FAMILY OF COSTIMULATORY LIGANDS AND ADA

FIELD OF THE DISCLOSURE

The present invention relates to cancer therapies. More specifically, the present invention relates to oncolytic adenoviral vectors and cells and pharmaceutical compositions comprising said vectors. The present invention also relates to a use of said vectors in the manufacture of a medicament for treating cancer in a subject and a method of treating cancer in a subject. Furthermore, the present invention relates to methods of producing a member of the B7 family of costimulatory ligands and adenosine deaminase (ADA) in a cell and increasing anti-tumor effect and induction of specific immune response in a subject, as well as uses of the oncolytic adenoviral vector of the invention for producing transgenes in a cell and increasing anti-tumor effect and generation of specific immune response in a subject.

BACKGROUND OF THE DISCLOSURE

During the last twenty years gene transfer technology has been under intensive examination. The aim of cancer gene therapies is to introduce a therapeutic gene into a tumor cell. These therapeutic genes introduced to a target cell may for example correct mutated genes, suppress active oncogenes or generate additional properties to the cell. Suitable exogenous therapeutic genes include but are not limited to immunotherapeutic, anti-angiogenic, chemoprotective and "suicide" genes, and they can be introduced to a cell by utilizing modified virus vectors or non-viral methods including electroporation, gene gun and lipid or polymer coatings.

Requirements of optimal viral vectors include an efficient capability to find specific target cells and express the viral genome in the target cells. Furthermore, optimal vectors have to stay active in the target tissues or cells. All these properties of viral vectors have been developed during the last decades and for example retroviral, adenoviral and adeno- associated viral vectors have been widely studied in biomedicine.

To further improve tumor penetration and local amplification of the anti-tumor effect, selectively oncolytic agents, e.g. conditionally replicating adenoviruses, have been constructed. Oncolytic adenoviruses are a promising tool for treatment of cancers. Tumor cells are killed by oncolytic adenoviruses due to a replication of the virus in a tumor cell, the last phase of the replication resulting in a release of thousands of virions into the surrounding tumor tissues for effective tumor penetration and vascular re-infection. Tumor cells allow replication of the virus while normal cells are spared due to engineered changes in the virus genome, which prevent replication in non-tumor cells.

In addition to replication mediated cell killing, oncolytic adenoviruses can also be armed with different therapeutic transgenes. This approach combines the advantages of conventional gene delivery with the potency of replication competent agents. One goal of arming viruses is induction of an immune reaction towards the cells that allow virus replication. Virus replication alone, although immunogenic, is normally not enough to induce effective anti-tumor immunity. To strengthen induction of therapeutic immunity, viruses can be armed with stimulatory proteins such as cytokines for facilitation of the introduction of tumor antigens to antigen presenting cells such as dendritic cells, and their stimulation and/or maturation. Introduction of immunotherapeutic genes into tumor cells and furthermore, translation of the proteins, leads to activation of the immune response and efficient destruction of tumor cells. The most relevant immune cells in this regard are natural killer celis (NK), T helper cells and cytotoxic CD8+ T-cells.

More than 50 different serotypes of adenoviruses have been found in humans. Adenovirus serotype 5 (Ad5) is known to cause respiratory diseases and it is the most common serotype studied in the field of gene therapy. In the first Ad5 vectors E1 and/or E3 regions were deleted enabling insertion of foreign DNA to the vectors. Furthermore, deletions of other regions as well as further mutations have provided extra properties to viral vectors, indeed, various modifications of adenoviruses have been suggested for achieving efficient antitumor effects.

ICOS - Inducible T-cell co-stimulator is an immune checkpoint protein. Is a CD28- superfamily costimulatory molecule that is expressed on activated T cells. Plays an important role in cell-cell signalling, immune responses and regulation of cell proliferation (Hutloff et al., 1999).

The B7 family of immune-regulatory ligands comprises inducible costimulator ligand (ICOSL), programmed death-1 ligand (PD-L1 ), programmed death-2 ligand (PD-L2), B7- H3, and B7-H4). In humans, cell surface expression of ICOS-L has been described on B cells, dendritic cells, monocytes/macrophages, and T cells. Instead, ICOS-L binds to ICOS, a T cell-specific costimulatory molecule homologous to CD28 and CTLA-4. ICOS- ligand costimulates Th1 and Th2 cytokine secretion by memory CD4+ T cells (Khayyamian et al., 2002).

ADA (adenosine deaminase (also known as adenosine aminohydrolase, or ADA) is an enzyme involved in purine metabolism. It is needed for the breakdown of adenosine from food and for the turnover of nucleic acids in tissues; including 2 isoforms of ADA: ADA1 and ADA2.

ICOS/ICOSL pathway is necessary for the optimal therapeutic effect of anti-check point inhibitors, thus implicating this pathway as a target of future combinatorial strategies to improve the efficacy of anti-cancer therapies. It has been shown the ICOS/ICOSL pathway is critical for optimal anti-tumour activity of anti-CTLA-4 (Fu et al., 201 1 ).

EP2379586B1 discloses an oncolytic adenoviral vector comprising an adenovirus serotype 5 (Ad5) nucleic acid backbone, a 24 bp deletion (D24) in the Rb binding constant region 2 of adenoviral E1 and a nucleic acid sequence encoding a granulocyte- macrophage colony-stimulating factor (GM-CSF) in the place of the deleted gp19k/6.7K in the adenoviral E3 region. WO2016126608A1 relates to adenoviruses. Exemplary oncolytic viruses include a conditionally replicating oncolytic serotype 5 adenovirus (Ad5) in which human E2F-1 promoter drives expression of the essential E1 a viral genes, thereby restricting viral replication and cytotoxicity to Rb pathway-defective tumor cells and DNX- 2401 (formerly named D24-RGD), which is an adenovirus that has been engineered to replicate selectively in retinoblastoma (Rb)-pathway deficient cells and to infect cells that express certain RGD-binding integrins more efficiently.

Still, more efficient and accurate gene transfer as well as increased specificity and sufficient tumor killing ability of gene therapies are warranted. Safety records of therapeutic vectors must also be excellent. The present invention provides a cancer therapeutic tool with these aforementioned properties by utilizing both oncolytic and immunotherapeutic properties of adenoviruses in a novel and inventive way.

BRIEF DESCRIPTION OF THE DISCLOSURE

The object of the invention is to provide novel methods and means for achieving the above- mentioned properties of adenoviruses and thus, solving the problems of conventional cancer therapies. More specifically, the invention provides novel methods and means for gene therapy.

Adenosine - Derivatives of adenosine are widely found in nature and play an important role in biochemical processes (ATP, ADP). Multiple immunosuppressive mechanisms impede anti-tumour immunity. Among them, the accumulation of extracellular adenosine is a potent and widespread strategy exploited by tumours to escape immunosurveillance through the activation of purinergic receptors. In tumours, this pathway is hijacked and hinders anti-tumour immunity, promoting cancer progression (Allard et al., 2016). Adenosine signalling downregulates functions of most immune effector cells but enhances expansion and activity of immune cells responsible for suppression of anti-tumour immune responses. Adenosine facilitates tumour escape from the immune control. It can be considered for therapeutics targeting the adenosinergic pathway for benefiting cancer patients (Muller-Haegele et al., 2014).

Adenosine deaminase (ADA), is an enzyme involved in purine metabolism that can inhibit tumour progression by metabolizing adenosine (Kutryb-Zajac et al., 2018, Londoho-R et al., 2018). This indicates a potential use of the inhibitors of adenosine pathway for cancer immunotherapy (Ohta, 2016).

The present inventors hypothesized that the combined expression of a member of the B7 family of costimulatory ligands and ADA would have beneficial effects for tumor eradication. More specifically, the present inventors hypothesized that the double transgene construct (TGX-1 1 ) coding for ICOSL and ADA1 utilizes the properties of oncolytic viruses - oncolyses due to the fact of 24bp deletion in E1 A region for cancer cell restricted replication (Kuryk et al., 2016) and also enhanced tropism due to fiber knob modifications (chimeric virus5/3 knob) (Kuryk et al., 2018). In addition to oncolytic properties, due to the presence of two transgenes the construct can exhibit enhanced anti tumour effect by increasing T cell activation by to the interactions of ICOSL with its receptor on T cells and reduction of the suppressive tumour microenvironment by the inhibition of adenosine (by ADA), leading to enhance cross presentation of tumour antigens, priming of cancer specific T cells and modulation of tumour microenvironment. The construct can improve infiltration with activated T cells in both virus-injected and distant tumours. Also, immunomodulation with an oncolytic virus TGX-1 1 expressing ICOSL and ADA1 can be an effective strategy to drive systemic efficacy of immune checkpoint blockade.

Produced transgenes along with the oncolytic properties of the construct can result in synergistic anti-cancer effect and improved clinical outcome.

The double transgene virus was found to be a more efficient model compared to that where both transgenes were encoded by separate viruses but used together. The double transgene simplifies the production process especially in clinical studies over combined viruses expressing transgenes separately.

