FIELD OF INVENTION

[0001] The present invention concerns the treatment of cancer and more particularly relates to the use of adenovirus as adjuvants.

BACKGROUND OF INVENTION

[0002] A large proportion of human tumors are deregulated for the expression of the APOBEC3B gene and express high levels of the APOBEC3B protein. The abnormal expression of this protein has two negative consequences. First, APOBECB protein fuels the genetic diversity of the tumors, contributing to metastasis and drug resistance. Second, the expression of APOBEC3B by the tumor cells limits the efficacy of oncolytic viruses.

[0003] There is thus an unmet medical need to find efficient ways to downregulate APOBEC3B in tumors, thereby decreasing the emergence of drug-resistant clones and to make the tumor cells more permissive for oncolytic viruses.

[0004] Adenoviruses are a large family of DNA viruses, counting more than 100 genotypes. Adenoviruses are increasingly used as backbone for vaccines and oncolytic viruses.

[0005] The present invention relates to the use of adenoviruses to antagonize the APOBEC3B protein produced by the tumor cells.

SUMMARY

[0006] The present invention relates to an adenovirus or a fragment thereof for use as an adjuvant for the treatment of cancer in a subject in need thereof. In some embodiments, the adenovirus is of a species selected from the group comprising or consisting of B, C, D, E and G. [0007] In some embodiments, the adenovirus is of a species selected from the group comprising or consisting of B and C. In some embodiments, the adenovirus is of a subtype selected from the group comprising or consisting of B3 and C2, preferably B3.

[0008] In some embodiments, the adenovirus is a modified adenovirus comprising at least one nucleic acid mutation compared to a wild type adenovirus.

[0009] The present invention further relates to a pharmaceutical composition comprising the adenovirus or a fragment thereof for use according to the invention, and a pharmaceutically acceptable carrier.

[0010] In some embodiments, the pharmaceutical composition further comprises an anticancer agent as an active ingredient.

[0011] In some embodiments, the adenovirus or a fragment thereof is in an amount sufficient to enhance the activity of the anticancer agent. In some embodiments, the adenovirus or a fragment thereof is administered at a dose from about 10 ⁴ to about IO ²⁰ viral particles.

[0012] In some embodiments, the anticancer agent is an oncolytic virus.

[0013] In some embodiments, the adenovirus or a fragment thereof potentiates the replication of said oncolytic virus.

[0014] In some embodiments, the oncolytic virus is selected from the group comprising or consisting of adenovirus, herpes virus, picornavirus, poliovirus, coxsackie virus, poxvirus, vaccina virus, paramyxovirus, measles virus, reovirus, rhabdovirus, vesicular stomatitis virus and senecavirus.

[0015] In some embodiments, the oncolytic virus as adenovirus is distinct from the adenovirus or fragment thereof.

[0016] In some embodiments, the pharmaceutical composition is for use in a method for the treatment of cancer. In some embodiments, the cancer is selected from the group comprising or consisting of gastric cancer, gastrointestinal cancer, lung cancer, cervical cancer, colorectal cancer, colon cancer, breast cancer, prostate cancer, melanoma, liver cancer, hepatic carcinoma, pancreatic cancer, kidney cancer, ovarian cancer, blood cancer, lymphoma, leukemia, thyroid cancer, squamous cell cancer, brain cancer, glioblastoma, endometrial carcinoma, salivary gland carcinoma, vulvar cancer, and bladder cancer.

[0017] The present invention further relates to the use of an adenovirus or a fragment thereof for the manufacture of an adjuvant for the treatment of cancer.

[0018] The present invention further relates to a combination kit comprising (i) an adenovirus or fragment according to the invention, or the pharmaceutical composition according to the invention, and (ii) an anticancer agent, for use for treating cancer in a subject in need thereof.

DEFINITIONS

[0019] In the present invention, the following terms have the following meanings:

[0020] “About” preceding a figure means plus or less 10% of the value of said figure.

[0021] “And/Or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0022] "Comprising", "comprises" and "comprised of" are used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. These terms also encompass “consisting of’.

[0023] “Fragment”, as used herein, refers to any part of the adenovirus for use according to the present invention.

[0024] “Subject” or “individual” refers to an animal individual, preferably a mammalian individual, more preferably a human individual. In some embodiments, an individual may be a mammalian individual. Mammalians include, but are not limited to, all primates (human and non-human), cattle (including cows), horses, pigs, sheep, goats, dogs, cats, and any other mammal which is awaiting the receipt of, or is receiving medical care or was/is/will be the object of a medical procedure, or is monitored for the development of a disease or condition, preferably cancer. In some embodiments, an individual may be a “patient”, /.< ., a warm-blooded animal, more preferably a human, who/which is awaiting the receipt of, or is receiving medical care or was/is/will be the object of a medical procedure, or is monitored for the development of a disease or condition, preferably cancer. In some embodiments, the individual is an adult (e.g., an individual above the age of 18). In some embodiments, the individual is a child (e.g., an individual below the age of 18). In some embodiments, the individual is a male. In some embodiments, the individual is a female.

[0025] “Pharmaceutically acceptable carrier” refers to a carrier that does not produce any adverse, allergic or other unwanted reactions when administered to an animal individual, preferably a human individual. It includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. For human administration, preparations should meet sterility, pyrogenicity, general safety, quality and purity standards as required by regulatory Offices, such as, e.g., the Food and Drug Administration (FDA) in the United States or the European Medicines Agency (EMA) in the European Union.

[0026] “Treating” or “treatment” refers to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder, preferably cancer. Those in need of treatment include those already with the disorder, preferably cancer, as well as those prone to have the disorder or those in whom the disorder is to be prevented. An individual or mammal is successfully “treated” for the disorder if, after receiving a therapeutic amount of an adenovirus vector according to the present invention, the individual shows observable and/or measurable reduction in or absence of one or more of the following: reduction in the number of pathogenic cells (i.e., cancer cells); reduction in the percent of total cells that are pathogenic; and/or relief to some extent, one or more of the symptoms associated with the specific disease or disorder, preferably cancer; reduced morbidity and mortality, and improvement in quality of life issues. The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.

[0027] “Therapeutically effective amount” refers to the level or amount of an agent that is aimed at, without causing significant negative or adverse side effects to the target, (1) delaying or preventing the onset of the disease or condition, preferably cancer; (2) slowing down or stopping the progression, aggravation, or deterioration of one or more symptoms of the disease or condition, preferably cancer; (3) bringing about ameliorations of the symptoms of the disease or condition, preferably cancer; (4) reducing the severity or incidence of the disease or condition, preferably cancer; or (5) curing the disease or condition, preferably cancer. A therapeutically effective amount may be administered prior to the onset of the disease or condition, for a prophylactic or preventive action. Alternatively, or additionally, the therapeutically effective amount may be administered after initiation of the disease or condition, preferably cancer, for a therapeutic action.

DETAILED DESCRIPTION

[0028] The present invention relates to an adenovirus or a fragment thereof for use as an adjuvant for the treatment of cancer in a subject in need thereof.

[0029] In some embodiments, the adenovirus or fragment thereof for use according to the invention is of a species selected from the group comprising or consisting of A, B, C,

D, E, F and G.

[0030] In some embodiments, the adenovirus or fragment thereof for use according to the invention is of a species selected from the group comprising or consisting of B, C, D,

E, F and G.

[0031] In a preferred embodiment, the adenovirus or fragment thereof for use according to the invention is of a species selected from the group comprising or consisting of B, C, D, E and G. In certain embodiments, the adenovirus or fragment thereof for use according to the invention is of a species selected from the group comprising or consisting of B, C, D and E, or from the group comprising or consisting of B, C, D and G, or from the group comprising or consisting of B, C, E and G, or from the group comprising or consisting of

B, D, E and G, or from the group comprising or consisting of C, D, E and G.

[0032] In some embodiments, the adenovirus or fragment thereof for use according to the invention is of a species selected from the group comprising or consisting of B, C, D,

E. In certain embodiments, the adenovirus or fragment thereof for use according to the invention is of a species selected from the group comprising or consisting of B, C, and D, or from the group comprising or consisting of B, C and E, or from the group comprising or consisting of B, D and E, or from the group comprising or consisting of C, D and E.

[0033] In some embodiments, the adenovirus or fragment thereof for use according to the invention is of a species selected from the group comprising or consisting of B, C, D. In certain embodiments, the adenovirus or fragment thereof for use according to the invention is of a species selected from the group comprising or consisting of B and C, or from the group comprising or consisting of B and D, or from the group comprising or consisting of C and D.

[0034] In a preferred embodiment, the adenovirus or fragment thereof for use according to the invention is of a species selected from the group comprising or consisting of B and

C. In a more preferred embodiment, the adenovirus or fragment thereof for use according to the invention is of species B. In a more preferred embodiment, the adenovirus or fragment thereof for use according to the invention is of species C. In another embodiment, the adenovirus or fragment thereof for use according to the invention is of species D. In another embodiment, the adenovirus or fragment thereof for use according to the invention is of species E. In another embodiment, the adenovirus or fragment thereof for use according to the invention is of species G.

[0035] In another embodiment, the adenovirus or fragment thereof for use according to the invention is of a species selected from the group comprising or consisting of A, C and

F. In another embodiment, the adenovirus or fragment thereof for use according to the invention is of a species selected from the group comprising or consisting of A and C. In one embodiment, the adenovirus or fragment thereof for use according to the invention is of species A. In one embodiment, the adenovirus or fragment thereof for use according to the invention is of species C. In one embodiment, the adenovirus or fragment thereof for use according to the invention is of species F.

[0036] In another embodiment, the adenovirus or fragment thereof for use according to the invention is of a species selected from the group comprising or consisting of B, A and C. In one embodiment, the adenovirus or fragment thereof for use according to the invention is of a species selected from the group comprising or consisting of B and A. In one embodiment, the adenovirus or fragment thereof for use according to the invention is of a species selected from the group comprising or consisting of B and C.

