral replication in cancer cells. Telomerase is responsible for maintaining the lengths of telomeres and is not active in adult cells but is highly active in cancer cells. This telomerase activity in cancer cells allows cells to replicate and proliferate in an uncontrolled manner. The hTERT promoter therefore confers neoplasm selective replication by only becoming active and driving expression of viral genes needed for replication in cancer cells with activated telomerase.

[0082] The present invention relates to approaches for treating cancer in a subject. In one embodiment of the invention, the subject is a human or a mammal, specifically a mammal or human patient, more specifically a human or a mammal suffering from cancer.

[0083] The approach can be used to treat any cancers or tumors, including both malignant and benign tumors, both primary tumors and metastases may be targets of the approach. In one embodiment of the invention the cancer features tumor-infiltrating lymphocytes. The tools of the present invention are particularly appealing for treatment of metastatic solid tumors featuring tumor-infiltrating lymphocytes.

[0084] As used herein, the term “treatment” or “treating” refers to administration of at least oncolytic adenoviral vectors and antibiotics, probiotics, other microbiome-targeting compositions, and / or fecal transplant, preferably a mammal or human subject, for purposes which include not only complete cure but also prophylaxis, amelioration, or alleviation of disorders or symptoms related to a cancer or tumor. Therapeutic effect may be assessed by monitoring the symptoms of a patient, tumor markers in blood, or for example a size of a tumor, or the length of survival of the patient, or the length of time in which the patient is free of tumor progression.

[0085] In another embodiment of the invention, the cancer or tumor is selected from a group consisting of nasopharyngeal cancer, synovial cancer, hepatocellular cancer, renal cancer, cancer of connective tissues, melanoma, lung cancer, bowel cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, throat cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, von Hippel- Lindau disease, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, ureter cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, carcinoid of gastrointestinal tract, fibrosarcoma, breast cancer, Paget’s disease, cervical cancer, esophagus cancer, gall bladder cancer, head and neck cancer, eye cancer, kidney cancer, Wilms’ tumor, Kaposi’s sarcoma, prostate cancer, testicular cancer, Hodgkin's disease, non-Hodgkin’s lymphoma, skin cancer, mesothelioma, multiple myeloma, ovarian cancer, endocrine pancreatic cancer, glucagonoma, parathyroid cancer, penis cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus cancer, thyroid cancer, trophoblastic cancer, hydatidiform mole, uterine cancer, endometrial cancer, vagina cancer, vulva cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gum cancer, heart cancer, lip cancer, meninges cancer, mouth cancer, nerve cancer, palate cancer, parotid gland cancer, peritoneum cancer, pharynx cancer, pleural cancer, salivary gland cancer, tongue cancer and tonsil cancer.

[0086] Before classifying a human or animal patient as suitable for the therapy of the present invention, the clinician may examine a patient. Based on the results deviating from the normal and revealing a tumor or cancer, the clinician may suggest treatment of the present invention for a patient. Administration

[0087] As used herein, the term "combination" refers to the use of more than one agent, e.g., i) an oncolytic vector and an antibiotic or ii) an oncolytic vector and a probiotic and / or other microbiome-targeting composition such as pre- or postbiotic, or ill) an oncolytic vector and a fecal transplant. The use of the term "combination" does not restrict the order in which therapies are administered to the patient, although it is preferable to administer the antibiotic, probiotic, other microbiome-targeting composition and / or fecal transplant prior to or simultaneously with the oncolytic vector. For example, the antibiotic, probiotic and / or other microbiome-targeting composition such as prebiotic or postbiotic, or fecal transplant can be administered prior to the oncolytic vector (e. g., 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), either punctually or several times (for example, each day) before the oncolytic vector treatment is administered. Advantageously, the antibiotic, probiotic and / or other microbiome-targeting composition such as prebiotic or postbiotic, or fecal transplant is administered before administration of the oncolytic vector, in order to modulate the patient's gut microbiota to optimize the effect of the oncolytic vector (such as those defined above). The present invention hence provides a method for treating a cancer patient, comprising administering one or more antibiotics, probiotic and / or other microbiome-targeting composition such as prebiotic or postbiotic, or fecal transplant prior to administering an oncolytic vector to said patient.

[0088] In one embodiment of the invention, the separate administration(s) of (a) an oncolytic vector, (b) one or more antibiotics, (c) one or more probiotics or other microbiometargeting composition and / or (d) one or more fecal transplant to a subject is (are) conducted simultaneously or consecutively, in any order. This means that (a), (b), (c) and (d) may be provided in a single unit dosage form for being taken together or as separate entities (e.g. in separate containers) to be administered simultaneously or with a certain time difference. In a preferred embodiment, the first administration of the adenoviral vector is conducted after the first administration of the antibiotic or the first administration of the probiotic or other microbiome-targeting composition. In addition, it is possible to administer the virus via another administration way than the antibiotic, the probiotic and / or other microbiome-targeting composition such as prebiotic or postbiotic or the fecal transplant. In this regard, it may be advantageous to administer either the oncolytic virus intratumorally and the antibiotic systemically, locally or orally. Probiotics and other microbiome-targeting compositions are preferably administered orally. Fecal transplants are preferably administered orally, or locally (e.g. via colonoscopy, enema, nasogastric tube). In one embodiment of the invention, the administration of oncolytic virus is conducted through an intratumoral, intranasal, intra-arterial, intravenous, intrapleural, intravesicular, intracavitary or peritoneal injection, or an oral administration. Any combination of administrations is also possible. The approach can give systemic efficacy despite local injection.

[0089] The effective dose of vectors depends on at least the subject in need of the treatment, tumor type and location of the tumor and stage of the tumor. The dose may vary for example from about 1x108viral particles (VP) to about 1x1014VP, specifically from about 1x109VP to about 1x1013VP and more specifically from about 3x109VP to about 5x1012VP. In one embodiment oncolytic adenoviral vectors coding for at least one cytokine are administered in an amount of 1 x1010- 1 x1014virus particles. In another embodiment of the invention the dose is in the range of about 5x1010- 5x1011VP.

[0090] Preferably, the oncolytic virus, the antibiotic, the probiotic and / or other microbiometargeting composition such as prebiotic or postbiotic and the fecal transplant are administered as separate compounds. Concomitant treatment with the agents is also possible.

[0091] As used herein “separate administration” or “separate” refers to a situation, wherein (a) an oncolytic vector, (b) one or more antibiotics, (c) one or more probiotics or other microbiome-targeting compositions, and (d) one or more fecal transplants are four different products or compositions distinct from each other.

