WO2018078220A1 - Interleukin 8 (il-8) as a prognostic and predictive biomarker and compositions and vectors for use in oncolytic immunotherapy - Google Patents

Abstract

The present invention relates to an oncolytic viral vector comprising a nucleic acid sequence encoding an anti-interleukin 8 neutralizing antibody.Further, the present invention relates to use of interleukin 8 as a prognostic and predictive biomarkerin oncolytic immunotherapy. In this respect, the present invention is directed to a method ofanalysing whether a subject suffering from a cancer is responsive or non-responsive to the treatment with an oncolytic viral vectorand to a method of monitoringefficacy of an oncolytic immunotherapy.

Description

INTERLEUKIN 8 (IL-8) AS A PROGNOSTIC AND PREDICTIVE BIOMARKER AND COMPOSITIONS AND VECTORS FOR USE IN ONCOLYTIC IMMUNOTHERAPY

FIELD OF THE INVENTION

The present invention relates to the fields of life sciences and medicine. Specifically, the invention relates to cancer therapies of humans. More specifically, the present invention relates to an oncolytic viral vector comprising a nucleic acid sequence encoding an anti-interleukin 8 neutralizing antibody. Further, the present invention relates to use of interleukin 8 as a prognostic and predictive biomarker in oncolytic immunotherapy. In this respect, the present invention is directed to a method of analysing whether a subject suffering from a cancer is responsive or non- responsive to the treatment with an oncolytic viral vector and to a method of monitoring efficacy of an oncolytic immunotherapy.

BACKGROUND OF THE INVENTION

The original discovery of the interleukin 8 (IL-8) pathway was made in human monocytes where it functioned as a neutrophil chemoattractant (Mukaida, Harada et al. 1998). Since then, the pathway has been identified also to be active in different cancers and one of its effects relates to angiogenesis (Xie 2001 ). In tumors, IL-8 has also been proposed to have a role in promotion of survival, cell proliferation, chemoresistance and metastasis formation (Waugh DJ, Wilson C 2008, Sanmamed MF, Carranza-Rua O et al. 2014). There have been several attempts to therapeutically target the IL-8 pathway in the context of cancer and other diseases, and some approaches have demonstrated promising efficacy (Mian BM, Dinney CP et al. 2003, Merritt WM, Lin YG et al. 2008, Mahler DA, Huang S et al. 2004, Skov L, Beurskens FJ et al. 2008). However, until now no therapies affecting IL-8 have been approved for clinical use.

A large number of different viruses have been shown to promote IL-8 production and it is a key mediator of neutrophil dependent pathogenesis in many viral infections (Murayama, Mukaida et al. 1998, Medin, Fitzgerald et al. 2005, Sato, Miura et al. 2005, Collins, Graham 2008). Some adenovirus serotypes, notably serotypes 7 and 19 (Natarajan, Rajala et al. 2003, Booth, Coggeshall et al. 2004, Rajaiya, Xiao et al. 2008), also upregulate IL-8 and recruit neutrophils to the infection site. Interestingly, considering the use of viruses in cancer immunotherapy, systemic neutrophil expansion has been recently linked to metastasis formation in murine tumor models (Coffelt, Kersten et al. 2015). To our knowledge, there are no human data about the effects of IL-8 in the context of oncolytic viruses. We have previously demonstrated that high levels of pre-existing activity of innate immune system can have harmful effects on the survival of the patients receiving oncolytic adenovirus (Taipale, Liikanen et al. 2016b). Additionally, high pre-treatment levels of HMGB1 , which regulates inflammatory response and is associated with autophagic cell death, have been linked to worse survival and therapeutic efficacy (Liikanen, Koski et al. 2015). Thus it is intriguing that HMGB1 has been linked to the IL-8 pathway in an immunologically relevant manner (Andersson, Wang et al. 2000) in the context of cancer (Dejean, Foisseau et al. 2012). A recent study indicated benefits for combining HMGB1 and IL-8 inhibition in experimental gastric cancer models (Chung, Jang et al. 2015). Similar combinations could yield increased anti-tumor effects also together with adenovirus treatments.

In addition to HMGB1 , we have found that the expression of TIM-3 in pre-treatment tumor samples is associated with worse prognosis in adenovirus treated patients (Taipale, Liikanen et al. 2016b). Previous studies on the function of TIM-3 in the context of cystic fibrosis have demonstrated first that TIM-3 activation induces IL-8 production (Vega-Carrascal, Reeves et al. 201 1 ), but also that IL-8 promotes expression of TIM-3 (Vega-Carrascal, Bergin et al. 2014). Thus, although the exact interdependencies of IL-8 and TIM-3 are still unclear, they may relate to phenomena that are important for the efficacy of oncolytic adenovirus specifically and anti-tumor immunotherapy more generally.