The present application describes construction of recombinant viral vectors, methods related to the vectors, and their use in tumor cells lines and animal models.

The present invention relates to an oncolytic adenoviral vector comprising an adenovirus serotype 5 (Ad5) nucleic acid backbone, a 24 bp deletion (D24) in the Rb binding constant region 2 of adenoviral E1 and a nucleic acid sequence encoding a member of the B7 family of costimulatory ligands and ADA transgenes. In addition, there may be an IRES in the place of the deleted 2.7 kbp in the adenoviral E3 region.

The present invention further relates to a cell comprising the adenoviral vector of the invention.

The present invention also relates to a pharmaceutical composition comprising the adenoviral vector of the invention.

The present invention also relates to a use of the adenoviral vector of the invention in the manufacture of a medicament for treating cancer in a subject.

The present invention also relates to a method of treating cancer in a subject, wherein the method comprises administration of the vector or pharmaceutical composition of the invention to a subject.

Furthermore, the present invention also relates to a method of producing a member of the B7 family of costimulatory ligands and ADA in a cell, wherein the method comprises: a) carrying a vehicle comprising an oncolytic adenoviral vector of the invention to a cell, and b) expressing a member of the B7 family of costimulatory ligands and ADA of said vector in the cell.

Furthermore, the present invention also relates to a method of increasing tumor specific immune response in a subject, wherein the method comprises: a) carrying a vehicle comprising an oncolytic adenoviral vector of the invention to a target cell or tissue, b) expressing a member of the B7 family of costimulatory ligands and ADA of said vector in the cell, and c) increasing amount of cytotoxic T cells and/or natural killer cells in said target cell or tissue.

Still, the present invention also relates to a use of the oncolytic adenoviral vector of the invention for producing a member of the B7 family of costimulatory ligands and ADA in a cell.

Still, the present invention also relates to a use of the oncolytic adenoviral vector of the invention for increasing tumor specific immune response in a subject.

The present invention provides a tool for treatment of cancers, some of which are refractory to current approaches. Also, restrictions regarding tumor types suitable for treatment remain few compared to many other treatments. In fact, all solid tumors may be treated with the proposed invention. Larger tumors by mass and more complex tumors can be treated by using the present invention. The treatment can be given intratumorally, intracavitary, intravenously and in a combination of these. The approach can give systemic efficacy despite local injection. The approach can also eradicate cells proposed as tumor initiating ("cancer stem cells").

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates the shuttle plasmid constructions for the shuttle plasmids for pTGX- 04/pTGX-05.

Figure 2 illustrates the shuttle plasmid constructions for the shuttle plasmid for pTGX-1 1 (CMV(CuO)-ICOSL-IRES-ADA-SV40pA).

Figure 3 illustrates the cosmid constructions for TGX-04 (AdV5/3-D24-ICOSL), TGX-05 (AdV5/3-D24-ADA), TGX-1 1 (AdV5/3-D24-CMV(CuO)-ICOSL-IRES-ADA-SV40pA).

Figure 4 shows TGX-04 (AdV5/3-D24-ICOSL) virus structure. ICOSL (functional expression cassette, location: 28,371 - 29,279bp) inserted into E3 region. The virus contains the 24-bp deletion in E1 A CR2 (E1 A Conserved Region 2 (CR2) with a 24-bp deletion that removed the strongest of two binding sites in E1 A for Rb protein (LTCHEAGF), 907 - 951 bp), expresses the Ad5/3 hybrid fiber (location: 30,967 - 32,730bp). Nucleotide sequence enclosed in attachment (TGX-04). Sequence identity for 3 genetic modifications in regions E1 A, E3 and fiber.

Figure 5 shows TGX-05 (AdV5/3-D24-ADA) virus structure. ADA (functional expression cassette, location: 28,371 - 30,476bp) inserted into E3 region. The virus contains the 24- bp deletion in E1 A CR2 (E1 A Conserved Region 2 (CR2) with a 24-bp deletion that removed the strongest of two binding sites in E1 A for Rb protein (LTCHEAGF), location: 907-951 bp), expresses the Ad5/3 hybrid fiber (location: 32,164 - 33,927bp). Nucleotide sequence enclosed in attachment (TGX-05). Sequence identity for genetic modifications in regions E1 A and fiber; silent mutation in ADA CDS.

Figure 6 shows TGX-1 1 (AdV5/3-D24-CMV(CuO)-ICOSL-IRES-ADA-SV40pA) virus structure. ICOSL (functional expression cassette, location: 28,904 - 29,812bp) -IRES (29,813 - 30,384bp) -ADA (functional expression cassette, location: 31 ,207 - 31 ,950) - SV40pA expression under the control of CMV promoter (28,172 - 28,705) and CuO inserted into E3 region. The virus contains the 24-bp deletion in E1 A CR2 (E1 A Conserved Region 2 (CR2) with a 24-bp deletion that removed the strongest of two binding sites in E1 A for Rb protein (LTCHEAGF), location: 907-951 bp), expresses the Ad5/3 hybrid fiber (location: 32,885 - 34,648bp). Nucleotide sequence enclosed in attachment (TGX-13). Sequence identity for genetic modifications in regions E1 A and fiber; - silent mutation in ADA CDS, 2 deletions in multiple cloning site. Figure 7a shows the effects of tested oncolytic adenoviruses on viability of human melanoma cell line A375 at the concentration of 1000 VP/cell after 72 hours post infection. Absorbance of untreated cells is expressed as 100% and absorbance of treated cells is expressed as a percentage of absorbance of untreated cells. Results are expressed as mean +/- SEM.

Figure 7b illustrates the effects tested oncolytic adenoviruses on viability of human melanoma cell line SK-MEL-2 at the concentration of 1 VP/cell after 72 hours post infection. Absorbance of untreated cells is expressed as 100% and absorbance of treated cells is expressed as a percentage of absorbance of untreated cells. Results are expressed as mean +/- SEM.

Figure 7c shows the effects of tested oncolytic adenoviruses on viability of human melanoma cell line SK-MEL-2 at the concentration of 1000 VP/cell after 72 hours post infection. Absorbance of untreated cells is expressed as 100% and absorbance of treated cells is expressed as a percentage of absorbance of untreated cells. Results are expressed as mean +/- SEM.

Figure 7d shows the effects of tested oncolytic adenoviruses on viability of human melanoma cell line A2058 at the concentration of 1000 VP/cell after 72 hours post infection. Absorbance of untreated cells is expressed as 100% and absorbance of treated cells is expressed as a percentage of absorbance of untreated cells. Results are expressed as mean +/- SEM.

Figure 8 shows the effect of tested oncolytic adenoviruses on cell death induction (apoptosis) of human cell line A2058 at the concentration of 1000 VP/cell at 72 hours post infection. Amount of apoptosis was assessed by Annexin V staining on the cell lines tested. Results are expressed as a percentage of untreated cells. Results are expressed as mean +/- SEM.

Figure 9a shows the effect of tested oncolytic adenoviruses on immunogenic cell death - induction of ATP release of human cell line A375 treated at the concentration of 0.1 VP/cell at 72 hours post infection. ATP quantity was evaluated using the ATP assay kit. Results are expressed as a percentage of untreated cells. Results are expressed as mean +/- SEM.

Figure 9b shows the effect of tested oncolytic adenoviruses on immunogenic cell death - induction of ATP release of human cell line SKMEL-2 treated at the concentration of 10VP/cell at 72 hours post infection. ATP quantity was evaluated using the ATP assay kit. Results are expressed as a percentage of untreated cells. Results are expressed as mean +/- SEM.

Figure 9c shows the effect of tested oncolytic adenoviruses on immunogenic cell death - induction of ATP release of human cell line SKMEL-28 treated at the concentration of 0.1 VP/cell at 72 hours post infection. ATP quantity was evaluated using the ATP assay kit. Results are expressed as a percentage of untreated cells. Results are expressed as mean +/- SEM.

Figure 10 shows the effect of tested oncolytic adenoviruses on immunogenic cell death - induction of Calreticulin exposure of human cell line A375 treated at the concentration of 1000 VP/cell at 72 hours post infection. Calreticulin was stained at the surface of the cells with an anti-calreticulin antibody. Results are expressed as a percentage of calreticulin positive cells. Results are expressed as mean +/- SEM.

Figure 11 shows the effect of tested oncolytic adenoviruses on immunogenic cell death - induction of HMGB1 release of human cell line A2058 treated at the concentration of 10 VP/cell at 72 hours post infection. Calreticulin was stained at the surface of the cells with an anti-calreticulin antibody. Results are expressed as ng/ml. Results are expressed as mean +/- SEM.

Figure 12a shows immune infiltration phenotype in the tumors at sacrifice. The % of human Granzyme B+ CD8 T cells; Immune infiltration was analyzed by flow cytometry in the tumors at sacrifice. N = 6-12 tumors per group. Individual data and mean +/- min & max whiskers are presented for each group. Anova test was used.