[0092] Any conventional method may be used for administration of the vector, antibiotic, probiotic and / or other microbiome-targeting composition such as prebiotic or postbiotic to a subject. The route of administration depends on the formulation or form of the composition, the disease, location of tumors, the patient, comorbidities and other factors. Accordingly, the dose amount and dosing frequency of each therapeutic agent in the combination depends in part on the particular therapeutic agent, the severity of the cancer being treated, and patient characteristics. Preferably, a dosage regimen maximizes the amount of each therapeutic agent delivered to the patient consistent with an acceptable level of side effects.

[0093] Any other treatment or combination of treatments may be used in addition to the therapies of the present invention. In a specific embodiment the method or use of the invention further comprises administration of concurrent or sequential radiotherapy, or other anti-cancer drugs or interventions (including surgery) to a subject. The terms “treat” or “increase”, as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete treatment or increase. Rather, there are varying degrees of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect.

[0094] Gut microbiome

[0095] The "gut microbiome" or “gut microbiota” designates the population of microorganisms living in the intestine of any organism belonging to the animal kingdom (human, animal, insect, etc.). While each individual has a unique microbiota composition (60 to 80 bacterial species are shared by more than 50% of a sampled population on a total of 400-500 different bacterial species / individual), it always fulfils similar main physiological functions and has a direct impact on the individual's health. Further, it is known that microbiome plays a role in cancer. Research has indicated that the presence of certain microorganisms can trigger cancer growth. For example, individuals with Helicobacter pylori are eight times more likely to develop stomach cancer. Other species, like Fusobacterium nucleatum are known to activate cancer-promoting genes and also some viruses, like human papillomavirus are known to induce cervical cancer [4], The microbiome is also important for the development of a proper immune system. It has been shown that a higher microbial diversity corresponds to a more “healthy” microbiome. In regards to cancer, immunotherapies showed higher success rates in patients with such a “healthy” microbiome and also immunotherapies seem to be more successful in the presence of certain bacteria like Bifidobacterium and Bacteroides [2],

[0096] Other embodiments

[0097] In an embodiment, a step of detecting the presence of Eggerthella lenta in the gut microbiome of the subject is performed before the onset of the treatment and / or during the treatment, wherein preferably the subject having said Eggerthella lenta in the gut is treated with an antibiotic and / or a bacteriophage targeting said Eggerthella lenta before the onset of the treatment with the oncolytic vector or during the treatment with the oncolytic vector of the present disclosure.

[0098] In an embodiment, a step of mapping the gut microbiome of the subject at least partly is performed before the onset of the treatment and / or during the treatment with the oncolytic vector of the present disclosure.

[0099] In an embodiment, a step of mapping the urine microbiome of the subject at least partly is performed before the onset of the treatment and / or during the treatment with the oncolytic vector of the present disclosure.

[0100] In an embodiment, a step of mapping the tumor microbiome of the subject at least partly is performed before the onset of the treatment and / or during the treatment with the oncolytic vector of the present disclosure.

[0101] In an embodiment, the subject is selected or not selected for the treatment with the oncolytic vector of the present disclosure based on the results of the above- mentioned mapping step(s).

[0102] In an embodiment, based on the results of the above-mentioned mapping(s), the subject is selected for the treatment with a probiotic comprising bacterial species belonging to Alistipes and / or Prevotella genus before the onset of the treatment with the oncolytic vector of the present disclosure and / or during the treatment with said oncolytic vector, preferably said bacterial species is Alistipes shahii.

[0103] In an embodiment, based on the results of above-mentioned the mapping(s), the subject is selected for the treatment with a prebiotic promoting the growth of bacterial species belonging to Alistipes and / or Prevotella genus before the onset of the treatment with the oncolytic vector of the present disclosure and / or during the treatment with said oncolytic vector.

[0104] In an embodiment, a probiotic, prebiotic or postbiotic agent increasing the amount of butyrate in the gut of the subject is administered to a subject before the onset of the treatment with the oncolytic vector of the present disclosure and / or during the treatment with said oncolytic vector.

[0105] In an embodiment, a probiotic, prebiotic or postbiotic agent increasing butyrate kinase activity in the gut of the subject is administered to a subject before the onset of the treatment with the oncolytic vector of the present disclosure and / or during the treatment said the oncolytic vector.

[0106] In an embodiment, a probiotic, prebiotic or postbiotic agent decreasing the amount of arginine in the gut of the subject is administered to a subject before the onset of the treatment with the oncolytic vector of the present disclosure and / or during the treatment with said oncolytic vector.

[0107] In an embodiment, a probiotic, prebiotic or postbiotic agent decreasing arginine deiminase activity in the gut of the subject is administered to a subject before the onset of the treatment with the oncolytic vector of the present disclosure and / or during the treatment with said oncolytic vector. Reference throughout this specification to one embodiment or an embodiment means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Where reference is made to a numerical value using a term such as, for example, about or substantially, the exact numerical value is also disclosed.

[0108] The verbs “to comprise” and “to include” are used in this document as open limitations that neither exclude nor require the existence of also un-recited features. The features recited in depending claims are mutually freely combinable unless otherwise explicitly stated. Furthermore, it is to be understood that the use of "a" or "an", that is, a singular form, throughout this document does not exclude a plurality.

[0109] It will be obvious to a person skilled in the art that, as the technology advances, the inventive concept can be implemented in various ways. The invention and its embodiments are not limited to the examples described below but may vary within the scope of the claims.

[0110] EXPERIMENTAL SECTION

[0111] EXAMPLE 1

[0112] Materials and methods

[0113] Animal experiments

[0114] A cocktail of 1 .67 g / l metronidazole and 833.3 mg / l vancomycin was given to the hamsters at day -14 via the drinking water, to deplete the hamster microbiome. Bottles were changed 3 times a week. At day -7, the hamsters were subcutaneously engrafted with HapT1 hamster pancreatic tumor cells. When the first tumor reached 5-6 mm in the longest diameter (day 0), hamsters were randomized and treated with 1x109VPs of oncolytic virus TILT-123 (with structure Ad5 / 3-E2F-D24-TNFa- IRES-IL2 as disclosed in WO2014170389) via intratumoral (IT) injections. Virus injections were repeated every 3 days after the first viral injection. Hamsters were sacrificed when tumors reached a diameter of 22 mm.