Past efforts to target IL-8 in cancer treatment have displayed promising results in preclinical models (Huang S, Mills L et al. 2002, Merritt WM, Lin YG et al. 2008, Mian BM, Dinney CP et al. 2003), but clinical trials for cancer with these agents have not been completed. One interesting approach utilized replication- incompetent oncolytic adenoviruses expressing short hairpin RNAs against IL-8 (Yoo, Kim et al. 2008). This treatment was shown to inhibit tumor progression and metastases in immunodeficient mouse xenograft models. Mice do not express IL-8 or any I L-8-I ike-protein (mice lack this cytokine completely), therefore the study was limited to the effect of the adenovirus construct on IL-8 expression inside the infected human tumor cells. One possible problem of previous IL-8 targeting treatments could be the focusing on tumor growth inhibition and antiangiogenic properties of the treatments, instead of their immunotherapeutic potential. In addition to cancer, IL-8 blocking antibodies have been studied in the context of other diseases, and they have thus far demonstrated a favorable safety profile in the first clinical trials for COPD and palmoplantar pustulosis (Mahler DA, Huang S et al. 2004, Skov L, Beurskens FJ et al. 2008). In the prior art, WO2004058797 describes isolated human monoclonal antibodies which bind to human IL-8. The human antibodies can be produced in a hybridoma, transfectoma or in a non-human transgenic animal, e.g., a transgenic mouse, capable of producing multiple isotypes of human monoclonal antibodies by undergoing V-D-J recombination and isotype switching. A method for treating or preventing cancer with human monoclonal IL-8 antibodies is also disclosed.

In WO2014137141 , an RNA aptamer binding to IL-8 is disclosed. An aptamer specifically binding to IL-8 was selected from an RNA library, and it was confirmed that the aptamer binds to IL-8, with high binding affinity, and thereby inhibits the movement and intracellular signal transduction of IL-8-induced neutrophil and the invasion of cancer cells. It was also confirmed that the aptamer binding to IL-8 can be effectively used for preventing and treating cancer or inflammatory diseases.

WO2014170389 relates to oncolytic adenoviral vectors alone or together with therapeutic compositions for therapeutic uses and therapeutic methods for cancer. A separate administration of adoptive cell therapeutic composition and oncolytic adenoviral vectors is disclosed. Adoptive cell therapies (ACT) are a potent approach for treating cancer but also for treating other diseases such as infections and graft versus host disease. Adoptive cell transfer is the passive transfer of ex vivo grown cells, most commonly immune-derived cells, into a host with the goal of transferring the immunologic functionality and characteristics of the transplant.

WO2016146894 discloses an oncolytic adenoviral vector encoding a bispecific monoclonal antibody.

After years of development, the oncolytic viruses are currently starting to be used as cancer therapeutics (Kaufman HL, Kohlhapp FJ et al. 2015). Although there have been some discoveries relating to the mechanisms of action and factors that influence the efficacy of the viruses (Russell, Peng et al. 2012, Lichty, Breitbach et al. 2014, Taipale, Liikanen et al. 2016a), there is still a need to identify pathways that determine the overall response to virotherapy. In clinical trials, oncolytic viruses have demonstrated a favorable safety profile and promising efficacy (Zamarin D, Pesonen S 2015). However, there is still room for improvement in the responses, especially in patients with a significant metastasis burden. Further characterization of pathways related to the activity of oncolytic viruses could reveal potential targets for improving the efficacy of virotherapy. Moreover, understanding the key molecular mechanisms would help selection of the right patients for therapy. Therefore, the efficacy of oncolytic viral vectors, either alone or together with other therapies, can still be improved. Increased specificity and sufficient tumor killing ability of therapies in general are warranted.

The present invention provides efficient tools and methods for cancer therapeutics by utilizing specific viral vectors, e.g. with adoptive cell therapies.

SUMMARY OF THE INVENTION

The aim of this invention was to examine how IL-8 activity shapes the responses to treatment with oncolytic immunotherapy. We analyzed baseline serum IL-8 in 103 patients treated with oncolytic adenovirus and measured post-treatment changes in IL-8. We then compared these findings to available survival and response data. In additional analyses, we compared serum IL-8 levels to tumor type and tumor load of the patients. We also assessed how different treatment characteristics affect IL-8 changes. Due to the close connection between IL-8 and neutrophils, their associations were investigated in peripheral blood. To evaluate the role of tumor-derived IL-8 on survival, expression levels of IL-8 and its receptors CXCR1 and CXCR2 were quantified by RNA microarrays from pre- and post- treatment tumor samples of oncolytic virus treated patients. We also analyzed the anti-tumor and immunostimulatory activity of a combination of IL-8 blockade and oncolytic adenovirus in tumors samples. Finally, based on the accumulated results, we describe a novel oncolytic virus coding for IL-8 neutralizing antibody.

The invention is based on the surprising findings that high baseline serum IL-8 concentration is independently associated with poor prognosis in an oncolytic immunotherapy and that a decrease in IL-8 concentration after treatment with oncolytic adenovirus also predicted better overall survival. Normal baseline IL-8 was also associated with improved prognostic potential of anti-tumor T cell activity and neutrophil-to-lymphocyte ratio. These results indicate a role for IL-8 as a biomarker in oncolytic virotherapy, but also provide a rationale for targeting IL-8 to improve the efficacy of an oncolytic virus treatment. Indeed, a combination treatment with adenovirus and anti-IL-8 antibody showed increased cell killing indicating that tumors are susceptible to the combination of oncolytic immunotherapy and IL-8 neutralizing antibody.

Accordingly, an object of the present invention is to provide simple methods and tools for overcoming the problems of inefficient, unsafe and unpredictable cancer therapies. In one embodiment, the invention provides novel methods and means for cell therapy. The objects of the invention are achieved by specific viral vectors, methods and arrangements, which are characterized by what is stated in the independent claims. The specific embodiments of the invention are disclosed in the dependent claims.

Specifically, the present invention provides an oncolytic viral vector comprising a nucleic acid sequence encoding an anti-interleukin 8 (IL-8) neutralizing antibody. The present invention also provides a pharmaceutical composition comprising said oncolytic vector and at least one of the following: a physiologically acceptable carrier, buffer, excipient and stabilizer. A particular aim of the present invention is to provide said oncolytic viral vector or pharmaceutical composition for use in the treatment of cancer.