[0037] In some embodiments, the adenovirus or fragment thereof for use according to the invention is of a subtype selected from the group comprising or consisting of A12, A18, A31, B3, B7, Bl l, B14, B16, B21, B34, B35, B50, B55, Cl, C2, C5, C6, C57, D8, D9, D10, D13, D15, D17, D19, D20, D22, D23, D24, D25, D26, D27, D28, D29, D30, D32, D33, D36, D37, D38, D39, D42, D43, D44, D45, D46, D47, D48, D49, D51, D53, D54, D56, D58, D59, D60, D62, D63, D64, D65, D67, D69, D70, D71, D72, D73, D74, D75, E4, F40, F41 and G52.

[0038] In some embodiments, the adenovirus or fragment thereof for use according to the invention is of a subtype selected from the group comprising or consisting of B3, B7, Bl l, B14, B16, B21, B34, B35, B50, B55, Cl, C2, C5, C6, C57, D8, D9, D10, D13, D15, D17, D19, D20, D22, D23, D24, D25, D26, D27, D28, D29, D30, D32, D33, D36, D37, D38, D39, D42, D43, D44, D45, D46, D47, D48, D49, D51, D53, D54, D56, D58, D59, D60, D62, D63, D64, D65, D67, D69, D70, D71, D72, D73, D74, D75, E4, F40, F41 and G52.

[0039] In some embodiments, the adenovirus or fragment thereof for use according to the invention is of a subtype selected from the group comprising or consisting of A12, A18, A31, B3, B7, Bl l, B14, B16, B21, B34, B35, B50, B55, Cl, C2, C5, C6 and C57. In a preferred embodiment, the adenovirus or fragment thereof for use according to the invention is of a subtype selected from the group comprising or consisting of A12, B3 and C2. In a more preferred embodiment, the adenovirus or fragment thereof for use according to the invention is of subtype B3. In another embodiment, the adenovirus or fragment thereof for use according to the invention is of subtype A12. In another embodiment, the adenovirus or fragment thereof for use according to the invention is of subtype C2.

[0040] In some embodiments, the adenovirus or fragment thereof for use according to the invention is of a subtype selected from the group comprising or consisting of B3, B7, Bl l, B14, B16, B21, B34, B35, B50, B55, Cl, C2, C5, C6 and C57. In a preferred embodiment, the adenovirus or fragment thereof for use according to the invention is of a subtype selected from the group comprising or consisting of B3 and C2. In a more preferred embodiment, the adenovirus or fragment thereof for use according to the invention is of subtype B3. In another embodiment, the adenovirus or fragment thereof for use according to the invention is of subtype C2.

[0041] The Inventors have demonstrated that the adenovirus or fragment thereof from the B3 and C12 decrease the quantity of ABOBEC2B with an enhanced efficacity than the other species of adenovirus. In particular, it appears from the results obtained by the Inventors that this effect is particularly significative with the B3 subtype.

[0042] In some embodiments, the adenovirus or fragment thereof for use according to the invention is of a subtype selected from the group comprising or consisting of B3, B7, Bl l, B14, B16, B21, B34, B35, B50, B55, D8, D9, D10, D13, D15, D17, D19, D20, D22, D23, D24, D25, D26, D27, D28, D29, D30, D32, D33, D36, D37, D38, D39, D42, D43, D44, D45, D46, D47, D48, D49, D51, D53, D54, D56, D58, D59, D60, D62, D63, D64, D65, D67, D69, D70, D71, D72, D73, D74, D75, E4 and G52.

[0043] In some embodiments, the adenovirus or fragment thereof for use according to the invention is of a subtype selected from the group comprising or consisting of Cl, C2, C5, C6, C57, D8, D9, D10, D13, D15, D17, D19, D20, D22, D23, D24, D25, D26, D27, D28, D29, D30, D32, D33, D36, D37, D38, D39, D42, D43, D44, D45, D46, D47, D48, D49, D51, D53, D54, D56, D58, D59, D60, D62, D63, D64, D65, D67, D69, D70, D71, D72, D73, D74, D75, E4, and G52. [0044] In some embodiment adenovirus or fragment thereof for use according to the invention is not from the A species. In some embodiment adenovirus or fragment thereof for use according to the invention is not of a subtype selected from the group comprising or consisting of A12, Al 8, A31. In some embodiment adenovirus or fragment thereof for use according to the invention is not the A12 subtype.

[0045] In some embodiments, the adenovirus or fragment thereof for use according to the invention is safe for mammalian administration, preferably human administration. In some embodiments, the adenovirus or fragment thereof for use according to the invention does not produce an adverse, allergic or other untoward reaction in the subject.

[0046] In some embodiments, the adenovirus or fragment thereof for use according to the invention has an increased tropism for cancer cells. As used herein, “increased tropism” means that the infection rate of the adenovirus or fragment thereof for cancer cells is 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 500-fold, 1,000-fold or more than healthy cells. In some embodiments, the adenovirus or fragment thereof for use according to the invention preferentially infects cancer cells. In some embodiments, the adenovirus or fragment thereof for use according to the invention exclusively infects cancer cells.

[0047] In some embodiments, the adenovirus or fragment thereof for use according to the invention inhibits or antagonizes the apolipoprotein B mRNA editing enzyme catalytic polypeptide-like (APOBEC) family of proteins. In some embodiments, the adenovirus or fragment thereof for use according to the invention inhibits or antagonizes an APOBEC 3 protein selected from the group comprising or consisting of APOBEC3 A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G and APOBEC3H.

[0048] In a preferred embodiment, the adenovirus or fragment thereof for use according to the invention inhibits or antagonizes APOBEC3B (or “A3B”).

[0049] In a preferred embodiment, the adenovirus or fragment thereof for use according to the invention that inhibits or antagonizes APOBEC3B (or “A3B”) is of a species selected from the group comprising or consisting from the B and C. [0050] In a preferred embodiment, the adenovirus or fragment thereof for use according to the invention that inhibits or antagonizes APOBEC3B (or “A3B”) is of a subtype selected from the group comprising or consisting from the B3 and C12 subtypes.

[0051] In a preferred embodiment, the adenovirus or fragment thereof for use according to the invention that inhibits or antagonizes APOBEC3B (or “A3B”) is not from the A species.

[0052] In some embodiments, the adenovirus or fragment thereof for use according to the invention reduces the quantity of A3B in a cell. As used herein, “reduces the quantity of A3B” means that the number of A3B proteins is decreased 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 500-fold, 1,000-fold or more compared to the number of A3B proteins in an uninfected cell. In certain embodiments, the adenovirus or fragment thereof for use according to the invention reduces the quantity of A3B in a cell to the levels normally found in a healthy cell, wherein the infected cell expresses higher levels of A3B compared to a healthy cell (e.g., a cancer cell). In certain embodiments, the adenovirus or fragment thereof for use according to the invention reduces the quantity of A3B in a cell to levels below those normally found in a healthy cell.

[0053] Methods for measuring the quantity of A3B in cells are well-known from the skilled person in the art. Measurements may be performed at the RNA level or the protein level.

[0054] In some embodiments, the adenovirus or fragment thereof for use according to the invention inhibits the enzymatic activity of A3B. In a preferred embodiment, the adenovirus or fragment thereof for use according to the invention inhibits the cytidine deaminase activity of A3B. As used herein, “inhibits the activity of A3B” means that the activity of A3B is decreased 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8- fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100- fold, 500-fold, 1,000-fold or more compared to A3B activity in an uninfected cell.

[0055] Methods for measuring the enzymatic activity of A3B in cells are well-known from the skilled person in the art. [0056] In some embodiments, the adenovirus or fragment thereof for use according to the invention has an increased tropism for cells overexpressing A3B and/or cells having an increased A3B activity.

[0057] In some embodiments, the adenovirus or fragment thereof for use according to the invention acts as a lytic virus and induces cell death in the infected cell. In a preferred embodiment, the adenovirus or fragment thereof for use according to the invention acts as an oncolytic virus and induces cell death in the infected cancer cell. In some embodiments, the adenovirus or fragment thereof for use according to the invention induces cell death in a population of cancer cells.

[0058] In some embodiment, the fragments of said adenovirus from the present invention retain at least the biological function of the adenovirus.

[0059] As used herein, the term “adenovirus” may refer interchangeably to both naturally occurring (or “wild type”) adenoviruses, and adenoviruses that have been genetically modified/engineered, /.< ., mutant adenoviruses.

[0060] In some embodiments, the adenovirus or fragment thereof for use according to the invention is a wild type adenovirus, or a modified adenovirus comprising at least one nucleic acid mutation compared to a wild type adenovirus.

[0061] In some embodiments, the adenovirus or fragment thereof for use according to the invention is a wild type adenovirus.

[0062] In some embodiments, the adenovirus or fragment thereof for use according to the invention is a modified adenovirus or fragment thereof comprising at least one nucleic acid mutation compared to a wild type adenovirus. As used herein, “at least one” means 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, 20 or more. In some embodiments, “nucleic acid mutation” comprises substitutions, deletions, insertions, inversions, point mutations, frameshift mutations and combination thereof.

[0063] In some embodiments, the mutation increases or decreases the ability of the adenovirus or fragment thereof for use according to the invention to infect cells. In one embodiment, the mutation increases the ability of the adenovirus or fragment thereof for use according to the invention to infect cells. In another embodiment, the mutation decreases the ability of the adenovirus or fragment thereof for use according to the invention to infect cells.

[0064] In some embodiments, the mutation increases or decreases the ability of the adenovirus or fragment thereof for use according to the invention to replicate. In one embodiment, the mutation increases the ability of the adenovirus or fragment thereof for use according to the invention to replicate. In another embodiment, the mutation decreases the ability of the adenovirus or fragment thereof for use according to the invention to replicate.

[0065] In some embodiments, the mutation increases the tropism for cancer cells of the adenovirus or fragment thereof for use according to the invention.