Figure 12b shows immune infiltration phenotype in the tumors at sacrifice. The % of human PD1 + CD8 T cells; Immune infiltration was analyzed by flow cytometry in the tumors at sacrifice. N = 6-12 tumors per group. Individual data and mean +/- min & max whiskers are presented.

Figure 12c shows immune infiltration phenotype in the tumors at sacrifice. The number of Human CD4 T cells/g of both tumors; Immune infiltration was analyzed by flow cytometry in the tumors at sacrifice. N = 6-12 tumors per group. Individual data and mean +/- min & max whiskers are presented.

SEQUENCE LISTING

Sequence ID NO: 1 . A nucleic acid sequence encoding an oncolytic adenoviral vector AdV5/3-D24-CMV(CuO)-ICOSL-IRES-ADA-SV40pA (TGX-1 1 ). ICOSL (functional expression cassette, location: 28,904 - 29,812bp) -IRES (29,813 - 30,384bp) -ADA (functional expression cassette, location: 31 ,207 - 31 ,950) -SV40pA expression under the control of CMV promoter (28,172 - 28,705) and CuO inserted into E3 region. The virus contains the 24-bp deletion in E1 A CR2 (E1 A Conserved Region 2 (CR2) with a 24-bp deletion that removed the strongest of two binding sites in E1 A for Rb protein (LTCHEAGF), location: 907-951 bp), expresses the Ad5/3 hybrid fiber (location: 32,885 - 34,648bp). Nucleotide sequence enclosed in attachment (TGX-13). Sequence identity for genetic modifications in regions E1 A and fiber; - silent mutation in ADA CDS, 2 deletions in multiple cloning site.

Sequence ID NO: 2. A nucleic acid sequence encoding an oncolytic adenoviral vector AdV5/3-D24-ICOSL (TGX-04). ICOSL (functional expression cassette, location: 28,371 - 29,279bp) inserted into E3 region. The virus contains the 24-bp deletion in E1 A CR2 (E1 A Conserved Region 2 (CR2) with a 24-bp deletion that removed the strongest of two binding sites in E1 A for Rb protein (LTCHEAGF), 907 - 951 bp), expresses the Ad5/3 hybrid fiber (location: 30,967 - 32,730bp). Nucleotide sequence enclosed in attachment (TGX-04). Sequence identity for 3 genetic modifications in regions E1 A, E3 and fiber.

Sequence ID NO: 3. A nucleic acid sequence encoding an oncolytic adenoviral vector AdV5/3-D24-ADA (TGX-05). ADA (functional expression cassette, location: 28,371 - 30,476bp) inserted into E3 region. The virus contains the 24-bp deletion in E1 A CR2 (E1 A Conserved Region 2 (CR2) with a 24-bp deletion that removed the strongest of two binding sites in EI A for Rb protein (LTCHEAGF), location: 907-951 bp), expresses the Ad5/3 hybrid fiber (location: 32,164 - 33,927bp). Nucleotide sequence enclosed in attachment (TGX- 05). Sequence identity for genetic modifications in regions E1 A and fiber; silent mutation in ADA CDS.

Sequence ID NO: 4. A binding site for Rb protein in E1 A.

Sequence ID NO: 5. CR2-D24 (in construct TGX-04).

Sequence ID NO: 6. Ad5/3 fiber (TGX-04).

Sequence ID NO: 7. ICOSL (TGX-04).

Sequence ID NO: 8. CR2-D24 (TGX-05).

Sequence ID NO: 9. Ad5/3 fiber (TGX-05).

Sequence ID NO: 10. ADA (TGX-05).

Sequence ID NO: 1 1 . CR2-D24 (TGX-1 1 ).

Sequence ID NO: 12. Ad5/3 fiber (TGX-1 1 ).

Sequence ID NO: 13. CMV promoter/enhancer (TGX-1 1 ). Sequence ID NO: 14. CUO (TGX-1 1 ).

Sequence ID NO: 15. ICOSL (TGX-1 1 ).

Sequence ID NO: 16. IRES (TGX-1 1 ).

Sequence ID NO: 17. ADARB1 (ADA) (TGX-1 1 ).

Sequence ID NO: 18. SV40 poly(A) signal (TGX-1 1 ).

Sequence ID NO: 19. Primer 288-17.

Sequence ID NO: 20. Primer 288-41 .

Sequence ID NO: 21 . Primer 288-18.

Sequence ID NO: 22. Primer 288-19.

Sequence ID NO: 23. Primer 288-20.

Sequence ID NO: 24. Primer 288-21 .

DETAILED DESCRIPTION OF THE DISCLOSURE

The double transgene oncolytic adenoviral vector of the present invention is based on an adenovirus serotype 5 (Ad5) nucleic acid backbone with a 24 bp deletion (D24) in the Rb binding constant region 2 (CR2) of adenoviral E1 . The vector comprises a nucleic acid sequence encoding a member of the B7 family of costimulatory ligands and a nucleic acid sequence encoding ADA in the place of the deleted 2.7kbp in the adenoviral E3 region. In a preferred embodiment of the invention, the adenoviral vector backbone is ONCOS-102 as disclosed in EP2379586B1 , which comprises of a member of the B7 family of costimulatory ligands and ADA transgenes connected with IRES or P2A sequence in the place of GM-CSF/E3 region.

Preferably, the B7 family of immune-regulatory ligands is ICOSL. Instead of ICOSL also other ligands of the B7 family of immune-regulatory ligands, such as programmed death- 1 ligand (PD-L1 ), programmed death-2 ligand (PD-L2), B7-H3, and B7-H4, can be used in the invention. Most preferably, a member of the B7 family of costimulatory ligands is human ICOSL.

Adenosine deaminase can be ADA1 or ADA2. Preferably ADA is human ADA1 .

The results of the experiments showed surprising superior effect of double transgene virus. Double transgene virus coding for ICOSL and ADA1 (TGX-1 1 ) showed anti-cancer superiority over single transgene viruses TGX-04 (coding for ICOSL), TGX-05 (coding for ADA1 ) and combinatory therapy of TGX-04 plus TGX-05 in several different cell types (Figures 7a-7d, Figure 8, Figures 9a-9c, Figure 10, Figure 1 1 , and Figures 12a-12c).

Compared to a wild type adenovirus genome, the adenoviral vector of the invention lacks 24 base pairs from CR2 in E1 region, specifically in E1 A region, and 2.7kbp in E3 region. 24 base pair deletion (D24) of E1 affects CR2 domain, which is responsible for binding the Rb tumor suppressor/cell cycle regulator protein and thus, allows the induction of the synthesis (S) phase i.e. DNA synthesis or replication phase. pRb and E1 A interaction requires eight amino acids 121 to 127 of the E1 A protein conserved region, which are deleted in the present invention. Viruses with the D24 are known to have a reduced ability to overcome the G1 -S checkpoint and replicate efficiently in cells where this interaction is not necessary, e.g. in tumor cells defective in the Rb-p16 pathway.

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 deletion in E3 refers to a deletion of 2.7k base pairs from the adenoviral E3A region. In a resulting adenoviral construct, where 2.7kbp along with gp19k and 6.7K genes are deleted. The gp19k gene product is known to bind and sequester major histocompatibility complex I (MHC1 ) molecules in the endoplasmic reticulum, and to prevent the recognition of infected cells by cytotoxic T-lymphocytes. Since many tumors are deficient in MFIC1 , deletion of gp19k 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.

An internal ribosome entry site, abbreviated IRES, is an RNA element that allows for translation initiation in a cap-independent manner, as part of the greater process of protein synthesis. In eukaryotic translation, initiation typically occurs at the 5' end of mRNA molecules, since 5' cap recognition is required for the assembly of the initiation complex.

Instead of these large IRES sequences, a much shorter, self-cleaving 2A peptide can be used. Virus-derived peptide sequences mediate a ribosome-skipping event enables generation of multiple separate peptide products from one mRNA. Peptides from porcine teschovirus-1 (P2A) facilitate expression of multiple transgenes.

In the present invention, a member of the B7 family of costimulatory ligands and ADA transgenes are placed into a 2.7kbp of deleted E3 region, under the E3 promoter or CMV(Cuo) 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 or endogenous promoter known in the art, preferably endogenous promoter. In a preferred embodiment of the invention, a nucleic acid sequence encoding a member of the B7 family of costimulatory ligands and ADA are under the control of the viral E3 promoter. CMV(CuO) promoter contains a copy of the cumate operator (CuO). In “regular” 293 cells and other cell lines, the CuO operator is inactive and transcription takes place from the CMV promoter. In 293-CymR cells, which express the CymR repressor, the CymR repressor binds onto the CuO operator, thereby preventing the transcription of the transgene.

The vectors of the invention may also comprise other modifications than partial deletions of CR2 and E3 and insertion of ICOSL and ADA sequences as mentioned above. In a preferred embodiment of the invention, all the other regions of the Ad5 vector are of a wild type. In another preferred embodiment of the invention, the E4 region is of a wild type. In a preferred embodiment of the invention, a wild type region is located upstream of the E1 region. "Upstream" refers to immediately before the E1 region in the direction of expression. E1 B region may also be modified in the vector of the invention.