Another aim of the present invention is to provide an anti-interleukin 8

(IL-8) neutralizing antibody and an oncolytic adenoviral vector for use in the treatment of cancer.

Another aim of the present invention is to provide a role for IL-8 as a biomarker in oncolytic virotherapy. Accordingly, the present invention is directed to a method of detecting and analysing whether a subject suffering from a cancer is responsive or non-responsive to the treatment with an oncolytic viral vector, the method comprising the steps of:

- providing a biological sample taken from the subject;

- measuring the level of interleukin 8 (IL-8) in said sample; and

- selecting the subject for the cancer therapy with said oncolytic viral vector, wherein the selection is based on the level or a change of the level of interleukin 8 (IL-8) in said sample.

The present invention is also directed to a method of monitoring efficacy of a cancer therapy with an oncolytic viral vector, the method comprising the steps of:

- providing a biological sample, preferably a blood or tumor sample, taken from a patient treated with an oncolytic viral vector; and

- measuring the level of interleukin 8 (IL-8) in said sample.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Overall survival and treatment responses in patients with normal and high pre-treatment IL-8 levels in blood. Patients were grouped based on the laboratory reference value for normal IL-8 (<62ng/l). Panel a: Overall survival was significantly longer in patients with normal IL-8 before treatment (n=53, median OS 147 days) when compared with high IL-8 patients (n=50, median OS 93 days, p<0.001 ). Panel b: Imaging response information was available for 59 patients, with baseline IL-8 measurements. No significant difference in disease control rate was observed between the IL-8 groups (p=0.182).

Figure 2. Overall survival in patients with low or high baseline GM- CSF, IL-10, IL-6 and TNFa. Panels a-d: Patients were grouped to high and low baseline groups using the median baseline concentration as cutoff. No significant differences in overall survival between the groups were found.

Figure 3. IL-8 levels in patients with different tumor characteristics and baseline leucocyte counts. Panel a: Mean pre-treatment IL-8 concentrations in patients with different tumor types. The differences were not considered significant. Error bars are shown as mean + SEM in all panels. Panel b: Scatter plot presenting IL-8 levels in patients with high and low tumor load scores. Vertical line indicates sample mean. The difference was not significant (p=0.187). Panel c: Mean IL-8 concentrations in patients with high and low lymphocyte/neutrophil counts. Patients were grouped based on median lymphocyte/neutrophil count. Difference between high and low neutrophil count patients was not significant (p=0.085).

Figure 4. Mean IL-8 concentrations 30 days after treatment in different IL-8 change groups. Patients were assigned to groups based on methodology detailed in materials and methods. Mean concentrations as percentage of the baseline value are presented for each group in different post- treatment timeframes.

Figure 5. Effect of IL-8 change status on overall survival and treatment responses. IL-8 change was determined from pre- and post-treatment blood IL-8 measurements as described in materials and methods. Panel a: Overall survival in different IL-8 change groups. Median survivals were 167, 71 and 120 days for decrease, increase and no change groups, respectively (p<0.001 ). Panel b: Disease control rates for patients with IL-8 decrease (n=9), increase (n=31 ) and no change (n=16). Fisher's exact test p=0.066 (after pooling no change and increase groups). Panel c: Separate disease control rates in IL-8 change groups for patients with normal or high baseline IL8.

Figure 6. Proportions of IL-8 changes in different treatment characteristics groups. Panels a-c: Patients were grouped based on viral capsid, viral transgene or concomitant treatment used in the first adenovirus treatment. The differences between the groups were not considered significant.

Figure 7. Tumor IL-8 and IL-8 receptor mRNA expression. Panel a: mRNA expression for IL-8 and its receptors CXC chemokine receptor 1 (CXCR1 ) and 2 (CXCR2) was quantified from pre- and post-treatment tumor samples. Expression is presented as log2-transformed values. Panel b: Overall survival in different IL-8 expression groups. Patients were grouped based on the pre-treatment IL-8 expression level. Median OS was 174 days in the low expression group and 72 days in the high expression group (n=15, p=0.058). Panel c: IL-8 expression change was determined as the difference between pre- and post-treatment expression values. No significant difference in survival between decrease and increase groups was found.

Figure 8. Overall survival in patients with different baseline T cell activity status, neutrophil-to-lymphocyte ratio (NLR) and IL-8 levels. Patients with increase or decrease in post-treatment anti-survivin were included in the T cell activity group, while patients with no change in the ELISPOT were assigned to anergy group. Panel a: Median overall survivals in high baseline IL-8 patients were 104 and 1 14 days in anti-survivin T cell anergy and activity groups, respectively. The difference was not considered significant. Panel b: for patients with normal baseline IL-8, median overall survivals were 104 and 1 14 days in anti-survivin T cell anergy and activity groups, respectively (p=0.052). Panel c: Pre-treatment NLR and IL-8 values were used to stratify patients into different groups. Overall survival was significantly increased in patients with low IL-8 and low NLR (p<0.001 ).

Figure 9. Results from cell killing (MTS) assay in ovarian tumor cell suspension obtained from patient OVCA1. Cell viability was measured at day 7 after the start of incubation. Viability is presented as percentage of mock group cell viability. Ad denotes Ad5/3-d24. Asterisks indicate the significance of findings: ^* (p<0.05), ^** (p<0.01 ), ^*** (p<0.001 ), ^**** (p<0.0001 ).