[0066] In some embodiments, the adenovirus or fragment thereof for use according to the invention comprises at least one heterologous sequence or peptide. By “heterologous sequence or peptide” is meant a sequence or peptide which is not endogenous or native to the adenovirus. In one embodiment, the nucleic acid of the heterologous peptide is inserted into the nucleic acid of the adenovirus.

[0067] In some embodiment, the adenovirus or fragment thereof is not a helperdependent adenovirus.

[0068] The present invention further relates to a pharmaceutical composition comprising the adenovirus or a fragment thereof for use according to the invention and a pharmaceutically acceptable carrier.

[0069] In some embodiments, the pharmaceutically acceptable carrier is selected in a group comprising or consisting of a solvent, a diluent, a carrier, an excipient, a dispersion medium, a coating, an antibacterial agent, an antifungal agent, an isotonic agent, an absorption delaying agent and any combinations thereof. The carrier, diluent, solvent or excipient must be “acceptable” in the sense of being compatible with the polypeptide, or derivative thereof, and not be deleterious upon being administered to a subject. Typically, the vehicle does not produce an adverse, allergic or other untoward reaction when administered to a subject, preferably a human subject.

[0070] For the particular purpose of human administration, the pharmaceutical compositions should meet sterility, pyrogenicity, general safety and purity standards as required by regulatory offices, such as, for example, the Food and Drugs Administration (FDA) Office or the European Medicines Agency (EMA).

[0071] In some embodiments, the carrier may be water or saline (e.g., physiological saline), which will be sterile and pyrogen free. Suitable excipients include mannitol, dextrose, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.

[0072] In some embodiments, the pharmaceutical composition for use according to the invention further comprises an anticancer agent as an active ingredient.

[0073] In some embodiments, the anticancer agent is an oncolytic virus or a commercially available anticancer medicament.

[0074] In some embodiments, the adenovirus or a fragment thereof for use according to the invention is in an amount sufficient to enhance the activity of the anticancer agent. In practice, the adenovirus or a fragment thereof increases the oncolytic effects and/or oncostatic effects, preferably oncolytic effects, of the anticancer agent are increased 1.2- fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4- fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 500-fold, 1,000-fold or more. In other words, the adenovirus or a fragment thereof for use according to the invention improves the anticancer effects of the anticancer agent. In some embodiments, the cancer cells are eliminated, and/or the tumor regresses, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 100 times, 500 times, 1,000 times faster or more.

[0075] It is to be understood that the adenovirus or a fragment thereof for use according to the invention acts as an adjuvant for the anticancer agent.

[0076] In a preferred embodiment, the anticancer agent is an oncolytic virus. [0077] In some embodiments, the oncolytic virus is selected from the group comprising or consisting of adenovirus, herpes virus, picornavirus, poliovirus, coxsackie virus, poxvirus, vaccina virus, paramyxovirus, measles virus, reovirus, rhabdovirus, vesicular stomatitis virus and senecavirus.

[0078] In some embodiments, the oncolytic virus is another adenovirus, wherein the other adenovirus is distinct from the adenovirus or fragment thereof for use according to the invention. In some embodiments, the oncolytic virus is a herpes virus. In some embodiments, the oncolytic virus is a picornavirus. In some embodiments, the oncolytic virus is a poliovirus. In some embodiments, the oncolytic virus is a coxsackie virus. In some embodiments, the oncolytic virus is a poxvirus. In some embodiments, the oncolytic virus is a vaccina virus. In some embodiments, the oncolytic virus is a paramyxovirus. In some embodiments, the oncolytic virus is a measles virus. In some embodiments, the oncolytic virus is a reovirus. In some embodiments, the oncolytic virus is a rhabdovirus. In some embodiments, the oncolytic virus is a vesicular stomatitis virus. In some embodiments, the oncolytic virus is a senecavirus.

[0079] In some embodiments, the adenovirus or a fragment thereof for use according to the invention potentiates the replication of the oncolytic virus. As used herein, “potentiates the replication” means that the oncolytic virus replicates 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6- fold, 7-fold, 8-fold, 9-fold, 10-fold faster, or more. In some embodiments, the adenovirus or a fragment thereof for use according to the invention promotes the late phase replication of the oncolytic virus.

[0080] It is to be understood that the adenovirus or a fragment thereof for use according to the invention acts as an adjuvant for the oncolytic virus.

[0081] In another embodiment, the anticancer agent is a commercially available anticancer medicament, e.g., a pharmacological agent, an antibody or fragment thereof, a nucleic acid for gene therapy or vector comprising thereof, and the like.

[0082] Commercially available anticancer medicament are known from the state of the art include, e.g., acalabrutinib, alectinib, alemtuzumab, anastrozole, avapritinib, avelumab, belinostat, bevacizumab, bleomycin, blinatumomab, bosutinib, brigatinib, carboplatin, carmustine, cetuximab, chlorambucil, cisplatin copanlisib, cytarabine, daunorubicin, decitabine, dexamethasone, docetaxel, doxorubicin, encorafenib, erdafitinib, etoposide, everolimus, exemestane, fludarabine, 5-fluorouracil, gemcitabine, ifosfamide, imatinib Mesylate, leuprolide, lomustine, mechlorethamine, melphalan, methotrexate, mitomycin, nelarabine, paclitaxel, pamidronate, panobinostat, pralatrexate, prednisolone, ofatumumab, rituximab, temozolomide, topotecan, tositumomab, trastuzumab, vandetanib, vincristine, vorinostat, zanubrutinib, and the likes.

[0083] In some embodiments, the adenovirus or a fragment thereof and/or the pharmaceutical composition is for use in a method for the treatment of cancer.

[0084] As used herein, the term “cancer" is intended to refer to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. Within the scope of the invention, the terms “cancer” and “cancerous” are intended to refer to, or to describe, the physiological condition in mammals that is typically characterized by unregulated cell growth or proliferation.

[0085] Non-limitative examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include, without limitation, breast cancer, prostate cancer, colon cancer, squamous cell cancer, lung cancer (including small-cell lung cancer and non-small cell lung cancer), gastrointestinal cancer, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, vulvar cancer, thyroid cancer, lymphoma, hepatic carcinoma and various types of head and neck cancer.

[0086] In some embodiments, the cancer is selected from the group comprising or consisting of gastric cancer, gastrointestinal cancer, lung cancer, cervical cancer, colorectal cancer, colon cancer, breast cancer, prostate cancer, melanoma, liver cancer, hepatic carcinoma, pancreatic cancer, kidney cancer, ovarian cancer, blood cancer, lymphoma, leukemia, thyroid cancer, squamous cell cancer, brain cancer, glioblastoma, endometrial carcinoma, salivary gland carcinoma, vulvar cancer, and bladder cancer. [0087] In some embodiments, the cancer is selected from the group comprising or consisting of gastric cancer, lung cancer, cervical cancer, colorectal cancer, breast cancer, prostate cancer, melanoma, liver cancer, pancreatic cancer, kidney cancer, ovarian cancer, lymphoma, leukemia, thyroid cancer and bladder cancer.

[0088] In some embodiment, the cancer cells have an increased expression of A3B and/or an increased A3B activity compared to healthy cells.

[0089] In some embodiment, the cancer cells have an increased expression of A3B compared to healthy cells. As used herein, “increased expression of A3B” means that the expression of A3B in cancer cells is increased 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6- fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, 100-fold, 500-fold, 1,000-fold or more compared to healthy cells.

[0090] In some embodiment, the cancer cells have an increased A3B activity compared to healthy cells. As used herein, “increased A3B activity” means that the enzymatic activity of A3B in cancer cells is increased 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10- fold, 100-fold, 500-fold, 1,000-fold or more compared to healthy cells.

[0091] In some embodiments, the adenovirus or fragment thereof for use according to the invention, or the pharmaceutical composition for use according to the invention is to be administered to an individual in need thereof by any suitable route, /.< ., by a dermal administration, by an oral administration, a topical administration or a parenteral administration, e.g., by injection, including a subcutaneous administration, a venous administration, an arterial administration, in intramuscular administration, an intraocular administration, an intracardiac administration and an intra-auricular administration.

[0092] In some embodiments, the adenovirus or fragment thereof for use according to the invention, or the pharmaceutical composition for use according to the invention is to be administered to an individual in need thereof by intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, intraocular or intracardiac injection. [0093] In some embodiments, the adenovirus or fragment thereof for use according to the invention, or the pharmaceutical composition for use according to the invention is to be administered to an individual in need thereof by a dermal administration. In some embodiments, the adenovirus or fragment thereof for use according to the invention, or the pharmaceutical composition for use according to the invention is to be administered to an individual in need thereof by an oral administration.

[0094] In some embodiments, the adenovirus or fragment thereof for use according to the invention is administered prior, concomitantly or after the anticancer agent. In some embodiments, the adenovirus or fragment thereof for use according to the invention is administered prior to the anticancer agent, preferably between about 1 month and about 1 minute prior to the anticancer agent. In some embodiments, the adenovirus or fragment thereof for use according to the invention is administered concomitantly with the anticancer agent. In some embodiments, the adenovirus or fragment thereof for use according to the invention is administered after the anticancer agent, preferably between about 1 minute and about 1 month after the anticancer agent.

[0095] In some embodiments, the adenovirus or fragment thereof for use according to the invention, or the pharmaceutical composition for use according to the invention, is to be administered in one or multiple administrations. In some embodiments, the adenovirus or fragment thereof for use or the pharmaceutical composition for use according to the invention is to be administered in 1, 2, 3, 4, 5, 6, 7, 8, 9 administrations, ore more. In certain embodiments, the adenovirus or fragment thereof for use or the pharmaceutical composition for use according to the invention is to be administered until the cancer is treated or cured.

[0096] In some embodiments, the adenovirus or fragment thereof for use or the pharmaceutical composition for use according to the invention is to be administered in a therapeutically effective amount.