As used herein, "Ad5/3 chimerism" of the capsid 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.

Expression cassettes are used for expressing transgenes in a target, such as a cell, by utilizing vectors. As used herein, the expression "expression cassette" refers to a DNA vector or a part thereof comprising nucleotide sequences, which encode cDNAs or genes, and nucleotide sequences, which control and/or regulate the expression of said cDNAs or genes. Similar or different expression cassettes may be inserted to one vector or to several different vectors. Ad5 vectors of the present invention may comprise either several or one expression cassettes. However, only one expression cassette is adequate. In a preferred embodiment of the invention, the oncolytic adenoviral vector comprises at least one expression cassette. In a preferred embodiment of the invention, the oncolytic adenoviral vector comprises only one expression cassette.

A cell comprising the adenoviral vector of the invention may be any cell such as a eukaryotic ceil, bacterial cell, animal cell, human cell, mouse cell etc. A cell may be an in vitro, ex vivo or in vivo cell. For example, the cell may be used for producing the adenoviral vector in vitro, ex vivo or in vivo, or the cell may be a target, such as a tumor cell, which has been infected with the adenoviral vector.

In a method of producing a member of the B7 family of costimulatory ligands and ADA transgenes in a cell, a vehicle comprising the vector of the invention is carried into a cell and furthermore a member of the B7 family of costimulatory ligands and ADA transgenes are expressed, and the protein is translated and secreted in a paracrine manner. "A vehicle" may be any viral vector, plasmid or other tool, such as a particle, which is able to deliver the vector of the invention to a target cell. Any conventional method known in the art can be used for delivering the vector to the cell.

Tumor specific immune response may be increased in a subject by the present invention. Helper T cells, cytotoxic T cells and/or natural killer cells are stimulated, produced and targeted as a consequence of the construct properties.

The recombinant Ad5 vectors of the invention have been constructed for replication competence in cells, which have defects in the Rb-pathway, specifically Rb-p16 pathway. These defective cells include all tumor cells in animals and humans. In a preferred embodiment of the invention, the vector is capable of selectively replicating in cells having defects in the Rb-pathway. 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.

Any cancers or tumors, including both malignant and benign tumors as well as primary tumors and metastasis may be targets of gene therapies. In a specific embodiment of the invention the cancer is any solid tumor. In a preferred embodiment of the invention, the cancer 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, brain cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, bone cancer, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, carcinoid of gastrointestinal tract, fibrosarcoma, breast cancer, Paget's disease, cervical cancer, colorectal cancer, rectal cancer, esophagus cancer, gall bladder cancer, head cancer, eye cancer, neck cancer, kidney cancer, Wilms' tumor, liver cancer, Kaposi's sarcoma, prostate cancer, lung cancer, testicular cancer, Hodgkin's disease, non-Hodgkin's lymphoma, oral cancer, skin cancer, mesothelioma, multiple myeloma, ovarian cancer, endocrine pancreatic cancer, glucagonoma, pancreatic cancer, 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, Hp 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.

A pharmaceutical composition of the invention comprises at least one type of the vectors of the invention. Furthermore, the composition may comprise at least two or three different vectors of the invention. In addition to the vector of the invention, a pharmaceutical composition may also comprise any other vectors, such as other adenoviral vectors, other therapeutically effective agents, any other agents such as pharmaceutically acceptable carriers, buffers, excipients, adjuvants, antiseptics, filling, stabilising or thickening agents, and/or any components normally found in corresponding products.

The pharmaceutical composition may be in any form, such as solid, semisolid or liquid form, suitable for administration. A formulation can be selected from a group consisting of, but not limited to, solutions, emulsions, suspensions, tablets, pellets and capsules.

The effective dose of vectors depends on at least the subject in need of the treatment, tumor type, location of the tumor and stage of the tumor. The dose may vary for example from about 10e8 viral particles (VP) to about 10e14 VP, preferably from about 5x10e9 VP to about 10e13 VP and more preferably from about 8x10e9 VP to about 10e12 VP. In one specific embodiment of the invention the dose is in the range of about 5x10e10 - 5x10e1 1 VP.

The vector or pharmaceutical composition of the invention may be administered to any eukaryotic subject selected from a group consisting of plants, animals and human beings, in a preferred embodiment of the invention, the subject is a human or an animal. An animal may be selected from a group consisting of pets, domestic animals and production animals.

Any conventional method may be used for administration of the vector or composition 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. In a preferred embodiment of the invention, the administration is conducted through an intratumoral, intramuscular, intra-arterial, intravenous, intrapleural, intravesicular, intracavitary or peritoneal injection, or an oral administration. Only one administration of oncolytic adenoviral vectors of the invention may have therapeutic effects. However, in a preferred embodiment of the invention, oncolytic adenoviral vectors or pharmaceutical compositions are administered several times during the treatment period. Oncolytic adenoviral vectors or pharmaceutical compositions may be administered for example from 1 to 10 times in the first 2 weeks, 4 weeks, monthly or during the treatment period. In one embodiment of the invention, administration is done three to seven times in the first 2 weeks, then at 4 weeks and then monthly. In a specific embodiment of the invention, administration is done four times in the first 2 weeks, then at 4 weeks and then monthly. The length of the treatment period may vary, and for example may last from two to 12 months or more.

The gene therapy of the invention is effective alone, but combination of adenoviral gene therapy with any other therapies, such as traditional therapy, may be more effective than either one alone. For example, each agent of the combination therapy may work independently in the tumor tissue, the adenoviral vectors may sensitize cells to chemotherapy or radiotherapy and/or chemotherapeutic agents may enhance the level of virus replication or effect the receptor status of the target cells. The agents of combination therapy may be administered simultaneously or sequentially.

In a preferred embodiment of the invention, the method or use further comprises administration of concurrent radiotherapy to a subject. In another preferred embodiment of the invention, the method or use further comprises administration of concurrent chemotherapy to a subject. As used herein "concurrent" refers to a therapy, which has been administered before, after or simultaneously with the gene therapy of the invention. The period for a concurrent therapy may vary from minutes to several weeks. Preferably the concurrent therapy lasts for some hours.

As used herein, the term "immune checkpoint inhibitor" or“checkpoint inhibitor” refers to molecules that totally or partially reduce, inhibit, interfere with or modulate one or more checkpoint proteins. Checkpoint proteins regulate T-cell activation or function. Central to the immune checkpoint process are the cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed death 1 (PD-1 ) immune checkpoint pathways. The CTLA-4 and PD-1 pathways are thought to operate at different stages of an immune response. CTLA-4 is considered the “leader” of the immune checkpoint inhibitors, as it stops potentially autoreactive T cells at the initial stage of naive T-cell activation, typically in lymph nodes. The PD-1 pathway regulates previously activated T cells at the later stages of an immune response, primarily in peripheral tissues. Inhibition of the immune checkpoint pathways has led to the approval of several new drugs: ipilimumab (anti-CTLA-4; Yervoy®), pembrolizumab (anti-PD-1 ; Keytruda®), and nivolumab (anti-PD-1 ; Opdivo®). Also, PD-L1 inhibitors, such as Atezolizumab (MPDL3280), Avelumab (MSB0010718C) and Durvalumab (MEDI4736), are available. These antagonistic antibodies have been associated with objective clinical responses in cancer patients. Antibodies targeting CTLA-4 are already marketed (e.g. Ipilimumab, Yervoy, Bristol-Myers Squibb, BMS) for metastatic melanoma. Antibody therapies with anti PD-L1 (e.g. MPDL3280A, Roche), anti PD-1 (e.g. Nivolumab, BMS) are also ongoing.

Other immune-checkpoint inhibitors include lymphocyte activation gene-3 (LAG-3) inhibitors, such as IMP321 , a soluble Ig fusion protein. Other immunecheckpoint inhibitors include B7 inhibitors, such as B7-H3 and B7-H4 inhibitors. In particular, the anti-B7-H3 antibody MGA271 . Also included are TIM3 (T-cell immunoglobulin domain and mucin domain 3) inhibitors. In certain embodiments the PD-1 blockers include anti-PD-L1 antibodies. In certain other embodiments the PD-1 blockers include anti-PD-1 antibodies and similar binding proteins such as nivolumab (MDX 1 106, BMS 936558, ONO 4538), a fully human lgG4 antibody 30 that binds to and blocks the activation of PD-1 by its ligands PD-L1 and PD-L2; lambrolizumab (MK-3475 or SCH 900475), a humanized monoclonal lgG4 antibody against PD-1 ; CT-01 1 a humanized antibody that binds PD-1 ; AMP-224 is a fusion protein of B7-DC; an antibody Fc portion; BMS-936559 (MDX- 1 105-01 ) for PD- L1 (B7-H1 ) blockade. Further examples of PD-L1 inhibitors that can be used in certain embodiments are Atezolizumab (MPDL3280), Avelumab (MSB0010718C) and Durvalumab.