Figure 10. Results from cell killing (MTS) assay in ovarian tumor cell suspension obtained from patient OVCA2. Cell viability was measured from two time points after the start of incubation. Viability is presented as percentage of mock group cell viability. Ad denotes Ad5/3-d24. Asterisks indicate the significance of findings: ^* (p<0.05), ^** (p<0.01 ), ^*** (p<0.001 ), ^**** (p<0.0001 ). Panel a: Viability at day 7. Panel b: Viability at day 13.

Figure 11. Results from cell killing (MTS) assay in ovarian tumor cell suspension obtained from patient OVCA4. Cell viability was measured at day 12 after the start of incubation. Viability is presented as percentage of mock group cell viability. Asterisks indicate the significance of findings: ^* (p<0.05), ^** (p<0.01 ), ^*** (p<0.001 ), ^**** (p<0.0001 ).

Figure 12. Results from cell killing (MTS) assay in ovarian tumor cell suspension obtained from patient OVCA5. Cell viability was measured from two time points after the start of incubation. Viability is presented as percentage of mock group cell viability. Asterisks indicate the significance of findings: ^* (p<0.05), ^** (p<0.01 ), ^*** (p<0.001 ), ^**** (p<0.0001 ). Panel a: Viability at day 7. Panel b: Viability at day 1 1 .

Figure 13. Results from cell killing (MTS) assay in ovarian tumor cell suspension obtained from patient OVCA6. Cell viability was measured from two time points after the start of incubation. Viability is presented as percentage of mock group cell viability. Ad denotes Ad5/3-d24. Asterisks indicate the significance of findings: ^* (p<0.05), ^** (p<0.01 ), ^*** (p<0.001 ), ^**** (p<0.0001 ). Panel a: Viability at day 8. Panel b: Viability at day 12.

Figure 14. Results from cell killing (MTS) assay in ovarian tumor cell suspension obtained from patient OVCA7. Cell viability was measured from two time points after the start of incubation. Viability is presented as percentage of mock group cell viability. Ad denotes Ad5/3-d24. Asterisks indicate the significance of findings: ^* (p<0.05), ^** (p<0.01 ), ^*** (p<0.001 ), ^**** (p<0.0001 ). Panel a: Viability at day 7. Panel b: Viability at day 10.

Figure 15. T cell proliferation in ovarian tumor derived TIL and TAN cultures obtained from human patients. Panels a-f: T cell proliferation was measured after 6 day incubation of TIL-TAN co-cultures or TILs alone. Bars represent the percentage of T cells in the culture. Ad = Ad5/3-d24. rlL8 = recombinant IL-8. Asterisks indicate the significance of findings: ^* (p<0.05), ^** (p<0.01 ), ^*** (p<0.001 ), ^**** (p<0.0001 ).

Figure 16. Helper T cell activation in ovarian tumor derived TIL and TAN cultures obtained from human patients. Helper T cell activation was measured after 6 day incubation of TIL-TAN co-cultures or TILs alone. Bars represent the percentage of T cells in the culture. Ad = Ad5/3-d24. rlL8 = recombinant IL-8. Asterisks indicate the significance of findings: ^* (p<0.05), ^** (p<0.01 ), ^*** (p<0.001 ), ^**** (p<0.0001 ). Panel a: Activation marker CD25 was used for samples from patient OVCA1 Panels b-f: Activation marker CD69 was used for samples from other patients.

Figure 17. Cytotoxic T cell activation in ovarian tumor derived TIL and TAN cultures obtained from human patients. Cytotoxic T cell activation was measured after a 6 day incubation of TIL-TAN co-cultures or TILs alone. Bars represent the percentage of activated CD25-positive T cells in the culture. Ad = Ad5/3-d24. rlL8 = recombinant IL-8. Asterisks indicate the significance of findings: ^* (p<0.05), ^** (p<0.01 ), ^*** (p<0.001 ), ^**** (p<0.0001 ). Panel a: Activation marker CD25 was used for samples from patient OVCA1 Panels b-f: Activation marker CD69 was used for samples from other patients. Figure 18. IL-8 concentrations in ovarian tumor derived TIL and TAN cultures obtained from patient OVCA1. IL-8 concentration was measured after a 24 hour incubation from TIL-TAN co-cultures or TILs/TANs alone. Ad = Ad5/3-d24. rlL8 = recombinant IL-8. Asterisks indicate the significance of findings: ^* (p<0.05), ^** (p<0.01 ), ^*** (p<0.001 ), ^**** (p<0.0001 ).

Figure 19. IL-8 concentrations in ovarian tumor derived TIL and TAN cultures obtained from patient OVCA2. IL-8 concentration was measured after a 24 hour incubation from TIL-TAN co-cultures or TILs/TANs alone. Ad = Ad5/3-d24. rlL8 = recombinant IL-8. Asterisks indicate the significance of findings: ^* (p<0.05), ^** (p<0.01 ), ^*** (p<0.001 ), ^**** (p<0.0001 ).

Figure 20. Illustration of the structure of the novel adenovirus constructs TILT-801 and TILT-802.

Figure 21. Illustration of the cloning procedure of TILT-801 and

TILT-802.

Figure 22. Hypothetical results from MTS assay with adenovirus coding for IL-8 antibody. Antibody-coding virus is tested together with backbone Ad5/3-d24 virus and Ad5/3-luc1 , which codes for luciferase. Viruses are tested in different VP/cell ratios. Cell viability is shown as percentage of 0 VP/cell viability (first markers). Panel a: Results from an assay using a cancer cell line that is not dependent on IL-8 pathway activity. Panel b: Results from an assay using a cancer cell line that is sensitive for IL-8 blockade.