[0097] Within the scope of the instant invention, the therapeutically effective amount of the adenovirus or fragment thereof for use or the pharmaceutical composition for use according to the invention, to be administered may be determined by a physician or an authorized person skilled in the art and can be suitably adapted within the time course of the treatment.

[0098] In certain embodiments, the therapeutically effective amount to be administered may depend upon a variety of parameters, including the material selected for administration, whether the administration is in single or multiple doses, and the individual’s parameters including age, physical conditions, size, weight, gender, and the severity of the age-related disease to be treated.

[0099] In some embodiments, the adenovirus or fragment thereof for use or the pharmaceutical composition for use according to the invention is administered at a dose from about 10 ³ to about IO ²⁰ pfu. The term pfu ("plaque forming unit") corresponds to the infectivity of a virus solution, and is determined by infection of a suitable cell culture, and measurement, generally after 15 days, the number of areas of infected cells. The techniques for determining the pfu titer of a viral solution are well documented in the art.

[0100] In some embodiments, the adenovirus or fragment thereof for use or the pharmaceutical composition for use according to the invention is administered at a dose from about 10 ⁴ to about IO ²⁰ pfu, from about 10 ⁵ to about IO ²⁰ pfu, from about 10 ⁶ to about IO ²⁰ pfu, from about 10 ⁷ to about IO ²⁰ pfu, from about 10 ⁸ to about IO ²⁰ pfu, from about 10 ⁹ to about IO ²⁰ pfu, from about 10 ¹⁰ to about IO ²⁰ pfu, from about 10 ¹¹ to about IO ²⁰ pfu, from about 10 ¹² to about IO ²⁰ pfu, from about 10 ¹³ to about IO ²⁰ pfu, from about 10 ¹⁴ to about IO ²⁰ pfu, from about 10 ¹⁵ to about IO ²⁰ pfu, from about 10 ¹⁶ to about IO ²⁰ pfu, from about 10 ¹⁷ to about IO ²⁰ pfu, from about 10 ¹⁸ to about IO ²⁰ pfu, from about 10 ¹⁹ to about IO ²⁰ pfu. In some embodiments, the adenovirus or fragment thereof for use or the pharmaceutical composition for use according to the invention is administered at a dose from about 10 ³ to about 10 ¹⁹ pfu, from about 10 ³ to about 10 ¹⁸ pfu, from about 10 ³ to about 10 ¹⁷ pfu, from about 10 ³ to about 10 ¹⁶ pfu, from about 10 ³ to about 10 ¹⁵ pfu, from about 10 ³ to about 10 ¹⁴ pfu, from about 10 ³ to about 10 ¹³ pfu, from about 10 ³ to about 10 ¹² pfu, from about 10 ³ to about 10 ¹¹ pfu, from about 10 ³ to about 10 ¹⁰ pfu, from about 10 ³ to about 10 ⁹ pfu, from about 10 ³ to about 10 ⁸ pfu, from about 10 ³ to about 10 ⁷ pfu, from about 10 ³ to about 10 ⁶ pfu, from about 10 ³ to about 10 ⁵ pfu, from about 10 ³ to about 10 ⁴ pfu. [0101] In some embodiments, the adenovirus or fragment thereof for use or the pharmaceutical composition for use according to the invention is administered at a dose from about 10 ³ to about IO ²⁰ viral particles.

[0102] In some embodiments, the adenovirus or fragment thereof for use or the pharmaceutical composition for use according to the invention is administered at a dose from about 10 ⁴ to about IO ²⁰ viral particles, from about 10 ⁵ to about IO ²⁰ viral particles, from about 10 ⁶ to about IO ²⁰ viral particles, from about 10 ⁷ to about IO ²⁰ viral particles, from about 10 ⁸ to about IO ²⁰ viral particles, from about 10 ⁹ to about IO ²⁰ viral particles, from about 10 ¹⁰ to about IO ²⁰ viral particles, from about 10 ¹¹ to about IO ²⁰ viral particles, from about 10 ¹² to about IO ²⁰ viral particles, from about 10 ¹³ to about IO ²⁰ viral particles, from about 10 ¹⁴ to about IO ²⁰ viral particles, from about 10 ¹⁵ to about IO ²⁰ viral particles, from about 10 ¹⁶ to about IO ²⁰ viral particles, from about 10 ¹⁷ to about IO ²⁰ viral particles, from about 10 ¹⁸ to about IO ²⁰ viral particles, from about 10 ¹⁹ to about IO ²⁰ viral particles. In some embodiments, the adenovirus or fragment thereof for use or the pharmaceutical composition for use according to the invention is administered at a dose from about 10 ³ to about 10 ¹⁹ viral particles, from about 10 ³ to about 10 ¹⁸ viral particles, from about 10 ³ to about 10 ¹⁷ viral particles, from about 10 ³ to about 10 ¹⁶ viral particles, from about 10 ³ to about 10 ¹⁵ viral particles, from about 10 ³ to about 10 ¹⁴ viral particles, from about 10 ³ to about 10 ¹³ viral particles, from about 10 ³ to about 10 ¹² viral particles, from about 10 ³ to about 10 ¹¹ viral particles, from about 10 ³ to about 10 ¹⁰ viral particles, from about 10 ³ to about 10 ⁹ viral particles, from about 10 ³ to about 10 ⁸ viral particles, from about 10 ³ to about 10 ⁷ viral particles, from about 10 ³ to about 10 ⁶ viral particles, from about 10 ³ to about 10 ⁵ viral particles, from about 10 ³ to about 10 ⁴ viral particles.

[0103] In some embodiments, the adenovirus or fragment thereof for use or the pharmaceutical composition for use according to the invention is administered at a dose from about 0.001 mg to about 10,000 mg.

[0104] In some embodiments, the adenovirus or fragment thereof for use or the pharmaceutical composition for use according to the invention is administered at a dose from about 0.01 mg to about 10,000 mg, from about 0.1 mg to about 10,000 mg, from about 1 mg to about 10,000 mg, from about 10 mg to about 10,000 mg, from about 100 mg to about 10,000 mg, from about 1,000 mg to about 10,000 mg. In some embodiments, the adenovirus or fragment thereof for use or the pharmaceutical composition for use according to the invention is administered at a dose from about 0.001 mg to about 1,000 mg, from about 0.001 mg to about 100 mg, from about 0.001 mg to about 10 mg, from about 0.001 mg to about 1 mg, from about 0.001 mg to about 0.1 mg, from about 0.001 mg to about 0.01 mg.

[0105] The present invention further relates to the use of an adenovirus or a fragment thereof, or a pharmaceutical composition comprising the same, for the manufacture of an adjuvant for the treatment of cancer.

[0106] In some embodiments, the adjuvant comprises a therapeutically effective amount of the adenovirus or a fragment thereof, or of the pharmaceutical composition, as described hereinabove.

[0107] In some embodiments, the adjuvant is a medicament for the treatment of cancer.

[0108] The present invention further relates to a combination kit comprising (i) the adenovirus or fragment according to the invention, the pharmaceutical composition according to the invention, or the adjuvant according to the invention, and (ii) means for administering the first part, for use for treating cancer in a subject in need thereof.

[0109] In some embodiments, the combination kit further comprises an anticancer agent. In some embodiments, the combination kit further comprises means for administering the anticancer agent.

[0110] The present invention further relates to a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of an adenovirus or fragment thereof as an adjuvant, or a pharmaceutical composition comprising the same.

[0111] In one embodiment, the method further comprises the administration of an anticancer agent as an active ingredient. In certain embodiments, the anticancer adjuvant according to the invention and the anticancer agent are comprised in the same composition. [0112] In another embodiment, the anticancer adjuvant according to the invention and the anticancer agent are comprised in different compositions.

[0113] In some embodiments, the adenovirus or fragment thereof is of a subtype selected from the group comprising or consisting of A12, Al 8, A31, B3, B7, Bl 1, B14, B16, B21, B34, B35, B50, B55, Cl, C2, C5, C6 and C57. In a preferred embodiment, the adenovirus or fragment thereof is of a subtype selected from the group comprising or consisting of A12, B3 and C2. In a more preferred embodiment, the adenovirus or fragment thereof is of a subtype selected from the group comprising or consisting of B3 and C2.In an even more preferred embodiment, the adenovirus or fragment thereof is of subtype B3. In some embodiments, the adenovirus or fragment thereof is a modified adenovirus.

[0114] In some embodiments, the adenovirus or fragment thereof does not encode for a cytokine. In some embodiments, the adenovirus or fragment thereof does not encode for degradation factor for the extracellular matrix. In some embodiments, the adenovirus or fragment thereof does not encode for any adjuvant. In some embodiments, the adenovirus or fragment thereof does not encode for an active agent. In some embodiments, the adenovirus or fragment thereof does not encode for an immunomodulatory factor. In some embodiments, the adenovirus or fragment thereof does not encode for a therapeutic molecule. In a certain embodiment, the adenovirus or fragment thereof does not encode for a protein. In a preferred embodiment, the adenovirus or fragment thereof is an empty vector.

[0115] In some embodiments, the adenovirus or fragment thereof, or the pharmaceutical composition comprising the same, is to be administered by any suitable route, /.< ., by a dermal administration, by an oral administration, a topical administration or a parenteral administration, e.g., by injection, including a subcutaneous administration, a venous administration, an arterial administration, in intramuscular administration, an intraocular administration, an intracardiac administration and an intra-auricular administration.

[0116] In some embodiments, the adenovirus or fragment thereof, or the pharmaceutical composition comprising the same, is to be administered at a dose from about 10 ³ to about IO ²⁰ pfu. In some embodiments, the adenovirus or fragment thereof, or the pharmaceutical composition comprising the same, is to be administered at a dose from about 10 ³ to about IO ²⁰ viral particles. In some embodiments, the adenovirus or fragment thereof, or the pharmaceutical composition comprising the same, is to be administered at a dose from about 0.001 mg to about 10,000 mg.

[0117] The present invention further relates to a method for inhibiting the protein APOBEC3B (A3B) in a subject in need thereof, comprising administering to the subject an effective amount of an adenovirus or fragment thereof, or a composition comprising the same.