[0115] During the animal experiments, tumors were measured three times a week. Tumor volumes were calculated with the formula: (length*widthA2) / 2 in which length was the largest of the two.

[0116] Fecal samples were collected at multiple time points; at the start of the antibiotics (day -14), before the start of the first TILT-123 therapy treatment (day 0) and three weeks after the start with TILT-123 therapy (day 21 ). In addition, samples were taken from the intestine immediately after hamsters were euthanized.

[0117] Patient sample collection

[0118] Fecal samples from two distinct phase 1 clinical trials TILT-T115 (TILT-123 monotherapy in solid tumors) and TILT-T563 (TILT-123 and aPD-1 combination therapy in patients with ovarian cancer) were collected. Fecal samples are obtained using a 70 ml sample tube and snap frozen at <-70°C, for further analysis.

[0119] Sample processing

[0120] DNA was extracted from the samples using the Qiagen MagAttract PowerSoil DNA KF kit and a KingFisher robot. Hamster fecal DNA was extracted using repeated bead beating and the KingFisher™ Flex purification system 96 (Thermo Fisher Scientific) using a previously described method [3] and DNA was shipped for sequencing.

[0121] Sequencing libraries were assembled using the Illumina Nextera library preparation kit for both hamster and human fecal DNA samples and they performed the sequencing run using the Illumina NextSeq system, generating 150bp paired-end reads.

[0122] Shedding sample analysis

[0123] From both human and hamster fecal samples, we received the raw metagenomics sequencing data and used nf-core / taxprofiler for pre-processing, including the removal of human / hamster DNA-sequences and bad quality reads. Subsequently, clean reads were aligned to the Greengenes2 and MetaPhlan4 databases for taxonomic annotation. In addition, the reads were aligned to the Humann3 database for functional annotation. Taxonomic and the functional data tables were analyzed in Rstudio.

[0124] Results

[0125] Depleted microbiome decreases efficacy of TILT-123 in vivo

[0126] To assess the effect of the microbiome on TILT-123 therapy, an in vivo hamster experiment was performed, in which the gut microbiome of the hamsters was depleted using an antibiotic cocktail. After the microbiome depletion, the hamsters were engrafted with HapT1 tumor cells and treated with TILT-123 therapy every three days (Figure 1a). Tumor control was measured and showed that TILT-123 therapy provided significantly lower tumor growth control in hamsters with a depleted microbiome (antibiotics + TILT-123) compared to hamsters without a depleted microbiome (TILT-123) (Figurelb).

[0127] Correlation between the gut microbiome at baseline and TILT-123 treatment outcome in cancer patients

[0128] Fecal samples have been collected from phase 1 clinical trials in which TILT-123 therapy was used, to determine the effect of the microbiome on TILT-123 treatment outcome. The fecal samples have been sequenced using shallow shotgun metagenomics and analyzed using bioinformatic pipelines. During the downstream processing, the microbiome at day 1 of TILT-123 treatment is compared between patients who experienced progressive disease (PD) and stable disease (SD). Of note, PD is defined as an increase of > 20% in tumor size at day 78 compared to day 0, measured with CT scan. SD is defined as an increase of < 20% in tumor size at day 78 compared to day 0, measured with CT scan. The data shows that there are significant differences between the baseline microbiome of patients experiencing PD and SD. For example, our data shows that at genus levels the Alistipes and / or Prevotella genus is significantly more abundant in patients experiencing SD as opposed to the other group (Figure 2a). Whereas, at species levels, the species Eggerthella lenta is significantly more abundant in patients experiencing PD compared with its counterpart (Figure 2b). Moreover, functional data analysis shows that differences in gut microbiome species, for example in Eggerthella lenta, also result in differences in functional profiling (Figure 2c). This shows that not only the microbial taxa but also the correlating pathways or metabolites can be of high impact on the disease control provided by oncolytic viral therapy.

[0129] EXAMPLE 2

[0130] Materials and methods

[0131] Animal experiments

[0132] Hamsters received a cocktail of 1 .67 g / l metronidazole and 833.3 mg / l vancomycin for 14 days, while mice received a cocktail of 0.6 g / l ciprofloxacin, 1 g / l metronidazole and 0.5 g / l Vancomycin for 9 days via the drinking water, to deplete the microbiome. Bottles were changed 3 times a week. Hamsters were subcutaneously engrafted with HapT1 hamster pancreatic tumor cells, while mice were subcutaneously engrafted with B16.OVA mouse melanoma cells. When the first tumor reached 3-4 mm (mice) or 5-6 mm (hamster) in the longest diameter, animals were randomized and treated. Hamsters were treated with 1x109VPs of oncolytic virus TILT-123 (with structure Ad5 / 3-E2F-D24-TNFa-IRES-IL2 as disclosed in WO2014170389) via intratumoral (IT) injections. Mice were treated IT treated with 1x107VPs of a cytokine-armed murine adenoviruses Ad5-CMV-mlL2 and Ad5-CMV-mTNFa, supposedly being a TILT-123 mouse equivalent. Virus injections were repeated every 3 days after the first viral injection. Animals were sacrificed when HEP was reached or at day 20 (mice) or 25 (hamster) when the experiment was ended. During the experiments, tumors were measured three times a week. Tumor volumes were calculated with the formula: (length*widthA2) / 2 in which length was the largest of the two.

[0133] Patient sample collection

[0134] Fecal samples from two distinct phase 1 clinical trials TILT-T115 (TILT-123 monotherapy in solid tumors) and TILT-T563 (TILT-123 and aPD-1 combination therapy in patients with ovarian cancer) were collected. Fecal samples were obtained using a 70 ml sample tube and snap frozen at <-70°C, for further analysis.

[0135] Urine samples from three distinct phase 1 clinical trials TILT-T 115 (TILT-123 monotherapy in solid tumors), TILT-T563 (TILT-123 and aPD-1 combination therapy in patients with ovarian cancer) and TILT-T215 (TILT-123 and TILs combination therapy in patients with melanoma cancer) were collected. Urine samples were obtained using a 10 ml sample tube and snap frozen at <-70°C, for further analysis.

[0136] Tumor samples from two distinct phase 1 clinical trials TILT-T115 (TILT-123 monotherapy in solid tumors), and TILT-T215 (TILT-123 and TILs combination therapy in patients with melanoma cancer) were collected. Samples were taken from injected and non-injected lesions. DNA was extracted and send for microbiome analysis.