Figure 23. Hypothetical results from nude mice experiment with human tumor xenografts. Nude mice are implanted with IL-8 sensitive human cancer cells. After the tumors have grown to an injectable size, they are injected with Ad5/3-d24, Ad5/3-d24-alL8 or phosphate buffered saline (mock). Tumor growth is measured and reported as percentage of the original size.

Figure 24. Hypothetical results from experiment on cytotoxic T cell activation in ovarian cancer derived TIL and TAN cultures obtained from a human patient. Cytotoxic T cell activation is measured after a 6 day incubation of TIL-TAN co-cultures or TILs alone. Bars represent the percentage of activated CD25-positive cytotoxic T cells in the culture. rlL8 = recombinant IL-8.

DETAILED DESCRIPTION OF THE INVENTION

Viral vectors

Oncolytic viral vectors are therapeutically useful anticancer viruses that can selectively infect and destroy cancer cells. Most current oncolytic viruses are adapted or engineered for tumour selectivity, although there are viruses, such as reovirus and Mumps virus, having natural preference for cancer cells. Many engineered oncolytic viral vectors take advantage of tumor-specific promoter elements making them replication competent only in cancer cells. Surface markers expressed selectively by cancer cells can also be targeted by using them as receptors for virus entry. A number of viruses including adenovirus, reovirus, measles, herpes simplex, Newcastle disease virus and vaccinia have now been clinically tested as oncolytic agents.

Preferably, the oncolytic vector used in the present invention is an adenoviral vector suitable for treating a human or animal. As used herein "an oncolytic adenoviral vector" refers to an adenoviral vector capable of infecting and killing cancer cells by selective replication in tumor versus normal cells.

In one embodiment of the invention, the adenoviral vectors are vectors of human viruses. In one embodiment the adenoviral vectors are selected from the group consisting of Ad5, Ad3 and Ad5/3 vectors. As used herein, expression "adenovirus serotype 5 (Ad5) nucleic acid backbone" refers to the genome of Ad5. Similarly "adenovirus serotype 3 (Ad3) nucleic acid backbone" refers to the genome of Ad3. "Ad5/3 vector" refers to a chimeric vector comprising or having parts of both Ad5 and Ad3 vectors. In a specific embodiment a backbone of the adenoviral vector is an adenovirus serotype 5 (Ad5) or serotype 3 (Ad3) nucleic acid backbone with specific mutations. E.g. fiber areas of the vector can be modified. In one embodiment the backbone is Ad5 nucleic acid backbone further comprising an Ad3 fiber knob. In other words the construct has the fiber knob from Ad3 while the remainder or the most of the remainder of the genome is from Ad5.

The adenoviral vectors may be modified in any way known in the art, e.g. by deleting, inserting, mutating or modifying any viral areas. The vectors are made tumor specific with regard to replication. For example, the adenoviral vector may comprise modifications in E1 , E3 and/or E4 such as insertion of tumor specific promoters (e.g. to drive E1 ), deletions of areas (e.g. the constant region 2 of E1 as used in "Δ24", E3/gp19k, E3/6.7k) and insertion of a transgene or transgenes.

One approach for generation of a tumor specific oncolytic adenovirus is engineering a 24 base pair deletion (Δ24) affecting the constant region 2 (CR2) of E1 . In wild type adenovirus CR2 is responsible for binding the cellular Rb tumor suppressor/cell cycle regulator protein for induction of the synthesis (S) phase i.e. DNA synthesis or replication phase. The interaction between pRb and E1A requires amino acids 121 to 127 of the E1A protein conserved region. The vector may comprise a deletion of nucleotides corresponding to amino acids 122-129 of the vector according to Heise C. et al. (2000, Nature Med 6, 1 134-1 139) and Fueyo J. et al. (2000, Oncogene 19(1 ):2-12). Viruses with the Δ24 are known to have a reduced ability to overcome the G1 -S checkpoint and replicate efficiently only in cells where this interaction is not necessary, e.g. in tumor cells defective in the Rb- p16 pathway, which includes most if not all human tumors. In one embodiment of the invention the vector comprises a 24 bp deletion (Δ24) in the Rb binding constant region 2 of adenoviral E1 (See figure 20).

It is also possible to replace E1 A endogenous viral promoter for example by a tumor specific promoter. For instance, E2F1 (e.g. in Ad5 based vector) or hTERT (e.g. in Ad3 based vector) promoter can be utilized in the place of E1A endogenous viral promoter. The vector may comprise E2F1 promoter for tumor specific expression of E1A.

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. In one embodiment of the invention the deletion of a nucleic acid sequence in the E3 region of the oncolytic adenoviral vector is a deletion of viral gp19k and 6.7k reading frames. The gp19k/6.7K deletion in E3 refers to a deletion of 965 base pairs from the adenoviral E3A region. In a resulting adenoviral construct, both gp19k and 6.7K genes are deleted (Kanerva A et al. 2005, Gene Therapy 12, 87-94). The gp19k gene product is known to bind and sequester major histocompatibility complex I (MHC1 , known as HLA1 in humans) molecules in the endoplasmic reticulum, and to prevent the recognition of infected cells by cytotoxic T-lymphocytes. Since many tumors are deficient in HLA1/MHC1 , deletion of 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.