Preferably, anti-PD-1 antibodies pembrolizumab (Keytruda), and nivolumab (Opdivo) are used in the invention. Also, PD-L1 inhibitors, such as durvalumab can be used in combination with anti-PD-1 -antibodies. The preferred checkpoint inhibitors of the present invention are thus those for PD-1 and PD-L1 .

Agents suitable for combination therapy include but are not limited to All-trans retinoic acid, Azacitidine, Azathioprine, Bleomycin, Carboplatin, Capecitabme, Cisplatin, Chlorambucil, Cyclophosphamide, Cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone, Etoposide, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Imattnib, Mechlorethamine, Mercaptopurine, Methotrexate, Mitoxantrone, Oxaliplatin, Paclitaxel, Pemetrexed, Temozolomide, Teniposide, Tioguanine, Valrubicin, Vinblastine, Vincristine, Vindesine and Vinorelbine.

In a preferred embodiment of the invention, the method or use further comprises administration of verapamil or another calcium channel blocker to a subject. "Calcium channel blocker" refers to a class of drugs and natural substances which disrupt the conduction of calcium channels, and it may be selected from a group consisting of verapamil, dihydropyridines, gallopamil, diltiazem, mibefradil, bepridil, fluspirilene and fendiline.

In a preferred embodiment of the invention, the method or use further comprises administration of autophagy inducing agents to a subject. Autophagy refers to a catabolic process involving the degradation of a cell's own components through the lysosomal machinery. "Autophagy inducing agents" refer to agents capable of inducing autophagy and may be selected from a group consisting of, but not limited to, mTOR inhibitors, P13K inhibitors, lithium, tamoxifen, chloroquine, bafilomycin, temsirolimus, sirolimus and temozolomide. In a specific embodiment of the invention, the method further comprises administration of temozolomide to a subject. Temozolomide may be either oral or intravenous temozolomide.

In one embodiment of the invention, the method or use further comprises administration of chemotherapy or anti-CD20 therapy or other approaches for blocking of neutralizing antibodies. "Anti-CD20 therapy" refers to agents capable of killing CD20 positive cells and may be selected from a group consisting of rituximab and other anti-CD20 monoclonal antibodies. "Approaches for blocking of neutralizing antibodies" refers to agents capable of inhibiting the generation of anti-viral antibodies that normally result from infection and may be selected from a group consisting of different chemotherapeutics, immunomodulatory substances, corticoids and other drugs. These substances may be selected from a group consisting of, but not limited to, cyclophosphamide, cyclosporin, azathioprine, methylprenisolone, etoposide, CD40L, CTLA4lg4, FK506 (tacrolismus), IL- 12, IFN-gamma, interleukin 10, anti-CD8, anti-CD4 antibodies, myeloablation and oral adenoviral proteins.

The oncolytic adenoviral vector of the invention induces virion mediated oncolysis of tumor cells and activates human immune response against tumor cells. In a preferred embodiment of the invention, the method or use further comprises administration of substances capable to downregulating regulatory T-cells in a subject. "Substances capable to down- regulating regulatory T-cells" refers to agents that reduce the number of cells identified as T-suppressor or Regulatory T-cells. These cells have been identified as consisting one or many of the following immunophenotypic markers: CD4+, CD25+, FoxP3+, CD127- and GITR+. Such agents reducing T- suppressor or Regulatory T-cells may be selected from a group consisting of anti-CD25 antibodies or chemotherapeutics. An object of the invention is an oncolytic adenoviral vector comprising an adenovirus serotype 5 (Ad5) nucleic acid backbone, a 24 bp deletion (D24) in the Rb binding constant region 2 of E1 , a nucleic acid sequence encoding a member of the B7 family of costimulatory ligands and a nucleic acid sequence encoding ADA. A more preferred object of the invention is the oncolytic adenoviral vector, wherein a member of the B7 family of costimulatory ligands and ADA are placed under E3 promoter. Still another preferred embodiment is the oncolytic adenoviral vector, wherein a member of the B7 family of costimulatory ligands and ADA are placed under CMV(Cuo) promoter. Still a further preferred object is the oncolytic adenoviral vector, wherein there is a 2.7kbp deletion in the E3. Further, the oncolytic adenoviral vector, wherein the B7 family of costimulatory ligand is ICOSL and ADA is ADA1 , is a preferred aspect of the invention. Also, the oncolytic adenoviral vector, wherein the nucleic acid sequence encoding a member of the B7 family of costimulatory ligands and the nucleic acid sequence encoding ADA are connected with IRES is a preferred aspect of the invention. The oncolytic adenoviral vector, wherein the nucleic acid sequence encoding a member of the B7 family of costimulatory ligands and the nucleic acid sequence encoding ADA are connected with P2A is also a preferred aspect of the invention. The oncolytic adenoviral vector comprising a native E1 A promoter is still another preferred aspect of the invention. Another object of the invention is this oncolytic adenoviral vector further comprising an Ad5/3 chimerism as a capsid modification. In still another object of the invention the oncolytic adenoviral vector comprises SEQ ID NO: 1 .

One object of the invention is the oncolytic adenoviral vector comprising at least one expression cassette. In another object of the invention, the oncolytic adenoviral vector is capable of selectively replicating in cells having defects in the Rb-pathway.

Also, a cell comprising the adenoviral vector is one aspect of the invention.

Another aspect of the invention is a pharmaceutical composition comprising the adenoviral vector of the invention. The adenoviral vector for use in the treatment of cancer or in the manufacture of a medicament for treating cancer in a subject is also an aspect of the invention. The cancer can be 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, brain cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, bone cancer, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, carcinoid of gastrointestinal tract, fibrosarcoma, breast cancer, Paget's disease, cervical cancer, colorectal cancer, rectal cancer, esophagus cancer, gall bladder cancer, head cancer, eye cancer, neck cancer, kidney cancer, Wilms' tumor, liver cancer, Kaposi's sarcoma, prostate cancer, lung cancer, testicular cancer, Hodgkin's disease, non- Hodgkin's lymphoma, oral cancer, skin cancer, mesothelioma, multiple myeloma, ovarian cancer, endocrine pancreatic cancer, glucagonoma, pancreatic cancer, 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.

The subject of the treatment can be a human or an animal.

The treatment of cancer with adenoviral vector in a subject can be conducted through an intratumoral, intramuscular, intra-arterial, intravenous, intrapleural, intravesicular, intracavitary or peritoneal injection, or an oral administration. The injection or the administration can be conducted several times during the treatment period. Also, an oncolytic adenoviral vector having a different fiber knob of the capsid compared to the vector of the earlier treatment can be used. The treatment of cancer in a subject can further comprise an administration of concurrent radiotherapy and/or concurrent chemotherapy to a subject. The treatment of cancer in a subject can further comprise an administration of a checkpoint inhibitor, verapamil or another calcium channel blocker, autophagy inducing agents, temozolomide, chemotherapy or anti-CD20 therapy or other approaches for blocking of neutralizing antibodies, and/or substances capable to downregulating regulatory T-cells in a subject.

A method of producing a member of the B7 family of costimulatory ligands and ADA in a cell in vitro, wherein the method comprises:

a. carrying a vehicle comprising an oncolytic adenoviral vector to a cell, and b. expressing a member of the B7 family of costimulatory ligands and ADA of said vector in the cell,

is an aspect of the present invention. Also, a use of the oncolytic adenoviral vector for producing a member of the B7 family of costimulatory ligands and ADA in a cell in vitro is an aspect of the invention.

A further object of the invention is a method of treating cancer in a human subject in need thereof, wherein a therapeutically effective amount of a pharmaceutical formulation comprising an oncolytic adenovirus comprising SEQ ID NO: 1 and a diluent or carrier is administered. Another object of the invention is a method, wherein a therapeutically effective amount of a pharmaceutical formulation comprising a combination of oncolytic adenovirus comprising SEQ ID NO: 2 and oncolytic adenovirus comprising SEQ ID NO: 3 is administered. Said methods can further comprise administering an effective amount of an oncolytic adenoviral vector or a pharmaceutical composition comprising the oncolytic adenoviral vector to the subject through intratumoral injection, wherein the oncolytic adenoviral vector comprises the adenovirus according to the invention. In said methods the effective amount of the oncolytic adenoviral vector contains between 5X10e10 - 5X10e1 1 viral particles. They also induce a specific antitumor immune response, including activation of CD8+ T cells, in tumors expressing a member of the B7 family of costimulatory ligands and ADA from the oncolytic adenoviral vector. In said methods for treatment, the oncolytic adenoviral vector can be administered three to seven times in the first two weeks of treatment.