[0137] Sample processing

[0138] Fecal samples were shipped in the original collection tubes to Microbiome Insights. DNA was extracted from the samples using the Qiagen MagAttract PowerSoil DNA KF kit and a KingFisher robot. Sequencing libraries were assembled using the Illumina Nextera library preparation kit and metagenomic shotgun sequencing was done using the Illumina NextSeq system, generating 150bp paired-end reads.

[0139] Urine samples were shipped in the original collection tubes to Microbiome Insights. DNA was extracted from the samples using MO BIO Powersoil DNA extraction kit and a KingFisher robot. Libraries were sequenced using Illumina Miseq, using primers for the V4 region (short read 16S rRNA sequencing).

[0140] Tumor samples were originally collected from injected and uninjected lesion. DNA was extracted at BioAgilityx using the MagMAX microbiome ultra nucleic acid isolation kit and DNA samples were shipped to Microbiome Insights for sequencing. Sequencing was done using 16S rRNA, Oxford Nanopore technology (long read sequencing).

[0141] Shedding sample analysis

[0142] Raw sequencing data was received from Microbiome Insights and analyzed using the nf-core / taxprofiler pipeline for pre-processing, including the removal of host DNA-sequences and bad quality reads. Subsequently, clean reads were aligned to the Greengenes2 and MetaPhlan4 databases (shotgun metagenomics, fecal samples), Kraken2 and GreenGenes2 (short read sequencing, urine samples) or Kraken2 and Centrifuge (long read sequencing, tumor samples) for taxonomic annotation. In addition, shotgun metagenomic reads (fecal) were aligned to the Humann3 database for functional annotation. Taxonomic and the functional data tables were analyzed in Rstudio.

[0143] Results

[0144] Fecal, urine and tumor samples have been collected from phase 1 clinical trials in which TILT-123 therapy was used, to determine the effect of the microbiome on TILT-123 treatment outcome. The samples have been sequenced and analyzed and we compared the baseline microbiome between patients who experienced Disease control (DC) and No_Disease control (No_DC). Of note, No_DC is defined as an increase of > 20% in tumor size at day 36 compared to day 0, measured with CT scan. DC is defined as an increase of < 20% in tumor size at day 36 compared to day 0, measured with CT scan. The fecal data shows that there are significant differences between the baseline gut microbiome of DC and No_DC patients. For example, our data shows that at genus levels the Alistipes genus is significantly more abundant in DC patients as opposed to the No_DC patients (Figure 3A). Alistipes bacteria are known short chain fatty acid (SCFA) producers, of which butyrate is the mainly produced. This was also observed by the functional data analysis of the baseline fecal samples, showing increased butyrate kinase activity in DC patients (Figure 3B). Contrary, at species levels, the species Eggerthella lenta is significantly more abundant in No_DC patients compared with its counterpart (Figure 3C). Eggerthella lenta bacteria are known to use arginine for growth, which was also shown by upregulated arginine pathways in the No_DC patients, after functional analysis (Figure 3D). Furthermore, to show the possibilities of using pre-, pro or postbiotics on TILT-123 treatment, we performed an animal experiment in which we investigated the addition of the beneficial Alistipes shahii bacteria (probiotics) to mice which were subcutaneously engrafted with B16.OVA melanoma tumors and were treated with TILT-123 therapy. This showed that mice which received Alistipes shahii bacteria, via oral gavage, has significantly better tumor growth control compared to mice that did not receive these bacteria (Figure 3E). Importantly, the combination of Alistipes shahii and TILT-123 showed significant better results compared to TILT-123 monotherapy. Altogether, this shows that the use of pre-, pro-, postbiotics or a combination of these, are of high interest to modify the host microbiome and consequently increase the efficacy of TILT-123 therapy.

[0145] Similarly, urine samples have been sequenced and analyzed and we compared the baseline urine microbiome (urobiome) between patients who experienced Disease control (DC) and No_Disease control (No_DC). This shows significant differences between the baseline microbiome of DC and No_DC patients at genus level (Figure 4A). In addition, our data shows that numerous Prevotella species are significantly more abundant in DC patients, including Prevotella oralis, Prevotella marshii, Prevotella bucallis en Prevotella enoeca (Figure 4B). Interestingly, Prevotella bacteria are also SCFA producers, highlighting the interest of using pre-, pro- and postbiotics for increasing SCFA production to enhance therapeutic response of TILT-123. In addition, these results suggest that the urine microbiome could be used as a biomarker and predict the response of patients to TILT-123 therapy.

[0146] Lastly, injected and uninjected tumor samples have been sequenced and analyzed. Again, differences are observed between the baseline tumor microbiome (oncobiome) of DC and No_DC patients. At genus level, the topmost abundant genus bacteria are shown for DC and No_DC patients and showing the presence of Desulfitobacterium in the topmost bacteria in the No Disease control patients (Figure 5A), but not in the topmost genus in the Disease control patients (Figure 5B). Moreover, the Alistipes bacteria, detected in the gut microbiome of DC patients, and the Prevotella bacteria, detected in the urine microbiome of DC patients, were also observed in the tumor microbiome of DC patients (Figure 3C). This suggests a systemic role of the microbiome where bacteria can travel to metastatic tumors and effect the response to therapy. Based on this, we believe that modifying the microbiome using pre-, pro- and postbiotics or a combination of these, is of high importance for systemic efficacy of TILT-123. References

[0147] [1] ‘Commensal Bifidobacterium promotes antitumor immunity and facilitates anti-PD-L1 efficacy - PMC’. Accessed: May 17, 2024. [Online]. Available: https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC4873287 /

[0148] [2] M. Vetizou et al., ‘Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota’, Science, vol. 350, no. 6264, p. 1079, Nov. 2015, doi:

[0149] 10.1126 / SCIENCE.AAD1329.

[0150] [3] A. Salonen et al., ‘Comparative analysis of fecal DNA extraction methods with phylogenetic microarray: Effective recovery of bacterial and archaeal DNA using mechanical cell lysis’, J Microbiol Methods, vol. 81 , no. 2, pp. 127-134, May 2010, doi: 10.1016 / J.MIMET.2010.02.007.

[0151] [4] Sahu SC, Hayes AW. The Human Microbiome: History and Future [Internet], Vol. 23, J Pharm Pharm Sci (www.cspsCanada.org). 2020. Available from: www.cspsCanada.org

[0152] [5] Hemminki O, Dos Santos JM, Hemminki A. Oncolytic viruses for cancer immunotherapy. J Hematol Oncol. 2020 Jun 29;13(1 ):84. doi: 10.1186 / s13045-020- 00922-1 .