In one embodiment of the invention, the transgene, i.e. a gene encoding an anti-interleukin 8 (IL-8) neutralizing antibody, is placed into a gp19k/6.7k deleted E3 region, under the E3 promoter. This restricts transgene expression to tumor cells that allow replication of the virus and subsequent activation of the E3 promoter. In a specific embodiment a nucleic acid sequence encoding an anti-interleukin 8 (IL-8) neutralizing antibody, preferably comprising a single chain variable fragment (scFv), is inserted into the place of the deleted nucleic acid sequence of viral gp19k and 6.7k reading frames. In another embodiment of the invention E3 gp19k/6.7k is kept in the vector but one or many other E3 areas have been deleted (e.g. E3 9-kDa, E3 10.2 kDa, E3 15.2 kDa and/or E3 15.3 kDa).

E3 promoter may be any exogenous (e.g. CMV or E2F promoter) or endogenous promoter known in the art, specifically the endogenous E3 promoter. Although the E3 promoter is chiefly activated by replication, some expression occurs when E1 is expressed. As the selectivity of Δ24 type viruses occurs post E1 expression (when E1 is unable to bind Rb), these viruses do express E1 also in transduced normal cells. Thus, it is of critical importance to regulate also E1 expression to restrict E3 promoter mediated transgene expression to tumor cells.

Specific embodiments of the invention include oncolytic adenoviral vectors (e.g. Ad5 or Ad3 vectors) whose replication is restricted to the p16/Rb pathway by dual selectivity devices: an E2F (e.g. E2F1 ) tumor specific promoter placed in front of the adenoviral E1A gene which has been mutated in constant region 2, so that the resulting E1A protein is unable to bind Rb in cells. Furthermore, the fiber is modified by 5/3 chimerism to allow efficient entry into tumor cell.

In a specific embodiment of the invention the oncolytic adenoviral vector comprises:

1 ) a 24 bp deletion (Δ24) in the Rb binding constant region 2 of adenoviral E1 ;

2) a nucleic acid sequence deletion of viral gp19k and 6.7k reading frames; and

3) a nucleic acid sequence encoding an anti-interleukin 8 (IL-8) neutralizing antibody in the place of the deleted nucleic acid sequence as defined in point 2).

The term "antibody" in the context of the present invention refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, which has the ability to specifically bind to an antigen, i.e. anti-interleukin 8 (IL-8). The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen. The constant regions of the antibodies (Abs) may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as C1 q, the first component in the classical pathway of complement activation. Antibodies may also be bispecific antibodies, diabodies, or similar molecules. The term antibody herein, unless otherwise stated or clearly contradicted by context, includes fragments of an antibody that retain the ability to specifically bind to the antigen. It has been shown that the antigen-binding function of an antibody may be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antibody" include (i) a Fab' or Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting essentially of the VH and CH1 domains; (iv) a Fv fragment consisting essentially of the VL and VH domains of a single arm of an antibody, (v) camelid or nanobodies, or (vi) single chain antibodies or single chain Fv (scFv). Such single chain antibodies are encompassed within the term antibody unless otherwise noted or clearly indicated by context. It also should be understood that the term antibody, unless specified otherwise, also includes polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides, such as chimeric antibodies and humanized antibodies, and antibody fragments retaining the ability to specifically bind to the antigen (antigen-binding fragments) provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques. An antibody as generated can possess any isotype. A "neutralizing antibody" is defined herein as an antibody that neutralizes any effect that the antigen has biologically. In the broadest sense, the term "antibody" also refers herein to aptamers and other peptide molecules that specifically bind to interleukin 8 or that are specifically engineered to bind to interleukin 8.

In an embodiment, the present invention is directed to an oncolytic viral vector, preferably an oncolytic adenoviral vector, comprising a nucleic acid sequence encoding an anti-interleukin 8 (IL-8) neutralizing antibody.

In a preferred embodiment, the backbone of the oncolytic adenoviral vector is an adenovirus serotype 5 (Ad5) or serotype 3 (Ad3) nucleic acid backbone.

In a more preferred embodiment, said nucleic acid sequence encoding an anti-interleukin 8 (IL-8) neutralizing antibody is in the place of a deleted nucleic acid sequence in the E3 region of said oncolytic adenoviral vector. Most preferably, the deletion of a nucleic acid sequence in the E3 region is a deletion of viral gp19k and 6.7k reading frames.

In another preferred embodiment, the vector also comprises a 24 bp deletion (Δ24) in the adenoviral E1 sequence of said oncolytic adenoviral vector.

In another preferred embodiment, the vector also comprises an Ad5/3 fiber knob.

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

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

Adoptive cell therapy

One approach of the present invention is the development of a treatment for patients with cancer using the transfer of immune lymphocytes that are capable of reacting with and destroying the cancer. Isolated tumor infiltrating lymphocytes are grown in culture to large numbers and infused into the patient. In the present invention oncolytic vectors encoding an anti-interleukin 8 (IL-8) neutralizing antibody may be utilized for increasing the effect of lymphocytes. As used herein "increasing the efficacy of adoptive cell therapy" refers to a situation, wherein the oncolytic vector of the invention is able to cause a stronger therapeutic effect in a subject when used together with an adoptive cell therapeutic composition compared to the therapeutic effect of the adoptive cell therapeutic composition alone. A specific embodiment of the invention is a method of treating cancer in a subject, wherein the method comprises administration of an oncolytic vector of the invention to a subject, said method further comprising administration of adoptive cell therapeutic composition to the subject. Adoptive cell therapeutic composition and the vectors of the invention are administered separately. Separate administrations of an adoptive cell therapeutic composition and adenoviral vectors may be preceded by myeloablating or non-myeloablating preconditioning chemotherapy and/or radiation. The adoptive cell therapy treatment is intended to reduce or eliminate cancer in the patient.