[0118] As used herein, “inhibiting the protein A3B” means that the activity of A3B is decreased 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1,000-fold or more.

[0119] In a preferred embodiment, the method is a method for inhibiting the cytidine deaminase activity of A3B.

[0120] In some embodiments, the decrease of activity is caused by a decrease of the quantity of A3B protein.

[0121] The present invention further relates to an in vitro method for inhibiting the protein APOBEC3B (A3B) in a population of cells, comprising administering to the population of cells an effective amount of an adenovirus or fragment thereof or composition comprising the same.

BRIEF DESCRIPTION OF THE DRAWINGS

[0122] Figure 1A-1C is a combination of photographs and histograms showing the establishment of bronchial epithelial cells over-expressing A3B. Immortalized human bronchial epithelial cells HBEC3-KT were transduced to stably express the GFP, the APOBEC3B protein (A3B) or a catalytically inactive version of the APOBEC3B protein (A3B-DD for Deaminase Dead). (Fig. 1A) A3B mRNA levels were quantified by RTqPCR and expressed relative to the amount produced by wild type HBEC3-KT cells (WT). (Fig. IB) A3B protein levels were assessed by western blot, quantified by densitometry and expressed relative to the amount produced by WT cells. (Fig. 1C) Total proteins were extracted in a non-denaturing buffer and mixed with the substrate of the deamination assay. Deaminase activity, if present in the cell lysate, will allow the conversion of substrates into shorter products. Substrates and products were quantified by densitometry. Deamination activities of the cell lysates were expressed as the percentage of substrate converted into product. P-values were calculated by t-tests.

[0123] Figure 2A-2C is a set of histograms showing that A3B restricts adenoviruses by a deaminase-dependent mechanism. HBEC-WT, -GFP, -A3B and -A3B-DD were infected with HAdV-A12, -B3 or -C2 at a MOI=0.03 and analyzed at 6- and 24-hours post infection (6 and 24 hpi) and at 2-, 4- and 7-days post infection (2, 4 and 7 dpi). (Fig. 2A) Intracellular viral DNA levels were quantified by qPCR and expressed relative to the levels measured in WT cells. (Fig. 2B) Viral DNA levels in the supernatant were quantified by qPCR and expressed relative to the levels measured in the supernatant of WT cells. (Fig. 2C) The percentage of infected cells were quantified by flow cytometry and expressed relative to the percentages measured in WT cells.

[0124] Figure 3A-3C is a set of histograms showing that A3B reduces viral load, progression to late phase and production of infectious viral particles. HBEC-WT, -GFP, -A3B and -A3B-DD were infected with HAdV-A12, -B3 or -C2 at a MOI=3 and analyzed 24 hours post infection. (Fig. 3A) The levels of different viral mRNAs were quantified by RTqPCR and expressed relative to their amounts measured in WT cells. An immediate early transcript (E1A), an early transcript (E4orf6) and a late transcript (Penton) were quantified. (Fig. 3B) The levels of viral DNA were quantified by qPCR and expressed relative to the amount measured in WT cells. (Fig. 3C) The levels of infectious viral particles were quantified by fluorescent forming unit assay and expressed relative to the production of WT cells. P-values were calculated by t-tests.

[0125] Figure 4A-4E is a combination of photograph, schemes and histograms showing that A3B hypermutates the adenovirus genomes. HBEC-WT, -GFP, -A3B and -A3B-DD were infected with HAdV-A12, -B3 or -C2 and the intracellular DNAs were extracted 48 hours post infection. 3DPCR reactions were conducted on the E1B, L3 and E4 genes. (Fig. 4A) Agarose gels illustrating 3DPCRs specific for the E4 gene of the A12 strain. The dotted white line indicates the threshold between mutated and unmutated 3DPCR products. Low denaturation temperature amplicons were recovered in HBEC-A3B cells only. (Fig. 4B) Graphical representation of the 3DPCR results obtained for the 3 viral genes (E1B, L3 and E4), for the 3 viral strains (A12, B3 and C2) and for the 4 cell lines (WT, GFP, A3B and A3B-DD). The lowest denaturation temperatures allowing the production of the expected amplicons were represented on the gradients by colored circles. (Fig. 4C) 3DPCR products generated from HAdV-A12-infected WT and A3B- expressing cells were cloned and sequenced. Hypermutated sequences detected in A3B- expressing cells (A3B) were aligned against the reference viral genome (Ref) and against sequences isolated from WT infected cells (WT). Deamination occurred on the coding (C to T mutations) or the template strand (G to A mutations of the coding strand). The mutations highlighted in grey took place within an A3 -favored motif with a T in 5’ of the deaminated C. (Fig. 4D) Mutation types recorded on the E1B, L3 and E4 hyper-mutated sequences. The numbers in brackets indicate the number of bases sequenced. (Fig. 4E) 5’ nucleotide contexts of the deaminated Cs recorded on the E1B, L3 and E4 hypermutated sequences were reported by dark bars. 5’ nucleotide context expected values, based on the dinucleotide composition of the DNA sequences were represented by white bars. P-values were calculated by %2 -tests.

[0126] Figure 5A-5C is a combination of photographs and histograms showing the establishment of A3B knockout lung cancer cells. A549 lung cancer cells were transduced to stably express APOBEC3B-targeting shRNAs (shA3B) or a nontargeting control shRNA (scramble). (Fig. 5A) A3B mRNA levels were quantified by RTqPCR and expressed relative to the amount produced by wild type A549 cells (WT). (Fig. 5B) A3B protein levels were assessed by western blot, quantified by densitometry and expressed relative to the amount produced by WT cells. (Fig. 5C) Deamination activities of the cell lysates were expressed as the percentage of substrate converted into product and compared to the deamination activity of the WT cells. P-values were calculated by t-tests.

[0127] Figure 6A-6C is a combination of photograph, schemes and histograms showing that A3B knockout promotes replication of strain A12 and reduces the presence of hyper- mutated viral genomes. A549-WT, -scramble and -shA3B were infected with HAdV- A12, -B3 or -C2 at a MOI=0.03 and analyzed at 6- and 24-hours post infection (6 and 24 hpi) and at 2-, 4- and 7-days post infection (2, 4 and 7 dpi). (Fig. 6A) Intracellular viral DNA levels were quantified by qPCR and expressed relative to the levels measured in WT cells. (Fig. 6B) Viral DNA levels in the supernatant were quantified by qPCR and expressed relative to the levels measured in the supernatant of WT cells. (Fig. 6C) The percentage of infected cells were quantified by flow cytometry and expressed relative to the percentages measured in WT cells. P-values were calculated by t-tests.

[0128] Figure 7A-7B is a combination of photograph, schemes showing that hypermutated viruses are not detected in A3B-KO-infected cells. 3DPCR reactions were conducted on the E1B, L3 and E4 genes. (Fig. 7A) Agarose gels illustrating 3DPCRs specific for the L3 gene of the A12 strain. The dotted white line indicates the threshold between mutated and unmutated 3DPCR products. (Fig. 7B) Graphical representation of the 3DPCR results obtained for the 3 viral genes (E1B, L3 and E4), for the 3 viral strains (A12, B3 and C2) and for the 3 cell lines (WT, scramble and shA3B). The lowest denaturation temperatures allowing the production of the expected amplicons were represented on the gradients.

[0129] Figure 8A-8E is a combination of photographs and histograms showing that adenoviruses antagonize A3B with varying efficiencies between strains. A549 cells were infected with HAdV-A12, -B3, -C2 or mock control at a MOI=3 (Fig. 8A) A3B protein levels were assessed by western blot at 1- and 2-days post infection, quantified by densitometry and expressed relative to the amount produced by mock-infected cells. (Fig. 8B) A3B mRNA levels were quantified by RTqPCR at 1- and 2-days post infection and expressed relative to GAPDH mRNA. (Fig. 8C) Deamination activities of the cell lysates were expressed as the percentage of substrate converted into product and compared to the deamination activities of the mock-infected cells at 1- and 2-days post infection. (Fig. 8D) The viral replication centers as identified by EdU labelling and the A3B protein were imaged by fluorescence microscopy. (Fig. 8E) EdU and A3B fluorescence intensities were reported in cross-sections for colocalization analysis. P-values were calculated by t- tests. [0130] Figure 9 is a graph showing that adenoviruses of a given species share a similar A3 footprint. The NTC codon observed/expected ratios were calculated in 70 different adenoviruses spanning the 7 species.

EXAMPLES

[0131] The present invention is further illustrated by the following examples.