[0153] [6] Kaufman HL, Kohlhapp FJ, Zloza A. Oncolytic viruses: a new class of immunotherapy drugs. Nat Rev Drug Discov. 2016;15(9):660.

Claims

CLAIMS1 . A method for treating cancer in a subject in need of such treatment comprising administering to the subject an effective amount of an oncolytic vector with (a) one or more probiotics, (b) one or more other microbiome-targeting compositions, (c) one or more antibiotics and / or (d) one or more fecal transplant.

2. The method according to claim 1 , wherein said oncolytic vector is an adenoviral vector.

3. The method according to claim 2, wherein said oncolytic adenoviral vector comprises an adenovirus serotype 5 (Ad5) backbone or an adenovirus serotype 3 (Ad3) backbone.

4. The method according to claim 3, wherein said oncolytic adenoviral vector comprises an adenovirus serotype 5 (Ad5) backbone with the fiber knob of adenovirus serotype 3 (Ad3).

5. The method according to any one of claims 1-4, wherein said oncolytic vector or oncolytic adenoviral vector comprises nucleic acid sequence encoding one or more cytokine transgenes, wherein the one or more cytokine transgenes are preferably selected from a group consisting of interferon alpha, interferon beta, interferon gamma, complement C5a, IL-2, CD40L, TNFalpha, IL-7, IL-12, IL-23, IL-15, IL-17, CCL1 , CCL11 , CCL12, CCL13, CCL14-1 , CCL14-2, CCL14-3, CCL15-1 , CCL15-2, CCL16, CCL17, CCL18, CCL19, CCL19, CCL2, CCL20, CCL21 , CCL22, CCL23-1 , CCL23-2, CCL24, CCL25-1 , CCL25-2, CCL26, CCL27, CCL28, CCL3, CCL3L1 , CCL4, CCL4L1 , CCL5, CCL6, CCL7, CCL8, CCL9, CCR10, CCR2, CCR5, CCR6, CCR7, CCR8, CCRL1 , CCRL2, CX3CL1 , CX3CR, CXCL1 , CXCL10, CXCL11 , CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL9, CXCR1 , CXCR2, CXCR4, CXCR5, CXCR6, CXCR7 and XCL2.

6. The method according to claim 5, wherein said nucleic acid sequence encodes TNFalpha, IL-2, CD40L, or IL-7.

7. The method according to any one of claims 1-4, wherein said oncolytic vector or oncolytic adenoviral vector comprises nucleic acid sequence encoding a bi-specific monoclonal antibody as a transgene.

8. The method according to claim 7, wherein the bispecific monoclonal antibody comprises a single chain variable fragment (scFv) specific for a cell surface molecule on immunological effector cells and a scFv specific for a tumor antigen,wherein the tumor antigen is EpCAMI or MUC 1 and the cell surface molecule is CD3.

9. The method according to any one of claims 2-6, wherein said oncolytic adenoviral vector comprises an adenovirus serotype 5 (Ad5) backbone and said nucleic acid sequence encoding TNFalpha and / or IL-2 is in the place of a deleted nucleic acid sequence in the E3 region of said oncolytic adenoviral vector.

10. The method according to any one of claims 2-9, wherein the vector comprises a deletion of a nucleic acid sequence in the E3 region, preferably a deletion of viral gp19k aod 6.7k reading frames.11 . The method according to any one of claims 3-10, wherein said oncolytic adenoviral vector comprises an adenovirus serotype 5 (Ad5) backbone and the vector comprises a 24 bp deletion (A24) in the adenoviral E1 sequence of said oncolytic adenoviral vector.

12. The method according to any one of claims 1-11 , wherein said antibiotic is effective against a bacterial species which decreases the effect of the treatment with an oncolytic vector in a subject when said bacterial species is present in the gut microbiome of said subject.

13. The method according to claim 12, wherein said bacterial species is Eggerthella lenta.

14. The method according to any one of claims 1-13, wherein said probiotic comprises a bacterial species which improves the effect of the treatment with an oncolytic vector in a subject when said bacterial species is present in the gut microbiome of said subject.

15. The method according to claim 14, wherein said bacterial species belongs to Alistipes and / or Prevotella genus, preferably said bacterial species is Alistipes shahii.

16. The method according to any one of claims 1-15, wherein said other microbiome-targeting composition is a prebiotic or a postbiotic.

17. The method according to claim 16, wherein said prebiotic or postbiotic promotes the growth of bacteria improves the effect of the treatment with an oncolytic vector in a subject, said bacteria preferably being bacterial species belonging to Alistipes and / or Prevotella genus.

18. The method according to any one of claims 1-17, wherein the fecal transplantT1 comprises bacteria improving the effect of the treatment with an oncolytic vector, preferably said bacteria comprises bacterial species belonging to Alistipes and / or Prevotella genus.

19. The method according to claim 18, wherein the fecal transplant is from an individual having a stable cancer disease or a cured cancer disease, wherein said individual has preferably benefited from oncolytic virus treatment, or from a healthy individual.

20. The method according to any one of claims 1 -19, wherein the oncolytic vector or the oncolytic adenoviral vector is administered intratumorally, intravenously, intraarterially, or intraperitoneally.21 . The method according to any one of claims 1 -20, wherein the oncolytic virus vector is administered in an amount from about 106-1014VP, 106-1012VP, 108-1014VP, 108-1012VP, 101°-1012VP or 108-101°VP.

22. The method according to any one of claims 1 -21 , wherein said one or more antibiotics is administered systemically or orally.

23. The method according to any one of claims 1 -22, wherein said probiotic or other microbiome-targeting composition is administered orally.

24. The method according to any one of claims 1 -23, wherein the subject has a cancer selected from nasopharyngeal cancer, synovial cancer, hepatocellular cancer, renal cancer, cancer of connective tissues, melanoma, lung cancer, bowel cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, throat cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, ureter cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, Ewing’s sarcoma, cancer of unknown primary site, carcinoid, carcinoid of gastrointestinal tract, fibrosarcoma, breast cancer, Paget’s disease, cervical cancer, esophagus cancer, gall bladder cancer, head and neck cancer, eye cancer, kidney cancer, Wilms’ tumor, Kaposi’s sarcoma, prostate cancer, testicular cancer, Hodgkin’s disease, non-Hodgkin’s lymphoma, skin cancer, mesothelioma, multiple myeloma, ovarian cancer, endocrine pancreatic cancer, glucagonoma, parathyroid cancer, penis cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus cancer, thyroid cancer, trophoblastic cancer, hydatidiform mole, uterine cancer, endometrial cancer, vagina cancer, vulvacancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gum cancer, heart cancer, lip cancer, meninges cancer, mouth cancer, nerve cancer, palate cancer, parotid gland cancer, peritoneum cancer, pharynx cancer, pleural cancer, salivary gland cancer, tongue cancer and tonsil cancer.