A specific embodiment of the invention relates to therapies with adenoviral vectors and an adoptive cell therapeutic composition, e.g. tumor infiltrating lymphocytes, TCR modified lymphocytes or CAR modified lymphocytes. T-cell therapies in particular, but also any other adoptive therapies such as NK cell therapies or other cell therapies may be utilized in the present invention. Indeed, according to the present invention the adoptive cell therapeutic composition may comprise unmodified cells such as in TIL therapy or genetically modified cells. There are two common ways to achieve genetic targeting of T-cells to tumor specific targets. One is transfer of a T-cell receptor with known specificity (TCR therapy) and with matched human leukocyte antigen (HLA, known as major histocompatibility complex in rodents) type. The other is modification of cells with artificial molecules such as chimeric antigen receptors (CAR). This approach is not dependent on HLA and is more flexible with regard to targeting molecules. For example, single chain antibodies can be used and CARs can also incorporate costimulatory domains. However, the targets of CAR cells need to be on the membrane of target cells, while TCR modifications can utilize intracellular targets.

As used herein "adoptive cell therapeutic composition" refers to any composition comprising cells suitable for adoptive cell transfer. In one embodiment of the invention the adoptive cell therapeutic composition comprises a cell type selected from a group consisting of a tumor infiltrating lymphocyte (TIL), TCR (i.e. heterologous T-cell receptor) modified lymphocytes and CAR (i.e. chimeric antigen receptor) modified lymphocytes. In another embodiment of the invention, the adoptive cell therapeutic composition comprises a cell type selected from a group consisting of T-cells, CD8+ cells, CD4+ cells, NK-cells, dendritic cells, delta-gamma T-cells, regulatory T-cells and peripheral blood mononuclear cells. In another embodiment, TILs, T-cells, CD8+ cells, CD4+ cells, NK-cells, delta-gamma T-cells, regulatory T-cells or peripheral blood mononuclear cells form the adoptive cell therapeutic composition. In one specific embodiment of the invention the adoptive cell therapeutic composition comprises T cells. As used herein "tumor-infiltrating lymphocytes" or TILs refer to white blood cells that have left the bloodstream and migrated into a tumor. Lymphocytes can be divided into three groups including B cells, T cells and natural killer cells. In another specific embodiment of the invention the adoptive cell therapeutic composition comprises T-cells which have been modified with target-specific chimeric antigen receptors or specifically selected T- cell receptors. As used herein "T-cells" refers to CD3+ cells, including CD4+ helper cells, CD8+ cytotoxic T-cells and γδ T cells.

In addition to suitable cells, adoptive cell therapeutic composition used in the present invention may comprise any other agents such as pharmaceutically acceptable carriers, buffers, excipients, adjuvants, additives, antiseptics, filling, stabilising and/or thickening agents, and/or any components normally found in corresponding products. Selection of suitable ingredients and appropriate manufacturing methods for formulating the compositions belongs to general knowledge of a person skilled in the art.

The adoptive cell therapeutic 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 compositions are not limited to a certain formulation, instead the composition can be formulated into any known pharmaceutically acceptable formulation. The pharmaceutical compositions may be produced by any conventional processes known in the art.

A combination of an oncolytic adenoviral vector of the invention and an adoptive cell therapeutic composition refers to use of an oncolytic adenoviral vector and an adoptive cell therapeutic composition together but as separate compositions. It is clear to a person skilled in the art that an oncolytic adenoviral vector of the present invention and an adoptive cell therapeutic composition are not used as one composition. Indeed, adenoviral vectors are not used for modifying the adoptive cells but for modifying the target tumor, so that the tumor is more amenable to the desired effects of the cellular transplant. In particular, the present invention enhances recruitment of the adoptive transplant to the tumor, and increases its activity there. In a specific embodiment of the invention oncolytic adenoviral vectors and an adoptive cell therapeutic composition of a combination are for simultaneous or sequential, in any order, administration to a subject.

Cancer

The present invention relates to approaches for treating cancer in a subject. In one embodiment of the invention, the subject is a human or an animal, specifically an animal or human patient, more specifically a human or an animal suffering from cancer. The approach of the present invention can be used to treat any cancers or tumors, including both malignant and benign tumors, both primary tumors and metastases may be targets of the approach. In one embodiment of the invention the cancer features tumor infiltrating lymphocytes. The tools of the present invention are particulary appealing for treatment of metastatic solid tumors featuring tumor infiltrating lymphocytes. In another embodiment the T-cell graft has been modified by a tumor or tissue specific T-cell receptor of chimeric antigen receptor.

As used herein, the term "treatment" or "treating" refers to

administration of at least one oncolytic vector coding for an anti-interleukin 8 (IL-8) neutralizing antibody or at least one oncolytic vector and a composition comprising an anti-interleukin 8 (IL-8) neutralizing antibody to a subject, preferably a mammal or human subject, for purposes which include not only complete cure but also prophylaxis, amelioration, or alleviation of disorders or symptoms related to a cancer or tumor. Therapeutic effect may be assessed by monitoring the symptoms of a patient, tumor markers e.g. in blood or for example a size of a tumor or the length of survival of the patient. In an embodiment, the present invention provides a further method for monitoring efficacy of a cancer therapy with an oncolytic viral vector, the method comprising the steps of: providing a biological sample, preferably a blood or tumor sample, taken from a patient treated with an oncolytic viral vector; and measuring the level of interleukin 8 (IL-8) in said sample. Preferably, a change in the level of interleukin 8 (IL-8) compared to an initial level before the treatment is indicative of the efficacy of the cancer therapy.