Example 1 :

Materials and Methods

Cell lines

[0132] HBEC3-KT cells are normal human bronchial epithelial cells immortalized with the human TERT and mouse Cdk4 genes. HBEC3-KT cells were obtained from UT Southwestern, Dallas, Texas, USA. HBEC3-KT were cultured in keratinocyte-SFM medium supplemented with bovine pituitary extract (50 pg/mL) and human recombinant epidermal growth factor (5 ng/mL) (Gibco) on gelatin-coated flasks. HBEC3-KT constitutively expressing GFP (HBEC-GFP), A3B (HBEC-A3B) or A3B Deaminase Dead (HBEC-A3B-DD) were established by transduction with lentiviral vectors encoding for a GFP, A3B or A3B-DD protein (pLenti4-A3B and pLenti4-A3B-E68Q-E255Q encoding respectively the catalytically active and inactive A3B). The human lung carcinoma cell line A549 (American Type Culture Collection) and PKR-deficient A549 cell line were cultured in Dulbecco’s Modified Eagle’s medium (Gibco) supplemented with 10% fetal calf serum (Gibco) and 10 mM of L-glutamine. PKR-deficient A549 cells were obtained from UTMB, Galveston, Texas, USA. A549 cells knock-down for A3B (A549 A3B shRNAs) were established by transduction with lentiviral vectors encoding A3B-targeting shRNAs (pSicoR-MS2 plasmids). A549 cells were also transduced with a lentiviral vector encoding non-targeting shRNA (A549 NTC shRNA) and used as control. Of note, the puromycin markers originally present in the pSicoR-M2 and pLenti4-A3B plasmids have been replaced by a blasticidin resistance gene prior A549 or HBEC3-KT transduction. Blasticidin was used to select for a polyclonal population of effectively transduced cells. All cell lines were incubated at 37°C and 5% CO2. Virus preparation procedure

[0133] Handling of human adenoviruses was done in a biosafety level 2 laboratory. Adenovirus A12 (HAdV-12, ATCC VR863) was purchased at the American Type Culture Collection. Adenoviruses B3 and -C2 were obtained from KULeuven, Leuven, Belgium and strain identity was verified by genotyping as published in (Lejeune et al., 2021) and using primers described in (Wang et al., 2013). HAdV-B3 and -C2 viral stocks were produced in A549 cells and HAdV-A12 viral stock in PKR-deficient A549 cells. During viral stock production, A549 cells and PKR-deficient A549 cells were cultured in OPTLMEM®! Reduced-Serum Medium without phenol red (ThermoFisher Scientific). When 80% of the cells showed cytopathic effects, cells were scraped, collected with the culture medium and centrifuged at 3,500g for 15 minutes. The supernatant was collected and set aside. The cell pellet was resuspended in 2 mL of culture medium, submitted to 3 freeze/thaw cycles and centrifuged at 3,500g for 15 minutes. The supernatant was collected, pooled with the previous supernatant and treated with the endonuclease Benzonase® (1 U/mL) (Sigma-Aldrich) at 37°C for 30 minutes to degrade the unpackaged nucleic acids. Supernatant was then filtered on a 0.22 pm Steriflip® Filters (Merck Millipore) to remove residual cell debris. Eluate was collected and further filtered through Amicon® Ultra- 15 Centrifugal Filter Unit 100 KDa (Merck Millipore). Virions were retained on the filter, resuspended in PBS and stored at -80°C. Titration of the viral stocks were done by fluorescent forming unit (FFU) assay and by qPCR quantification of genome copies. HBEC3-KT cells were infected with the HAdV-A12, -B3 or -C2 strain at a multiplicity of infection (MOI) of 0.03 or 3 in keratinocyte-SFM medium supplemented with bovine pituitary extract (50 pg/mL) and human recombinant epidermal growth factor (5 ng/mL) (Gibco) on gelatin-coated flasks. A549 cells were infected with the HAdV-A12, -B3 or -C2 strain at a multiplicity of infection (MOI) of 0.03 or 3 in Dulbecco’s Modified Eagle’s medium (Gibco) supplemented with 2% fetal calf serum (Gibco) and 10 mM of L-glutamine.

Viral and cellular mRNA quantification by RT-qPCR

[0134] One or two-days post infection, cells were collected, washed in PBS and resuspended in 1ml of TRIzol reagent (Invitrogen) for RNA extraction. Two hundred (200) [il of chloroform were added and samples were centrifugated at 4°C at 12 000g for 15 minutes. Five hundred (500) pL from the aqueous phase was collected and added to 450 pL of isopropanol. Samples were centrifuged at 12,000g for 15 minutes. Pellets were then washed into 900 pL of 75% ethanol, centrifuged at 12,000g for 10 minutes and resuspended into 30 pL of RNAse free water. cDNA was obtained by reverse transcription of 1 pg of RNA using iScript cDNA Synthesis Kit (Bio-rad) following manufacturer’ s instructions. The cDNA was subj ected to qPCR using TakyonTM No Rox SYBR 2X MasterMix blue dTTP (Eurogentec) and CFX96TM lOOOTouch Real-Time PCR System (Biorad). The primers used to titrate A3B were designed by Refsland and colleagues (Refsland et al., 2010).

[0135] The A3B, E1A, E4orf6 and penton levels were expressed relatively to the abundance of GAPDH. Primers used for mRNA quantification by RTqPCR are listed in Table 1.

[0136] Table 1: Primers for cellular and viral mRNAs quantification

Viral copies quantification by qPCR

[0137] DNA was extracted from infected cells and subjected to PCR using Nucleospin Tissue (Macherey Nagels) according manufacturer’s instructions. In parallel, supernatant containing virions was inactivated 15 minutes at 70°C and viral copies were directly quantified. A fragment of the penton gene was amplified using TakyonTM No Rox SYBR

2X MasterMix blue DTTP (Eurogentec) and CFX96TM lOOOTouch Real-Time PCR System (Biorad). Serial dilutions of plasmids containing the target sequences were used as calibration curves. Primers used for vDNA quantification by qPCR are listed in Table 2. [0138] Table 2: Primers for adenovirus DNA quantification

Infectious viral particles quantification by foci forming unit assay

[0139] A549 cells were infected with serial dilutions of HAdV stocks or supernatants containing virions in DMEM supplemented with 2% FCS. After twenty-four hours, cells were fixed with a mix of methanol/acetone 1 : 1 and incubated 5 min at -20°C. Cells were then washed once with PBS and permeabilized using the permeabilization buffer (Invitrogen) according manufacturer’s instructions. Cells were then incubated one hour at 4°C with anti -HAdV antibody (MAB 8051, Sigma- Aldrich) used at a dilution of 1 :200 in permeabilization buffer supplemented with 2% of goat serum (Gibco). After 2 washes in permeabilization buffer supplemented with goat serum, cells were incubated 1 hour with an anti-mouse AlexaFluor 568 (Invitrogen). Cells were washed twice with permeabilization buffer supplemented with 2% of goat serum and once in PBS. Finally, slides were mounted in Fluoromount G (Invitrogen) and imaged using Leica SP5 microscope. Infected cells were counted to calculate the number of foci forming unit (FFU) and expressed per pl of the starting infectious material.

Percentage of infected cells by flow cytometry

[0140] Two-, four- or seven-days post infection, cells were collected and washed in PBS.

Cells were fixed and permeabilized using the eBiosciencesTM Foxp3/Transcri ption Factor Staining Buffer set (InvitrogenTM) according manufacturer’s instructions. Cells were then incubated 30 minutes at 4°C with anti-HAdV antibody (MAB 8051, Merck Millipore) used at a dilution of 1 :200 in permeabilization buffer supplemented with 2% of goat serum (Gibco). After 2 washes in permeabilization buffer supplemented with goat serum, cells were incubated 30 minutes with an anti-mouse antibody coupled with phycoerythrin (Miltenyi Biotec). Cells were washed two times with permeabilization buffer supplemented with 2% of goat serum and then resuspended in PBS. Percentage of HAdV-infected cells was quantified using BD FACSVerse Flow Cytometer and BD FACSuite vl.0.6 software (BD Biosciences).

A3B protein detection by immunoblotting

[0141] One or two-days post infection, cells were collected and washed in PBS. Cells were resuspended in HED buffer (20mM HEPES pH 7.4, 5mM EDTA, ImM DTT, 10% glycerol) supplemented with cOmplete™ Protease Inhibitor Cocktail (Roche). Cells were then submitted to one freeze/thaw cycle and sonicated 15 cycles of 30 sec ON / 30 sec OFF using Bioruptor Pico device (Diagenode) at 4°C. Cell lysates were spun down at 14,000 rpm for 15 minutes to remove cell debris. Proteins were quantified using PierceTM BCA Protein Assay Kit (ThermoFisher Scientific). Twenty-five pg of proteins were loaded on a 10% SDS-PAGE gel and transferred actively to PVDF membrane (GE healthcare Life Sciences). Membrane was blocked in TBS (Tris Buffered Saline) supplemented with 0.1% Tween 20 and 5% bovine serum albumin (BSA). A3B protein was detected with anti-Human APOBEC3B monoclonal antibody (5210-87-13, cat# 12397, obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH) used at a dilution of 1 : 1000 in TBS supplemented with 0.1% Tween 20 and 5% BSA. Hsp90 was used as loading control and was detected with an anti-HSP90ABl antibody (Sigma-Aldrich) at a dilution of 1 : 1000 in TBS supplemented with 0.1% Tween 20 and 5% BSA. An HRP-coupled anti -rabbit IgG secondary antibody (Dako) was used at a dilution of 1 :2000 in TBS with 0.1% Tween 20 and 5% BSA or 4% milk. Membranes were incubated with the chemoluminescent SuperSignal TM West Femto Maximun Sensitivity Substrate (ThermoFisher Scientific) and chemoluminescence was read using an ImageQuant LAS4000 (GE Healthare Life Sciences). Densitometry analysis was done using Imaged software. Deamination assay

[0142] One or two-days post infection, cells were collected and washed in PBS. Cells were resuspended in HED buffer (20mM HEPES pH 7.4, 5mM EDTA, ImM DTT, 10% glycerol) supplemented with cOmpleteTM Protease Inhibitor Cocktail (Roche). Cells were then submitted to one freeze/thaw cycle and sonicated 15 cycles of 30 sec ON / 30 sec OFF using Bioruptor Pico device (Diagenode) at 4°C. Cell lysates were spun down at 14,000 rpm for 15 minutes to remove cell debris. Proteins were quantified using Pierce TM BCA Protein Assay Kit (ThermoFisher Scientific). Forty pg of proteins were incubated overnight at 37°C with 1 pmol of a fluorescent oligo substrate (5’- ATTATTATTATTCAAATGGATTTATTTATTTATTTATTTATTT-Cy5-3’), ImM ZnC12, 0.025U uracil DNA glycosylase (NEB), 2 pl lOx UDG buffer (NEB) and 100 pg/ml RNAse A (Thermofisher Scientific). Reaction mixture was treated with 50 mM NaOH and heated to 95°C for 10 min to cleaved DNA probes at the abasic site. Reaction mixture was then neutralized with 50 mM HC1 and mixed with 1.25x formamide buffer. Substrates (43 bases-long) from products (30 bases-long) were separated on a 15% tris- borate-EDTA (TBE)-urea gel. The Cy5-labeled substrates and deamination products were detected using ImageQuant LAS4000 mini (GE Healthcare Life Sciences). Densitometry analysis was done using ImageJ software.