25. The method according to any one of claims 1 -24, wherein the subject is a human.

26. The method according to any one of claims 1 -25, further comprising a step of treating the subject with one or more additional therapy selected from radiotherapy, chemotherapy, antiangiogenic agents or targeted therapies, such as alkylating agents, nucleoside analogs, cytoskeleton modifiers, cytostatic agents, monoclonal antibodies such as checkpoint inhibitors, kinase inhibitors.

27. The method according to any one of claims 1 -26, wherein a first dose of the oncolytic vector and a first dose of the antibiotic are simultaneously administered to the subject.

28. The method according to any one of claims 1 -27, wherein a first dose of the oncolytic vector and a first dose of the probiotic or other microbiome-targeting composition are simultaneously administered to the subject.

29. The method according to any one of claims 1 -28, wherein a first dose of the antibiotic and a first dose of the probiotic or other microbiome-targeting composition are simultaneously administered to the subject.

30. The method according to any one of claims 1 -29, wherein a first dose of the oncolytic adenoviral vector and a first dose of one or more of the following: the antibiotic, the probiotic, the microbiome-targeting composition and the fecal transplant, are administered simultaneously or sequentially in any order.31 . The method according to any one of claims 1 -30 comprising a step of detecting the presence of Eggerthella lenta in the gut microbiome of the subject before the onset of the treatment.

32. The method according to any one of claims 1 -30 comprising a step of mapping the gut microbiome of the subject at least partly before the onset of the treatment.

33. The method according to any one of claims 1 -30 comprising a step of mapping the urine microbiome of the subject at least partly before the onset of the treatment.

34. The method according to any one of claims 1 -31 comprising a step of mappingthe tumor microbiome of the subject at least partly before the onset of the treatment.

35. The method according to any one of claims 32-34, wherein the subject is selected or not selected for the treatment based on the results of the mapping step.

36. The method according to any one of claims 32-34, wherein based on the results of the mapping, the subject is selected for the treatment with a probiotic comprising bacterial species belonging to Alistipes and / or Prevotella genus before the onset of the treatment with the oncolytic vector.

37. The method according to any one of claims 1-36 comprising a step of detecting the presence of Eggerthella lenta in the gut microbiome of the subject before the onset of the treatment and / or during the treatment, wherein preferably the subject having said Eggerthella lenta in the gut is treated with an antibiotic and / or a bacteriophage targeting said Eggerthella lenta before the onset of the treatment with the oncolytic vector or during the treatment with the oncolytic vector.

38. The method according to any one of claims 1 -36 comprising a step of mapping the gut microbiome of the subject at least partly before the onset of the treatment and / or during the treatment.

39. The method according to any one of claims 1 -38 comprising a step of mapping the urine microbiome of the subject at least partly before the onset of the treatment and / or during the treatment.

40. The method according to any one of claims 1 -39 comprising a step of mapping the tumor microbiome of the subject at least partly before the onset of the treatment and / or during the treatment.41 . The method according to any one of claims 38-40, wherein the mapping is performed before the onset of the treatment and the subject is selected or not selected for the treatment based on the results of the mapping step.

42. The method according to any one of claims 38-40, wherein based on the results of the mapping, the subject is selected for the treatment with a probiotic comprising bacterial species belonging to Alistipes and / or Prevotella genus before the onset of the treatment with the oncolytic vector and / or during the treatment with the oncolytic vector.

43. The method according to any one of claims 38-40, wherein based on the results of the mapping, the subject is selected for the treatment with a prebiotic promoting the growth of bacterial species belonging to Alistipes and / or Prevotella genus before the onset of the treatment with the oncolytic vector and / or during the treatment withthe oncolytic vector.

44. The method according to any one of claims 1-43 comprising administering to a subject a probiotic, prebiotic or postbiotic agent increasing the amount of butyrate in the gut of the subject before the onset of the treatment with the oncolytic vector and / or during the treatment with the oncolytic vector.

45. The method according to claim 44 comprising administering to a subject a probiotic, prebiotic or postbiotic agent increasing butyrate kinase activity in the gut of the subject before the onset of the treatment with the oncolytic vector and / or during the treatment with the oncolytic vector.

46. The method according to any one of claims 1-43 comprising administering to a subject a probiotic, prebiotic or postbiotic agent decreasing the amount of arginine in the gut of the subject before the onset of the treatment with the oncolytic vector and / or during the treatment with the oncolytic vector.

47. The method according to any one of claims 1-43 comprising administering to a subject a probiotic, prebiotic or postbiotic agent decreasing arginine deiminase activity in the gut of the subject before the onset of the treatment with the oncolytic vector and / or during the treatment with the oncolytic vector.

48. Pharmaceutical composition comprising an oncolytic vector with one or more of the following: a probiotic, an antibiotic, a microbiome-targeting composition and a fecal transplant.

49. The pharmaceutical composition of claim 48, wherein said oncolytic vector is an oncolytic adenoviral vector comprising- a 5 / 3 chimeric fiber knob,- E2F1 promoter for tumor specific expression of E1A,- a 24 bp deletion (D24) in the Rb binding constant region 2 of adenoviral E1 ,- a nucleic acid sequence deletion of viral gp19k and 6.7k reading frames, and- a nucleic acid sequence encoding at least one cytokine or a bi-specific monoclonal antibody as transgene(s) in the place of the deleted gp19k / 6.7K '\r\ the E3 region resulting in replication-associated control of transgene expression under the viral E3 promoter.

50. The pharmaceutical composition of claim 48 or 49, wherein said antibiotic is effective against a bacterial species which decreases the effect of the treatment withan oncolytic vector in a subject when said bacterial species is present in the gut microbiome of said subject.51 . The pharmaceutical composition of claim 48 or 49, wherein one or more of the following: a probiotic, a microbiome-targeting composition and a fecal transplant, is / are improving the effect of the treatment with an oncolytic vector.