In said treatment methods cancer can be 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, brain cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, bone cancer, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, carcinoid of gastrointestinal tract, fibrosarcoma, breast cancer, Paget's disease, cervical cancer, colorectal cancer, rectal cancer, esophagus cancer, gall bladder cancer, head cancer, eye cancer, neck cancer, kidney cancer, Wilms' tumor, liver cancer, Kaposi's sarcoma, prostate cancer, lung cancer, testicular cancer, Hodgkin's disease, non- Hodgkin's lymphoma, oral cancer, skin cancer, mesothelioma, multiple myeloma, ovarian cancer, endocrine pancreatic cancer, glucagonoma, pancreatic cancer, 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.

According to one embodiment, the administration of the adenoviral vector or vectors or pharmaceutical composition can be conducted through an intratumoral, intramuscular, intra-arterial, intravenous, intrapleural, intravesicular, intracavitary or peritoneal injection, or an oral administration. According to another embodiment, the adenoviral vector or vectors or pharmaceutical composition can be administered several times during a treatment period. According to a preferred embodiment, concurrent radiotherapy can be administered to the subject. According to a more preferred embodiment, concurrent chemotherapy can be administered to the subject. According to a still more preferred embodiment verapamil or another calcium channel blocker can be administered to the subject.

In one preferred object of the invention, is a method for treatment with the adenoviral vector or vectors further comprising administering substances capable to downregulating regulatory T-cells in the subject.

Any method or use of the invention may be either in vivo, ex vivo or in vitro method or use.

The present invention is illustrated by the following examples, which are not intended to be limiting in any way.

EXAMPLES

Cloning of oncolytic viruses

Oncolytic viruses were constructed using genetic engineering tools. The construction of oncolytic vectors included the following steps:

1 . Shuttle Plasmid Constructions:

For TGX-04/05, the transgenes were inserted into the E3 region at the exact same location as the GM-CSF coding sequence in vector Ad5/3-d24-GMCSF. In that vector, a 0.9 kb BsiWI-Mfel fragment encompassing the E3 6.7K ORF and gp19K ORF was deleted and replaced by the GM-CSF coding sequence. For this cloning we used shuttle plasmid pAd1 129-14, which contain the WT E3 region and a hybrid Ad5/3 fiber. The ICOSL ligand (ICOSL), ADA (ADARB1 ) sequences were inserted between the BsiWI and Mfel site of pAd1 129-14 using standard cloning methods (Figure 1 ). The identity of the shuttle plasmids was verified by restriction analysis and sequencing.

For TGX-1 1 , the dicistronic cassettes CMV-ICOSL-IRES-ADA-SV40pA was inserted in place of the E3 region deleted for most of its genes. We used shuttle plasmid pAd1 129- 27, which contains the Ad5 E3 region and a hybrid Ad5/3 fiber gene. A 2.7 kb Bglll fragment was deleted from the E3 region and replaced with a multiple cloning site. Both dicistronic cassettes were inserted into pAd1 129-27 using standard cloning methods (Figure 2). The identity of the plasmids was verified by restriction analysis and sequencing.

2. Cosmid Construction

The entire genomes of the recombinant adenoviruses were reconstituted in cosmids (Figure 3). T ransfection-grade cosmid DNAs were purified and their identity was confirmed by restriction analysis.

3. Virus rescue and characterization

Excision of the adenovirus genome from the cosmids obtained in step 2, and transfection into 293 or 293-Cymr or A549 cells. Three virus plaques per construct were harvested and amplified in small scale. Their identity will be verified by restriction analysis of their genome using 3 enzymes (Flirt supernatant).

4. Western Blot

Expression of the various transgenes from the viruses were assessed by infecting a reporter cell line with the various virus clones, lysing the cells 24 hours post-infection, separating the total protein content by PAGE, and performing western blotting (WB) by using anti-ICOSL and anti-ADA antibodies to detect the various transgene products by chemiluminescence. These experiments will enable us to select virus clones for amplification and purification.

5. Small-scale amplification

Small-scale amplification of the viral clones, purification on two CsCI gradients, dialysis against an isotonic buffer (GTS buffer: 2.5% glycerol, 25 mM NaCI, 20 mM T ris-HCI, pH 8.0), filtration through a low-protein binding 0.22 pm filter, and virus particle (VP) concentration determination by ultraviolet absorbance (Abs260-SDS method) was carried out.

6. Confirmation of virus identity by sequencing The identity of the purified recombinant adenoviruses was confirmed by sequencing regions of the virus genomes specific for these recombinant viruses, including the 24-bp deletion in the E1 A CDS, the entire mono- or dicistronic cassettes inserted into the E3 region, the Ad5/3 hybrid fiber, and the junctions between the DNA fragments ligated to each other during the cosmid construction. Experimental sequences were assembled into contigs and aligned with the expected sequences of the viruses. Viral genomic DNAs were extracted from CsCI-purified virus particles using proteinase K and resuspended into 30 mI_ TE pH 7.5. Their quality was assessed by spectrophotometry (Abs260/280/320 nm) and agarose gel electrophoresis. TGX viruses’ DNAs were sent to a third party for shotgun library making and sequencing. Libraries were generated using a Nextera XT kit (lllumina, Inc). They were pooled, quantified by qPCR, and sequenced on a 2x300 MiSeq run (lllumina, Inc) .fastq files were processed using the program“BWA Aligner”. The software Geneious v1 1 .1 .5 (Biomatters Ltd) was used to visualize the .bam files and calculate the consensus sequences. The consensus sequences were aligned to the predicted sequences of the virus genomes using the software SnapGene v4.2.6.

7. Titration of the viral suspension

Titration of the viral suspension: determination of the concentration of infectious units per mL virus suspension was performed by TCID50 assay.

Cloning of TGX-04 (Ad5 3-D24-ICOSL) in more detail

To construct, amplify, purify, titrate and characterize an oncolytic adenovirus vector characterized by the following features:

24-bp deletion in E1 A CR2

- a sequence encoding ICOSL embedded into the E3 region

- a hybrid Ad5/3 fiber

Recombinant adenovirus TGX04 was constructed by reconstituting its entire genome in a cosmid (pTGX04). The following 3 DNA fragments were ligated to each other:

1 . The 27 kb Pacl-Spel fragment from pAd5/3-d24-WTE3

2. The 8833 bp Spel-Pacl fragment from pTGX103

3. The 8 kb Pad fragment from pAd5dSfi

Where:

pAd5/3-d24-WTE3 (pAd5/3-d24-GMCSF) - pTGX103 is a plasmid that contains the right end of the virus genome (Spel-Pacl sequence from pAd5/3.d24-WTE3) with the ICOSL coding sequence inserted between the BsiWI and Mfel sites in the E3 region

- pAd5dSfil is a cosmid that contains the entire Ad5 genome. The 8 kb Pad fragment provides the cosmid backbone for pTGX04

pTGX103 was constructed by inserting the 1070 bp BsiWI-Notl DNA fragment from pAd1 129-ICOSL between the corresponding sites of pTGX101

Where:

- pTGX101 is a plasmid that contains the right end of the Ad5/3.d24-WTE3 genome (Spel-Pacl fragment from pAd5/3.d24-WTE3)

- pAd1 129-ICOSL is a shuttle plasmid that contains the ICOSL coding sequencer between the BsiWI and Mfel site in the E3 region

pAd1 129-ICOSL was constructed by inserting a 969 bp synthetic dsDNA encoding ICOSL between the BsiWI and Mfel sites of pAd1 129-14

Where:

- pAd1 129-14 is a shuttle plasmid containing the WT Ad5 E3 region and a hybrid Ad 5/3 fiber

- gBIock-ICOSL (transcript variant 1 , 909 bp, NM_ 015259.4)

Cloning of TGX-05 (Ad5 3-D24-ADA) in more detail

To construct, amplify, purify, titrate and characterize an oncolytic adenovirus vector characterized by the following features:

24-bp deletion in E1 A CR2

a sequence encoding ADARB1 embedded into the E3 region

- a hybrid Ad5/3 fiber

Recombinant adenovirus TGX05 was constructed by reconstituting its entire genome in a cosmid (pTGX05). The following 3 DNA fragments were ligated to each other:

1. The 27 kb Pacl-Spel fragment from pAd5/3-d24-WTE3

2. The 10030 bp Spel-Pacl fragment from pTGX104

3. The 8 kb Pad fragment from pAd5dSfi

Where:

pAd5/3-d24-WTE3 (pAd5/3-d24-GMCSF) - pTGX104 is a plasmid that contains the right end of the virus genome (Spel-Pacl sequence from pAd5/3.d24-WTE3) with the ADARB1 coding sequence inserted between the BsiWI and Mfel sites in the E3 region

- pAd5dSfil is a cosmid that contains the entire Ad5 genome. The 8 kb Pad fragment provides the cosmid backbone for pTGX05

pTGX104 was constructed by inserting the 2267 bp BsiWI-Notl DNA fragment from pAd1 129-ADARB1 between the corresponding sites of pTGX101 :

Where:

- pTGX101 is a plasmid that contains the right end of the Ad5/3.d24-WTE3 genome (Spel-Pacl fragment from pAd5/3.d24-WTE3)

pAd1 129-ADARB1 is a shuttle plasmid that contains the ADARB1 coding sequence between the BsiWI and Mfel site in the E3 region

pAd1 129-ADARB1 was constructed by inserting a 2166 bp synthetic dsDNA encoding ADARB1 between the BsiWI and Mfel sites of pAd1 129-14:

Where:

- pAd1 129-14 is a shuttle plasmid containing the WT Ad5 E3 region and a hybrid Ad 5/3 fiber

- gBIock-ADARBI (GenBank # NM_001 1 12.3)

Cloning of TGX-11 (Ad5 3-D24-ICOSL-ADA) in more detail

To construct, amplify, purify, titrate and characterize an oncolytic adenovirus vector characterized by the following features:

24-bp deletion in E1 A CR2

- a dicistronic cassette expressing ICOSL and ADARB1 in place of the E3 region (2.7 kb Bglll deletion)

- a hybrid Ad5/3 fiber

Recombinant adenovirus TGX1 1 was constructed by reconstituting its entire genome in a cosmid (pTGX1 1 ). The following 3 DNA fragments were ligated to each other:

1. The 27 kb Pacl-Spel fragment from pAd5/3-d24-WTE3

2. The 10751 bp Spel-Pacl fragment from pTGX1 10c

3. The 8 kb Pad fragment from pAd5dSfi

Where: - pAd5/3-d24-WTE3 (pAd5/3-d24-GMCSF)

- pTGXH Oc is a plasmid that contains the right end of the virus genome (Spel-Pacl sequence from pAd5/3.d24-WTE3) with the CMV-ICOSL-IRES-ADARB1 cassette inserted in place of the E3 region

- pAd5dSfil is a cosmid that contains the entire Ad5 genome. The 8 kb Pad fragment provides the cosmid backbone for pTGX1 1

pTGX1 10c was constructed by ligating the following 3 DNA fragments to each other:

1 . the 1349 bp Csil-Rsrll fragment from pTGX101

2. the 6491 bp Rsrll-Nhel fragment from pTGX101

3. the 5482 bp Nhel-Csil fragment from pTGX1 10b

Where:

- pTGX101 is a plasmid that contains the right end of the Ad5/3.d24-WTE3 genome (Spel-Pacl fragment from pAd5/3.d24-WTE3)

- pTGX1 10b is a plasmid that contains the CMV-ICOSL-IRES-ADARB1 -SV40pA signal cassette in place of the E3 region (2.7 kb Bglll deletion)

pTGX1 10b was constructed by digesting pTGX1 10 with Spel, filling-in the sticky ends with Klenow, and religating both fragments with each other, in order to destroy the Spel sites flanking the CMV-ICOSL-IRES-ADARB1 cassette.

Where:

- pTGX1 10 is a plasmid that contains the CMV-ICOSL-IRES-ADARB1 -SV40pA signal cassette in place of the E3 region (2.7 kb Bglll deletion)

pTGX1 10 was constructed by inserting the following 4 fragments between the Sacll and Acc65l sites of pAd1 129-GrB-F:

1 . the 945 bp ICOSL coding sequence amplified by PCR using pAd1 129-ICOSL as template and primers 288-17 and 288-41

2. the 645 bp IRES-att element amplified by PCR using pAd1 129-IRES-att as template and primers 288-18 and 288-19

3. the 21 12 bp ADARB1 coding sequence isolated from plasmid pAd1 129-ADARB1 by BsiWI/Mfel digestion

4. the 174 bp SV40pA signal amplified by PCR using pAd1 129-GrB-F as template and primers 288-20 and 288-21 . Where:

pAd1 129-ICOSL was made for the construction of TGX04

pAd1 129-ADARB1 was made for the construction of TGX05

pAd1 129-GrB-F was made for the construction of TGX01

- pAd1 129-IRES-att is a plasmid that contains an attenuated version of the EMCV Internal Ribosome Entry Site (IRES)

Primers:

288-17:

ACACCGGG ACCG AT CCAGCCT CCGCGGGCCGCCACCAT GCGGCTGGGCAGT CCT GGACTGC

288-41 : T CAAACGT GGCCAGT G AGCT CT GT C

288-18: AGCTCACTGGCCACGTTTGACCCCTCTCCCTCCCCCCCCT

288-19: T CTT CAT CTT CT AT AT CCAT GT CG ACT CT AG AGG AT CCCG

288-20 : AGTT CT CACT CACGCCCT G AAACTT GTTT ATT GCAGCTT A

288-21 : T AGTT AAAGGG AAT AAG AT CGGT ACC.

Cell lines

Melanoma cell lines, A2058 (ATCC® CRL-1 147™), A375 (ATCC® CRL-1619™) and SK- MEL-2 (ATCC® HTB-68™), SK-MEL-28 (ATCC® HTB-72™) were cultured in DMEM high glucose medium supplemented with 10% FCS and penicillin/streptomycin. Trypsin-EDTA was used to detach and sub-culture cells at 80% confluence for in vitro assays.

Melanoma mouse model - humanized xenograft mouse model

Immunomodulatory properties (ability to infiltrate various immune cells, TILs into the tumor) generated by the treatment with viruses was assessed in humanized melanoma mouse model. We have previously developed and tested humanized mouse model based on NOD/Shi-scid/IL-2Rynull immunodeficient (NOG) with CD34+ FISC cells from human cord blood (Kuryk et al., 2018). The NOG mouse were engrafted with mesenchymal and hematopoietic stem cells. This model generates a closer representative of patients’ antitumor response. The strength of this approach is the ability to generate multiple hematopoietic lineages including T and B cells, dendritic cells and NK cells. Mice were injected subcutaneously with A2058 human melanoma tumor cells on both flanks and randomized into different treatment groups (n=8/group). The viruses were administered intratumorally (locally). After the course of a treatment (5 injections of the virus), mice were sacrificed and tumor dissected, infiltrated leukocytes phenotype was analyzed by flow cytometry.

Data analyses

All parameters were analysed using GraphPad Prism software (version 7). Statistical analyses have been performed using RM-one-way ANOVA followed by a Tukey post-test or T test (Mann Whitney Test).

EXAMPLE 1 In vitro - cell viability assay (MTS)

Cell viability was evaluated by using the MTS Cell Proliferation Assay kit (Abeam) according to the manufacturer’s instructions with the following modifications. MTS assay kit is a colorimetric method for the sensitive quantification of viable cells. It can be used to assess cell proliferation, cell viability and cytotoxicity. The MTS assay is based on the reduction of the MTS tetrazolium compound by viable mammalian cells to generate a coloured formazan dye that is soluble in cell culture media. This conversion is thought to be carried out by NAD(P)H-dependent dehydrogenase enzymes in metabolically active cells. The formazan dye is quantified by measuring the absorbance at 490 nm. Cells were seeded on 96 well plate at a concentration of 3000 and 5000 cells/well. Cells were incubated with and without 0.1 , 1 , 10, 100 and 1000 viral particles (VP) of tested virus. Treatment was initiated in a final volume of 200pL. 72 hours later, MTS assay was performed according to manufacturer’s protocol. The plates were shaken briefly on a shaker and absorbance measured at 490 nm. Absorbance values for untreated cells were set to correspond to 100% viability, and viability of treated cells was expressed as a percentage of the untreated control absorbance value.

Double transgene virus coding for ICOSL and ADA (TGX-1 1 ) showed anti-cancer superiority when administered at the concentration of 1000VP/cell (respectively 33,51 % of untreated cells) over single transgene viruses TGX-04 (coding for ICOSL, 60,94% of untreated cells) and TGX-05 (coding for ADA, 64,71 % of untreated cells) in vitro in A375 cell line. Importantly, the double transgene virus TGX-1 1 showed stronger anti-cancer effect than the combinatory therapy of TGX-04 plus TGX-05 (94,88%) (Figure 7a).

Double transgene vector showed also anti-cancer superiority (TGX-1 1 ) over single transgene vectors (TGX-04 and TGX-05) and the combinatory therapy of TGX-04 plus TGX-05 in SK-MEL-2 cell line when tested at the concentration of 1 and 1000 VP/cell (respectively for 1 VP/cell: 79,69%; 1 14,22%; 100,73%, 122,85% and 1000 VP/cell: 45,83%; 69,19%; 91 ,56%; 1 16,27% of untreated cells), (Figure 7b and Figure 7c).

Enhanced anticancer effect of TGX-1 1 (56,29 % of untreated cells) was recorded also in A2058 cell line also at the concentration of 1000 VP/cell over single transgene vectors TGX-04 and TGX-05 and the combinatory therapy of TGX-04 plus TGX-05 (respectively 67,72; 75,5% and 85,64% of untreated cells), (Figure 7d).