Selective amplification of hypermutated viral sequences by 3DPCR

[0143] Total DNA from infected cells was extracted using the MasterPure complete DNA and RNA purification kit (Epicentre) and resuspended in 50 pl sterile water. All amplifications were performed using first-round standard PCR followed by nested 3DPCR. PCR was performed with 1U Taq DNA polymerase (Bioline) per reaction. After purification, PCR products were cloned into TOPO 2.1 vector (Life Technologies) and sequencing was outsourced to Eurofins Genomics. Primers used for classic PCR and 3DPCR are listed in Table 3. [0144] Table 3: Primers and cycling conditions for 3DPCR

Discrimination of the different A3B transcriptional isoforms by RT-PCR

[0145] Two-days post infection, cells were collected, washed in PBS and resuspended in 1 ml of TRIzol reagent (Invitrogen) for RNA extraction as described above. Samples were then treated with the TURBO DNA-free kit (Invitrogen) to remove DNA contaminants. cDNA was obtained by reverse transcription of 1 pg of mRNA using iScript cDNA Synthesis Kit (Bio-rad) following manufacturer’s instructions. cDNA was subjected to PCR using the Go Taq® G2 polymerase (Promega) using two sets of primers enabling discrimination of the four A3B transcriptional isoforms. Primers used for discriminations of the A3B transcriptional isoforms by RT-PCR are listed in Table 4. [0146] Table 4: Primers for the discrimination of the APOBEC3B mRNA isoforms

A3B and viral replication centers subcellular localization by immunofluorescence microscopy

[0147] Nineteen- or twenty-three-hours post-infection, A549 cells were treated with EdU (Click-iT™ Plus EdU Cell Proliferation Kit for Imaging, Invitrogen) at a final concentration of 10 pM according the manufacturer’s instructions. After one hour of incubation, cells were fixed with a mix of methanol/acetone 1 : 1 and incubated 5 min at - 20°C. Cells were then washed once with PBS and permeabilized using the permeabilization buffer (Invitrogen) according manufacturer’s instructions. Cells were then incubated overnight at 4°C with anti-Human APOBEC3B monoclonal antibody (5210-87-13, cat# 12397) used at a dilution of 1 :250 in permeabilization buffer supplemented with 2% of goat serum (Gibco). After 2 washes in permeabilization buffer supplemented with goat serum, cells were incubated 1 hour with an anti-rabbit AlexaFluor 488 (Invitrogen). Cells were washed twice with permeabilization buffer supplemented with 2% of goat serum. EdU labelling was then performed according manufacturer’s instructions. Cells were washed twice with permeabilization buffer supplemented with 2% of goat serum and once in PBS. Finally, slides were mounted in Fluoromount G (Invitrogen) and imaged using Zeiss LSM 900 Airyscan 2 Multiplex microscope. Images were then processed using Fiji software. APOBEC3 evolutionary footprint by bioinformatic analysis

[0148] Complete HAdV genomes were downloaded from “NCBI Nucleotides” database. Calculation of the NTC ratio was done as described in (Poulain et al., 2020). Briefly, the NTC ratio is given as the log2 ratio of the observed occurrence of the NTC codon to its expected occurrence. The NTC codon includes the ATC, CTC, GTC and TTC codons. To calculate the expected occurrence of the NTC codon, each coding sequence has been randomly shuffled a thousand times, retaining only the nucleotide composition. The expected count of NTC codons is calculated as the average of the occurrences of this codon over the thousand iterations. To calculate the NTC ratio for an entire viral genome, a “synthetic coding genome” was generated by concatenating the different coding sequences. The synthetic coding sequence is then randomly shuffled a thousand times and NTC ratio calculated as above. A NTC ratio less than zero indicates NTC under representation and a NTC ratio equal to zero means that no representation bias is observed.

Statistics and data representation

[0149] Error bars show standard deviations. P-values were calculated according to the indicated test. ND for stands for Not Detected, NS for Not Significant, * p< 0.05, ** p< 0.01, *** p< 0.001. The experiments were replicated at least three times and representative images are shown.

Results

A3B limits the propagation of adenoviruses by a deaminase -dependent mechanism

[0150] The human epithelial bronchial cells (HBEC3-KT, thereafter named HBEC-WT) were transduced by lentiviral vectors in order to induce a constitutive expression of A3B. Three different cell lines were established: the HBEC-A3B cells (expressing an enzymatically active A3B protein), the HBEC-A3B-DD cells (producing a Deaminase Dead A3B mutant) and the HBEC-GFP cells as control. The levels of A3B mRNA in the HBEC-A3B and HBEC-A3B-DD cells are increased by more than 100 folds compared to WT, whereas GFP expression does not impact A3B mRNA level (Figure 1A). The HBEC-A3B and A3B-DD express high and comparable levels of A3B protein (Figure

IB) but the deaminase activity can only be evidenced in the HBEC-A3B cells (Figure

IC)

[0151] HBEC-WT, HBEC-A3B, HBEC-A3B-DD and HBEC-GFP were infected with the different adenoviral strains A12, B3 and C2 at a low MOI (0.03) and monitor viral replication in the 4 cell lines. Figure 2A shows the viral DNA load detected in the cells by qPCR at different time-points post-infection and expressed relative to the viral load measured in the WT infected cells. The viral load is decreased in the HBEC-A3B cells but not in the GFP nor in the A3B-DD cells. The magnitude of the effect is stronger in the case of the HBEC-A3B cells infected with the A12 strain compared to the cells infected with the B3 or C2 strains. The decrease of the intracellular viral load appears earlier and is more prolonged in the case of the A12 strain. Figure 2B shows the amount of viral DNA in the supernatant measured by qPCR and expressed relative to the WT infected cells. This amount is decreased in the conditions where the HBEC-A3B cells were infected with the A12 strain. The decrease can also be observed for the B3 and C2 albeit with a much lower magnitude. Finally, Figure 2C shows the percentage of infected cells measured by flow cytometry and relative to the WT infected cells. A reduction of the percentage of infected cells was observed in the HBEC-A3B but not in the HBEC- GFP or HBEC expressing the deaminase dead A3B. Once again, the magnitude of the effect appears stronger for the A12 strain.

[0152] In conclusion, A3B restricts the replication of these different adenoviral strains, the deaminase activity of the protein is required, and the magnitude of the effect is influenced by the strain of the virus, some being more sensitive to the A3B-mediated restriction than others.

A3B reduces viral load, progression to late phase, and production of infectious viral particles

[0153] To better characterize the mechanism of A3B restriction, the HBEC-WT, -GFP, -A3B and -A3B-DD were infected with the 3 strains at a high MOI (MOI=3) to get a synchronous infection. Twenty-four hours post infection, the expression of immediate early, early and late transcripts was measured by RTqPCR, the replication of the viral DNA by qPCR and the production of infectious viral particles by foci forming assays. The expression of the immediate early gene E1A is not impacted in any conditions (Figure 3A), indicating that the virus enters the cell and starts its replication program regardless of the A3B status of the infected cell. A decrease in the abundance of the early transcript E4orf6 was observed in the HBEC-A3B cells infected with the A12 strain compared to WT infected cells (Figure 3B). This decrease can also be observed for the abundance of the penton transcripts for the 3 strains albeit being only significant for the A12 (Figure 3A). Figure 3B shows that viral DNA load is lower in cells expressing the enzymatically active A3B protein in the A12- and B3-infected conditions. Finally, virions have been isolated for each condition and their infectivity has been measured by foci forming unit assay. Figure 3C shows that the A12- and B3-infected cells expressing the enzymatically active A3B protein produce a lower amount of infectious viral particles than their WT counterpart. The lower production of infectious viral particles in the HBEC-A3B cells (Figure 3C) is certainly explained by the lower viral DNA load (Figure 3B)

[0154] In conclusion, A3B does not impact the entry of the virus, nor the expression of immediate early genes. Rather, A3B decreases viral DNA replication and delays the progression into the late phase resulting in a lower production of infectious viral particles.

APOBEC3B hyper-mutates the viral genome of the Al 2, B3 and C2 strains

[0155] The above results show that A3B restricts adenovirus replication in a deaminasedependent way and via the reduction of the viral genome replication. It was therefore questioned whether A3B hypermutates the viral DNA. HBEC-WT, -GFP, -A3B and - A3B-DD were infected with HAdV-A12, -B3 or -C2 and the intracellular DNAs were extracted 48 hours post infection. The selective amplification of the hyper-mutated viral sequences has been done by 3DPCR (Differential DNA Denaturation). It was previously reported that adenoviruses bear an A3 evolutionary footprint (Lejeune et al., 2021; Poulain et al., 2020). Importantly, from in-silico studies revealed that the A3 footprint appears stronger at the ends of the virus compared to the center of the viral genome (Poulain et al., 2020). Thus, 3DPCR reactions were designed on 3 different sections of the viral genome: at the ends (E1B and E4 genes) and in the middle (L3 gene) of the linear genome.