52. A kit which comprises a first container, a second container and a package insert, wherein the first container comprises at least one dose of a pharmaceutical composition containing an oncolytic vector, the second container containing one of the following: a probiotic, an antibiotic, a microbiome-targeting composition or a fecal transplant, and the package insert comprises instructions for treating a subject having cancer using the compositions of the first and second containers, wherein said probiotic, microbiome-targeting composition or fecal transplant comprises or promotes a bacterial species which improves the effect of the treatment with an oncolytic vector in a subject when said bacterial species is present in the gut microbiome of said subject, and wherein said antibiotic is effective against a bacterial species which decreases the effect of the treatment with an oncolytic vector in a subject when said bacterial species is present in the gut microbiome of said subject.

53. The kit according to claim 52, wherein said oncolytic vector is an oncolytic adenoviral vector comprising- a 5 / 3 chimeric fiber knob,- E2F1 promoter for tumor specific expression of E1A,- a 24 bp deletion (D24) in the Rb binding constant region 2 of adenoviral E1 ,- a nucleic acid sequence deletion of viral gp19k aod 6.7k reading frames, and- a nucleic acid sequence encoding at least one cytokine or a bi-specific monoclonal antibody as transgene(s) in the place of the deleted gp19k / 6.7K '\r\ the E3 region resulting in replication-associated control of transgene expression under the viral E3 promoter.

54. The kit according to claim 52 or 53, wherein said probiotic comprises a bacterial species belonging to Alistipes and / or Prevotella genus, preferably said bacterial species is Alistipes shahii, or wherein said microbiome-targeting composition is a prebiotic promoting the growth of bacterial species belonging to Alistipes and / or Prevotella genus.

55. An oncolytic vector together with (a) one or more probiotics, (b) one or more other microbiome-targeting compositions, (c) one or more antibiotics and / or (d) one or more fecal transplant for use in the treatment of cancer.

56. The oncolytic vector for use according to claim 55, wherein said oncolytic vector is an adenoviral vector.

57. The oncolytic vector for use according to claim 56, wherein said oncolytic adenoviral vector comprises an adenovirus serotype 5 (Ad5) backbone or an adenovirus serotype 3 (Ad3) backbone58. The oncolytic vector for use according to claim 57, wherein said oncolytic adenoviral vector comprises an adenovirus serotype 5 (Ad5) backbone with the fiber knob of adenovirus serotype 3 (Ad3).

59. The oncolytic vector for use according to any one of claims 55-58, wherein said oncolytic vector or oncolytic adenoviral vector comprises nucleic acid sequence encoding one or more cytokine transgenes, wherein the one or more cytokine transgenes are preferably selected from a group consisting of interferon alpha, interferon beta, interferon gamma, complement C5a, IL-2, CD40L, TNFalpha, IL-7, IL-12, IL-23, IL-15, IL-17, CCL1 , CCL11 , CCL12, CCL13, CCL14-1 , CCL14-2, CCL14-3, CCL15-1 , CCL15-2, CCL16, CCL17, CCL18, CCL19, CCL19, CCL2, CCL20, CCL21 , CCL22, CCL23-1 , CCL23-2, CCL24, CCL25-1 , CCL25-2, CCL26, CCL27, CCL28, CCL3, CCL3L1 , CCL4, CCL4L1 , CCL5, CCL6, CCL7, CCL8, CCL9, CCR10, CCR2, CCR5, CCR6, CCR7, CCR8, CCRL1 , CCRL2, CX3CL1 , CX3CR, CXCL1 , CXCL10, CXCL11 , CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL9, CXCR1 , CXCR2, CXCR4, CXCR5, CXCR6, CXCR7 and XCL2.

60. The oncolytic vector for use according to claim 59, wherein said nucleic acid sequence encodes at least TNFalpha, IL-2, CD40L, or IL-7.61 . The oncolytic vector for use according to any one of claims 55-58, wherein wherein said oncolytic vector or oncolytic adenoviral vector comprises nucleic acid sequence encoding a bi-specific monoclonal antibody as a transgene.

62. The oncolytic vector for use according to claim 61 , wherein the bispecific monoclonal antibody comprises a single chain variable fragment (scFv) specific for acell surface molecule on immunological effector cells and a scFv specific for a tumor antigen, wherein the tumor antigen is EpCAMI or MUC 1 and the cell surface molecule is CD3.

63. The oncolytic vector for use according to any one of claims 56-60, wherein said oncolytic adenoviral vector comprises an adenovirus serotype 5 (Ad5) backbone and said nucleic acid sequence encoding TNFalpha and / or IL-2 is in the place of a deleted nucleic acid sequence in the E3 region of said oncolytic adenoviral vector.

64. The oncolytic vector for use according to claim 56-63, wherein the deletion of a nucleic acid sequence in the E3 region is a deletion of viral gp19k and 6.7k reading frames.

65. The oncolytic vector for use according to any one of claims 56-64, wherein said oncolytic adenoviral vector comprises an adenovirus serotype 5 (Ad5) backbone and the vector comprises a 24 bp deletion (A24) in the adenoviral E1 sequence of said oncolytic adenoviral vector.

66. The oncolytic vector for use according to any one of claims 56-65, wherein said antibiotic is effective against a bacterial species which decreases the effect of the treatment with an oncolytic vector in a subject when said bacterial species is present in the gut microbiome of said subject.

67. The oncolytic vector for use according to claim 66, wherein said bacterial species is Eggerthella lenta.

68. The oncolytic vector for use according to any one of claims 55-67, wherein said probiotic comprises a bacterial species which improves the effect of the treatment with an oncolytic vector in a subject when said bacterial species is present in the gut microbiome of said subject.

69. The oncolytic vector for use according to claim 68, wherein said bacterial species belongs to Alistipes and / or Prevotella genus, preferably said bacterial species is Alistipes shahii.

70. The oncolytic vector for use according to any one of claims 55-67, wherein said other microbiome-targeting composition is a prebiotic or a postbiotic.71 . The oncolytic vector for use according to claim 70, wherein said prebiotic or postbiotic promotes the growth of bacteria improves the effect of the treatment with an oncolytic vector in a subject, said bacteria preferably being bacterial species belonging to Alistipes and / or Prevotella genus.

72. The oncolytic vector for use according to any one of claims 55-71 , wherein the fecal transplant comprises bacteria improving the effect of the treatment with an oncolytic vector, preferably said bacteria comprises bacterial species belonging to Alistipes and / or Prevotella genus.