EXAMPLE 2 In vitro - apoptotic cell death

Complementary analysis on treatment efficacy focusing on evaluation of early and late apoptotic cells were carried out as well. Staining with Annexin V conjugates were carried out in order to identify cell membrane changes associated with early apoptosis. Cells were seeded on a 24 well plate at a density of 1 .5x10M cells per well for A2058 cell line. Treatment was initiated on the same day of plating and the amount of apoptotic and necrotic cells was measured 72 hours after the beginning of the treatment by flow cytometry, using the dead cell apoptosis kit, according to the manufacturer’s instructions. Double transgene virus coding for ICOSL and ADA (TGX-1 1 ) showed anti-cancer superiority (induction of apoptotic cell death) when administered at the concentration of 1000 VP/cell (respectively 175,43% of untreated cells) over single transgene viruses TGX- 04 (coding for ICOSL, 120,76% of untreated cells), TGX-05 (coding for ADA, 159,17% of untreated cells) and the combinatory therapy of TGX-04 plus TGX-05 (162,75% of untreated cells) in vitro in A2058 cell line (Figure 8a).

EXAMPLE 3 In vitro - immunogenetic cell death (ICD)

It has been shown that cancer cell death can be immunogenic or non-immunogenic (Tesniere et al., 2008). Immunogenic cell death comprises changes in the structure of the cell surface and leads to the release of pro-immunogenic factors (Kroemer et al., 2013). Subsequently it attracts antigen presenting cells (APCs) to take up tumor antigens, process them, and finally elicit anti-tumor immune response (specific anti-tumor T cells) (Tesniere et al., 2008). The success of the cancer treatment relies on the induction of immunogenic tumor cell death and induction of anti-tumor immune responses (Kepp et al., 201 1 ). Cell death can be evaluated by the presence of immunogenic cell death biomarkers such as calreticulin (CRT) on the outer plasma membrane, followed by extracellular release of high-mobility group box 1 protein (HM-GB1 ) and adenosine triphosphate (ATP) (Kepp et al., 201 1 , Obeid et a!., 2007, Kroemer et al., 2013).

Cells were seeded on 24 well plate at a density of 2.5x10M cells per well for A375 and SK-MEL-2 cell lines and at a density of 1 .5x10M cells per well for A2058 and SK-MEL-28 cell lines. Treatment with the viruses was initiated on the day of the plating. 72 hours after the beginning of the treatment, supernatants from treated cells or untreated cells were harvested. Calreticulin was stained at the surface of the cells with an anti-calreticulin antibody according to the manufacturer’s instructions (Abeam). ATP quantity (from supernatant) was evaluated using the ATP assay kit according to the supplier instructions (Abeam). HMGB-1 was measured by ELISA (IBL international) according to manufacturer’s instruction.

Double transgene virus coding for ICOSL and ADA (TGX-1 1 ) showed enhanced immunogenic cell death (ATP release) when administered at the concentration of 0,1 VP/cell over single transgene viruses TGX-04 (coding for ICOSL), TGX-05 (coding for ADA) and the combinatory therapy of TGX-04 plus TGX-05 in A375 cell line (Figure 9a). Similar superiority of double transgene virus (ATP release) was observed when administered at the concentration of 10 and 0,1 and VP/cell in SK-MEL-2 and SK-MEL-28 cell lines, respectively (Figure 9b and Figure 9c).

Double transgene virus coding for ICOSL and ADA (TGX-1 1 ) showed enhanced immunogenic cell death (Calreticulin expression level, 5,31 % of untreated cells) when administered at the concentration of 1000 VP/cell over single transgene viruses TGX-04 (coding for ICOSL, 4,08% of untreated cells), TGX-05 (coding for ADA, 2,17% of untreated cells) and the combinatory therapy of TGX-04 plus TGX-05 (3,54% of untreated cells) in A375 cell line (Figure 10).

Double transgene virus coding for ICOSL and ADA (TGX-1 1 ) showed enhanced immunogenic cell death (HMGB1 release, 9,69 ng/ml of untreated cells) when administered at the concentration of 10 VP/cell over single transgene viruses TGX-04 (coding for ICOSL, 8,53 ng/ml of untreated cells), TGX-05 (coding for ADA, 6,59 ng/ml of untreated cells) and the combinatory therapy of TGX-04 plus TGX-05 A2058 (4,4 ng/ml of untreated cells) in A2058 cell line (Figure 1 1 ).

EXAMPLE 4 In vivo - humanized melanoma mouse model The treatment with double transgene virus coding for ICOSL and ADA (TGX-1 1 ) resulted in enhanced infiltration of Granzyme B CD8+ T, PD-1 CD8+T and CD4+ T cells into the tumor mass and was superior over the effect of the single transgene viruses TGX-04 (coding for ICOSL), TGX-05 (coding for ADA) and the combinatory therapy of TGX-04 plus TGX-05 in A2058 humanized melanoma mouse model (Figure 12a, Figure 12b and Figure 12c).

REFERENCES

Allard, B., Beavis, P. A., Darcy, P. K. & Stagg, J. 2016. Immunosuppressive Activities of Adenosine In Cancer. Curr Opin Pharmacol, 29, 7-16.

Fu, T., He, Q. & Sharma, P. 201 1 . The lcos/lcosl Pathway is Required for Optimal Antitumor Responses Mediated by Anti-Ctla-4 Therapy. Cancer Res, 71 , 5445-54.

Hutloff, A., Dittrich, A. M., Beier, K. C., Eljaschewitsch, B., Kraft, R., Anagnostopoulos, I. & Kroczek, R. A. 1999. Icos is an Inducible T-Cell Co-Stimulator Structurally and Functionally Related To Cd28. Nature, 397, 263-6.

Kepp O, Galluzzi L, Martins I, Schlemmer F, Adjemian S, Michaud M, Sukkurwala AQ, Menger L, Zitvogel L, Kroemer G. 201 1 . Molecular determinants of immunogenic cell death elicited by anticancer chemotherapy. Cancer Metastasis Rev, 30, 61 -9.

Khayyamian, S., Hutloff, A., Buchner, K., Grafe, M., Henn, V., Kroczek, R. A. & Mages, H. W. 2002. Icos-Ligand, Expressed on Human Endothelial Cells, Costimulates Th1 And Th2 Cytokine Secretion By Memory Cd4+ T Cells. Proc Natl Acad Sci U S A, 99, 6198-203.

Kroemer G, Galluzzi L, Kepp O, Zitvogel L. 2013. Immunogenic cell death in cancer therapy. Annu Rev Immunol, 31 , 51 -72.

Kuryk, L., Haavisto, E., Garofalo, M., Capasso, C., Hirvinen, M., Pesonen, S., Ranki, T., Vassilev, L. & Cerullo, V. 2016. Synergistic Anti-Tumor Efficacy of Immunogenic Adenovirus Oncos-102 (Ad5/3-D24-Gm-Csf) And Standard of Care Chemotherapy in Preclinical Mesothelioma Model. Int J Cancer, 139, 1883-93.

Kuryk, L., Moller, A.-S. W. & Jaderberg, M. 2018. Combination of Immunogenic Oncolytic Adenovirus Oncos-102 With Anti-Pd-1 Pembrolizumab Exhibits Synergistic Antitumor Effect in Humanized A2058 Melanoma Hunog Mouse Model. Oncoimmunology, 8, 1 -1 1 .

Kutryb-Zajac, B., Koszalka, P., Mierzejewska, P., Bulinska, A., Zabielska, M. A., Brodzik, K., Skrzypkowska, A., Zelazek, L., Pelikant-Malecka, I., Slominska, E. M. & Smolensk!, R. T. 2018. Adenosine Deaminase Inhibition Suppresses Progression Of 4t1 Murine Breast Cancer by Adenosine Receptor-Dependent Mechanisms. J Cell Mol Med, 22, 5939-5954.

Londono-R, L. M., Cowell, J., Wang, L., Zhao, Q., Huang, L., Thanos, C., Labarre, M. J., Li, X. & Cekic, C. 2018. Abstract 1755: Pegylated Adenosine Deaminase (Ada2) Prevents Adenosine-Mediated Increase in Tumor Growth and Improves Antitumor Immune Responses. Cancer Research, 78, 1755-1755. Muller-Haegele, S., Muller, L. & Whiteside, T. L. 2014. Immunoregulatory Activity of Adenosine and Its Role in Human Cancer Progression. Expert Rev Clin Immunol, 10, 897- 914.

Obeid M, Tesniere A, Ghiringhelli F, Fimia GM, Apetoh L, Perfettini JL, Castedo M, Mignot G, Panaretakis T, Casares N, Metivier D, Larochette N, van Ended P, Ciccosanti F, Piacentini M, Zitvogel L, Kroemer G. 2007. Calreticulin exposure dictates the immunogenicity of cancer cell death. Nat Med, 13, 54-61 .

Ohta, A. 2016. A Metabolic Immune Checkpoint: Adenosine in Tumor Microenvironment. Front Immunol, 7, 109.

Tesniere A, Panaretakis T, Kepp O, Apetoh L, Ghiringhelli F, Zitvogel L, Kroemer G.2008. Molecular characteristics of immunogenic cancer cell death. Cell Death Differ, 15, 3-12.