[0156] Figure 4A illustrates the E4-specific 3DPCR amplicons obtained during HAdV- A12 infection of WT-, GFP-, A3B- and A3B-DD-expressing cells. Low denaturation temperature amplicons were observed (Figure 4A) in the 3DPCR reactions done on the DNA extracted from A3B-expressing cells but not from the other cell lines. Figure 4B reports the 3DPCR results for the 3 viral genes (E1B, L3 and E4), the 3 viral strains (A12, B3 and C2) and the 4 cell lines (WT, GFP, A3B and A3B-DD). Low denaturation temperature amplicons were observed in A3B-expressing cells but not in the cells expressing the catalytically inactive form of A3B, nor in the WT or GFP cells. Importantly, low denaturation temperature amplicons were observed in A3B-expressing cells for the 3 strains. Amplicons obtained during HAdV-A12 infection of WT and A3B- expressing cells were cloned and sequenced. Figure 4C reports representative sequences for the E1B, L3 and E4 genes. Amplicons from WT cells were recovered at a denaturation temperature of 87°C, 82.8°C and 82.2°C for respectively the E1B, L3 and E4 3DPCR reactions. The WT sequences were not different from the reference genome. Amplicons from A3B-expressing cells were recovered at a denaturation temperature of 86, 82.2 and 81.3°C for respectively the E1B, L3 and E4 3DPCR reactions. Two different and representative sequences were reported for each gene. Deamination events were observed in both the coding (C to T mutations) and template strand (C to T mutations in the template strand resulting in G to A mutations in the coding strand) viral sequences. As expected, when a hypermutated sequence is recovered, the mutations are almost exclusively either C to T (mutations of the coding strand) or G to A (C to T mutations of the template strand). Figure 4D shows that the mutations recorded in the hyper-mutated clones are almost exclusively C to T or G to A (making more than 97% of the mutations). Lastly, the nucleotide context upstream the mutated C was investigated. Figure 4E shows an enrichment for a T in 5’ of the mutated C, both in the E1B, L3 and E4 hyper-mutated sequences. This context is typical for the A3 proteins (with the sole exception of A3G that favors deamination of a C following another C). [0157] Overall, these results demonstrate that A3B deaminates the viral genome of the three strains, on both strands and throughout the whole genome. Even though hypermutated viral sequences were retrieved for the three strains, the 3DPCR does not allow their quantification. Because the replication of the A12 strain is more impacted, it can be speculated that the proportion of hypermutated viruses is higher during infection with the A12 compared to the other two strains. It might be asked whether some low A3B activity might be benefit for the virus in vivo, like it is the case for HIV or BK Py V, to fuel genetic diversity and promote the emergence of drug- or antibody-resistant variants. One might also wonder whether the A3B protein expressed during HAdV infection could accidentally mutate the cellular genome and perhaps promote cell transformation. Indeed, mutations attributed to A3 A and/or A3B activities are found in many cancer types but the mechanisms leading to the A3A/A3B upregulations are still incompletely understood.

A3B knockout promotes replication of strain Al 2

[0158] It was next investigated whether knockdown of A3B could facilitate adenovirus replication. The A549 lung cancer cell line that constitutively expresses A3B was used (Figure 6B). The A549 cells were transduced with lentiviral vectors expressing shRNAs against A3B (shA3B) or scramble control (scramble). Figure 5A shows that A549-shA3B produces 26 times less mRNA that the WT or scramble control. Figure 5B shows that the A549-shA3B barely express the A3B protein and Figure 5C shows that A549-shA3B does not display deaminase activity on the contrary to the WT or scramble controls. Similarly with the previous experiment in HBEC3-KT cells, the A549-WT, -scramble and -shA3B were infected with the 3 HAdVs strains at a low MOI (0.03) and the viral replication was monitored during several days post infection. Figure 6A shows the viral DNA load detected in the cells by qPCR at different time-points post-infection and relative to the load measured in the WT infected cells. The viral load in the A549-shA3B cells infected with the A12 strain is increased at 2- and 4-days post infection. On the contrary, no difference was observed in the cells infected with the B3 nor with the C2 strain. Figure 6B shows the amount of viral DNA in the supernatant measured by qPCR and expressed relative to the amount present in the supernatant of WT infected cells. The viral load in the supernatant of the A549-shA3B cells infected with the A12 strain is increased at 4- and 7-days post infection. Once again, no difference was observed in the supernatant isolated from the cells infected with the B3 nor with the C2 strain. Finally, Figure 6C shows the percentage of infected cells measured by flow cytometry and relative to the WT conditions. The percentage of A12-infected cells is increased in the A549-shA3B cells at 4- and 7-days post infection whereas no difference was observed for the B3 and C2 strains.

[0159] To summarize, the downregulation of A3B speeds up the propagation of the A12 strain in cell culture, increasing its DNA replication and virions production. In other words, an endogenously-expressed A3B protein effectively restricts the propagation of the A12 strain. The replication of the two other strains, B3 and C2, is not impacted by the knockdown of the A3B gene.

Hyper-mutated viruses are not detected in A3B-KO-infected cells

[0160] It was then investigated whether the A3B knockdown would prevent the generation of hyper-mutated viruses. Figure 7A illustrates the L3-specific 3DPCR amplicons obtained during HAdV-A12 infection of WT, scramble, and shA3B cells. Low denaturation temperature amplicons were observed (Figure 7A) in the 3DPCR reactions done on the DNA extracted from WT and scramble cells but not from shA3B cells, indicating that A3B is necessary for the generation of hyper-mutated viruses. Figure 7B reports the 3DPCR results for the 3 viral genes (E1B, L3 and E4), the 3 viral strains (A12, B3 and C2) and the 3 cell lines (WT, scramble and shA3B). The dotted lines arbitrary separating mutated from unmutated amplicons were placed based on the results obtained on the HBEC-WT cells which do not constitutively express A3B (Figures IB and 4B). Amplicons from WT and scramble cells generally display a lower denaturation temperature than the amplicons generated in A3B KO cells. This demonstrates that the endogenously expressed A3B is responsible for the generation of hyper-mutated viruses.

The Al 2, B3 and C2 strains differently antagonize the A3B protein by reducing its quantity and/or its enzymatic activity

[0161] Albeit A3B hypermutates the genomes of the three strains, it impacts more strongly the replication of A12 strain than the B3 or the C2. To understand these differences, A3B was studied at the transcriptional and protein levels and its enzymatic activity and subcellular localization was measured in A549 cells infected with each of the three strains. A549 cells were infected at a high MOI (MOI=3).

[0162] Figure 8A shows that infection of A549 cells with the A12 strain does not modify the level of the A3B protein, whereas a significant decrease of the protein amount can be detected in the cells infected with the B3 and the C2 strains after 1- and 2-days post infection (lanes 3, 4, 7 and 8). Figure 8B shows that the levels of A3B mRNA transcripts were not impacted by the B3 nor the C2 infection, and a slight increase can be observed in the A12-infected cells at 2-days post infection as compared with mock-infected cells. Therefore, the decrease of the A3B protein observed in the B3- and to a lesser extent in the C2-infected cells is not due to transcriptional changes. Figure 8C shows that the deaminase activity of the cell lysates is significantly decreased for the 3 strains, the magnitude of the decrease being stronger in the B3- and C2-infected cells. The loss of deaminase activity of the cell lysates isolated from the B3- and C2-infected cells was expected as the amount of the A3B protein has been significantly reduced. Importantly, albeit the infection with the A12 did not significantly modify the total amount of the A3B protein, the deaminase activity in the A12 infected cells is being reduced (Figure 8C).

[0163] In conclusion, the three adenoviral strains antagonize the A3B protein by reducing its quantity and/or its enzymatic activity depending on the strain.

[0164] Finally, the subcellular distribution of A3B during the infection was studied (Figures 8D-8E). The A3B protein is distributed homogeneously in the nucleus of mock- infected cells. The A3B labelling is weak in C2- and ever weaker in B3-infected cells. On the contrary, the A3B protein forms dense clusters in the nucleus of A12-infected cells. Strikingly, these clusters are located at the center of the viral replication centers as labelled by the incorporation of EdU. Because adenoviruses replicate by strand displacement, the displaced strand represents the prototypical substrate for the editing by A3B. Adenoviruses of a given species share a similar A3 footprint

[0165] It was then investigated whether the ability to antagonize A3B is shared between strains of a given species. That question was tackled by looking at the genomic sequences of adenoviruses. It was previously reported the presence of an A3 evolutionary footprint on Human adenoviruses (Poulain et al., 2020), and that the A12 strain bears a stronger APOBEC3 footprint than the B3 and C2 strains (Lejeune et al., 2021). Next, it was asked whether the A12 was the sole strain showing such footprint or whether it is shared by other adenoviruses. As explained above, A3 proteins favor deamination of C when preceded by a T. Thus, the A3s will turn 5’TC motifs into 5’TU dinucleotides in the genome of exposed viruses. Depending on the position of the mutated C within the codon, the mutation can be synonymous or nonsynonymous. When the mutated C is at the third position of the codon, deamination of the C will turn the NTC codon into an NTT codon. This mutation will always be synonymous. When A3-related deamination intervenes on a C located at the first position of a codon, the NNTCNN motif will be converted into an NNTUNN motif and will produce a nonsynonymous mutation. Similarly, deamination of a C located at the second position of the codon will convert a TCN codon into a TUN codon and most likely introduce a nonsynonymous mutation. Because a synonymous mutation will be more likely conserved than a nonsynonymous, the A3 -driven natural selection should deplete more intensively NTC codons than TCN or NNTCNN motifs (as in those cases the C to U mutation will impact the encoded amino acid). Thus, the APOBEC3 footprint was defined as an under-representation of NTC codons because A3s favor 5’-TC motifs and because a C to U mutation in the third position of a codon is likely to be retained. Figure 9 shows that adenoviruses of the species A display an occurrence of NTC codons lower than expected. The level of NTC under-representation is similar between the four strains of adenoviruses A (namely A12, A18, A31 and A61) and is lower than any other species. It can be speculated that, like the A12, the other A strains are less able to antagonize A3B and therefore evolved to reduce the number of A3 -favored motifs. Within the different species, the A3 footprint is relatively homogenous. The B, D, E and G species show no NTC depletion on average for the whole viral genome, whereas the C and the F species display a moderate under-representation of NTC codon. These data corroborate the in vitro observations and suggest that the capacities to antagonize A3B are shared among members of a given species.

[0166] Importantly, the discovery that these adenoviral strains antagonize the A3B protein, as disclosed herein, strongly support the use of these adenoviruses for inhibiting A3B in cells overexpressing this protein, in particular in cancer cells overexpressing A3B in the context of anticancer treatment by oncolytic viruses.