73. The oncolytic vector for use according to claim 72, wherein the fecal transplant is from a healthy individual.

74. The oncolytic vector for use according to claim 72, wherein the fecal transplant is from an individual having a stable cancer disease or a cured cancer disease, wherein said individual has preferably benefited from oncolytic virus treatment.

75. The oncolytic vector for use according to any one of claims 55-64, wherein the oncolytic vector or the oncolytic adenoviral vector is administered intratumorally, intravenously, intra-arterially, or intraperitoneally.

76. The oncolytic vector for use according to any one of claims 55-75, wherein the oncolytic virus vector is administered in an amount from about 106-1014VP, 106-1012VP, 108-1014VP, 108-1012VP, 101°-1012VP or 108-101°VP.

77. The oncolytic vector for use according to any one of claims 55-76, wherein said one or more antibiotics is administered systemically or orally.

78. The oncolytic vector for use according to any one of claims 55-77, wherein said probiotic or other microbiome-targeting composition is administered orally.

79. The oncolytic vector for use according to any one of claims 55-78, wherein the subject has a cancer selected from nasopharyngeal cancer, synovial cancer, hepatocellular cancer, renal cancer, cancer of connective tissues, melanoma, lung cancer, bowel cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, throat cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, uretercancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, Ewing’s sarcoma, cancer of unknown primary site, carcinoid, carcinoid of gastrointestinal tract, fibrosarcoma, breast cancer, Paget’s disease, cervical cancer, esophagus cancer, gall bladder cancer, head and neck cancer, eye cancer, kidney cancer, Wilms’ tumor, Kaposi’s sarcoma, prostate cancer, testicular cancer, Hodgkin’s disease, non-Hodgkin’s lymphoma, skin cancer, mesothelioma, multiple myeloma, ovarian cancer, endocrine pancreatic cancer, glucagonoma, parathyroid cancer, penis cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus cancer, thyroid cancer, trophoblastic cancer, hydatidiform mole, uterine cancer, endometrial cancer, vagina cancer, vulva cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gum cancer, heart cancer, lip cancer, meninges cancer, mouth cancer, nerve cancer, palate cancer, parotid gland cancer, peritoneum cancer, pharynx cancer, pleural cancer, salivary gland cancer, tongue cancer and tonsil cancer.

80. The oncolytic vector for use according to any one of claims 55-79, wherein the subject is a human.81 . The oncolytic vector for use according to any one of claims 55-80, further comprising a step of treating the subject with one or more additional therapy selected from radiotherapy, chemotherapy, antiangiogenic agents or targeted therapies, such as alkylating agents, nucleoside analogs, cytoskeleton modifiers, cytostatic agents, monoclonal antibodies such as checkpoint inhibitors, kinase inhibitors.

82. The oncolytic vector for use according to any one of claims 55-81 , wherein a first dose of the oncolytic vector and a first dose of the antibiotic are simultaneously administered to the subject.

83. The oncolytic vector for use according to any one of claims 55-82, wherein a first dose of the oncolytic vector and a first dose of the probiotic or other microbiometargeting composition are simultaneously administered to the subject.

84. The oncolytic vector for use according to any one of claims 55-83, wherein a first dose of the antibiotic and a first dose of the probiotic or other microbiome-targeting composition are simultaneously administered to the subject.

85. The oncolytic vector for use according to any one of claims 55-84, wherein a first dose of the oncolytic adenoviral vector and a first dose of one or more of the following: the probiotic, the antibiotic, the microbiome-targeting composition and the fecal transplant, are administered simultaneously or sequentially in any order.

86. The oncolytic vector for use according to any one of claims 55-85 comprising a step of detecting the presence of Eggerthella lenta in the gut microbiome of the subject before the onset of the treatment and / or during the treatment, wherein preferably the subject having said Eggerthella lenta in the gut is treated with an antibiotic and / or a bacteriophage targeting said Eggerthella lenta before the onset of the treatment with the oncolytic vector or during the treatment with the oncolytic vector.

87. The oncolytic vector for use according to any one of claims 55-85 comprising a step of mapping the gut microbiome of the subject at least partly before the onset of the treatment and / or during the treatment.

88. The oncolytic vector for use according to any one of claims 55-85 comprising a step of mapping the urine microbiome of the subject at least partly before the onset of the treatment and / or during the treatment.

89. The oncolytic vector for use according to any one of claims 55-86 comprising a step of mapping the tumor microbiome of the subject at least partly before the onset of the treatment and / or during the treatment.

90. The oncolytic vector for use according to any one of claims 87-89, wherein the subject is selected or not selected for the treatment based on the results of the mapping step.91 . The oncolytic vector for use according to any one of claims 87-89, wherein based on the results of the mapping, the subject is selected for the treatment with a probiotic comprising bacterial species belonging to Alistipes and / or Prevotella genus before the onset of the treatment with the oncolytic vector and / or during the treatment with the oncolytic vector, preferably said bacterial species is Alistipes shahii.

92. The oncolytic vector for use according to any one of claims 87-91 , wherein based on the results of the mapping, the subject is selected for the treatment with a prebiotic promoting the growth of bacterial species belonging to Alistipes and / or Prevotella genus before the onset of the treatment with the oncolytic vector and / or during the treatment with the oncolytic vector.

93. The oncolytic vector for use according to any one of claims 55-85 comprising administering to a subject a probiotic, prebiotic or postbiotic agent increasing the amount of butyrate in the gut of the subject before the onset of the treatment with the oncolytic vector and / or during the treatment with the oncolytic vector.

94. The oncolytic vector for use according to claim 93 comprising administering to a subject a probiotic, prebiotic or postbiotic agent increasing butyrate kinase activity in the gut of the subject before the onset of the treatment with the oncolytic vector and / or during the treatment with the oncolytic vector.

95. The oncolytic vector for use according to any one of claims 55-85 comprising administering to a subject a probiotic, prebiotic or postbiotic agent decreasing the amount of arginine in the gut of the subject before the onset of the treatment with the oncolytic vector and / or during the treatment with the oncolytic vector.

96. The oncolytic vector for use according to any one of claims 55-85 comprising administering to a subject a probiotic, prebiotic or postbiotic agent decreasing arginine deiminase activity in the gut of the subject before the onset of the treatment with the oncolytic vector and / or during the treatment with the oncolytic vector.