In one 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, 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.

Before classifying a human or animal patient as suitable for the therapy of the present invention, the clinician may examine a patient. Based on the results deviating from the normal and revealing a tumor or cancer, the clinician may suggest treatment of the present invention for a patient. In an embodiment, the present invention provides a further method for analysing whether a subject suffering from a cancer is responsive or non-responsive to the treatment with an oncolytic viral vector, the method comprising the steps of: providing a biological sample taken from the subject; measuring the level of interleukin 8 (IL-8) in said sample; and selecting the subject for the cancer therapy with said oncolytic viral vector, wherein the selection is based on the level or a change of the level of interleukin 8 (IL-8) in said sample. Preferably, said biological sample is a blood or tumor sample. More preferably, a cutoff value between 50-80 ng IL-8/1 determines high and low status groups and subjects in the low status group are selected for the cancer therapy. Most preferably, the cutoff value is 62 ng IL-8/1.

The biological sample for the present methods is preferably obtained from a mammalian subject, such as a human subject. A biological sample is any solid or fluid sample obtained from, excreted by or secreted by the subject, including samples from a healthy human subject or a human patient affected by a condition or disease to be diagnosed or investigated, such as cancer. For example, a biological sample can be a biological fluid obtained from, for example, blood, plasma, serum, urine, bile, ascites, saliva, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion, or fluid obtained from a joint. A biological sample can also be a sample obtained from any organ or tissue, including a biopsy, such as a tumor biopsy. Preferably, the biological sample is a blood or tumor sample. Antibodies and ELISA kits for the detection of IL-8 from biological samples are known in the art.

Pharmaceutical composition

A pharmaceutical composition of the invention comprises at least one type of viral vectors of the invention. In one embodiment a pharmaceutical composition of the invention comprises an oncolytic adenoviral vector comprising a deletion of a nucleic acid sequence in the E3 region, and a nucleic acid sequence encoding an anti-interleukin 8 (IL-8) neutralizing antibody in the place of the deleted nucleic acid sequence in E3 region. Furthermore, the composition may comprise at least two, three or four different vectors. In addition to the vector, a pharmaceutical composition may also comprise other therapeutically effective agents, any other agents such as pharmaceutically acceptable carriers, buffers, excipients, adjuvants, additives, antiseptics, filling, stabilising and/or thickening agents, and/or any components normally found in corresponding products. Selection of suitable ingredients and appropriate manufacturing methods for formulating the compositions belongs to general knowledge of a person skilled in the art.

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 compositions of the current invention are not limited to a certain formulation, instead the composition can be formulated into any known pharmaceutically acceptable formulation. The pharmaceutical compositions may be produced by any conventional processes known in the art.

A pharmaceutical kit of the present invention may comprises oncolytic vectors encoding an anti-interleukin 8 (IL-8) neutralizing antibody or an adoptive cell therapeutic composition and an oncolytic vector encoding an anti-interleukin 8 (IL-8) neutralizing antibody. In a specific embodiment the adoptive cell therapeutic composition is formulated in a first formulation and the oncolytic vectors are formulated in a second formulation. In another embodiment of the invention the first and the second formulations are for simultaneous or sequential, in any order, administration to a subject.

Administration

The adenoviral vector or pharmaceutical composition of the invention may be administered to any eukaryotic subject selected from a group consisting of animals and human beings. In a specific 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 one embodiment of the invention both adenoviral vectors and adoptive cell therapeutic composition are administered to a subject. The administration(s) of adoptive cell therapeutic composition and oncolytic vectors coding for an anti- interleukin 8 neutralizing antibody to a subject may be conducted simultaneously or consecutively, in any order. In one embodiment of the invention the oncolytic viral vectors and an adoptive cell therapeutic composition are administered separately. As used herein "separate administration" or "separate" refers to a situation, wherein adoptive cell therapeutic composition and oncolytic vectors are two different products or compositions distinct from each other.

In another embodiment, a pharmaceutically effective amount of an anti- interleukin 8 (IL-8) neutralizing antibody and an oncolytic adenoviral vector is administered to a subject. Preferably, the administration(s) of an anti-interleukin 8 (IL-8) neutralizing antibody and an oncolytic adenoviral vector to a subject is(are) conducted simultaneously or consecutively, in any order.

Only one administration of adenoviral vectors of the invention or single administrations of an adoptive cell therapeutic composition and oncolytic vectors may have therapeutic effects. There may be any period between the administrations of oncolytic viruses or between the administrations of oncolytic viruses and adoptive cell therapeutic composition depending for example on the patient and type, degree or location of cancer. In one embodiment of the invention there is a time period of one minute to four weeks, specifically 1 to 10 days, more specifically 1 to five days, between the consecutive administration of adoptive cell therapeutic composition and oncolytic adenoviral vectors coding for an anti-interleukin 8 (IL-8) neutralizing antibody. Several administrations of adoptive cell therapeutic composition and oncolytic viral vectors are also possible. The numbers of administration times of adoptive cell therapeutic composition and oncolytic viral vectors may also be different during the treatment period. Oncolytic viral vectors or pharmaceutical or adoptive cell 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 of vectors or any compositions 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.

In a specific embodiment of the invention an adoptive cell therapeutic composition and oncolytic viral vectors are administered on the same day and thereafter oncolytic viral vectors are administered every week, two weeks, three weeks or every month during a treatment period which may last for example from one to 6 or 12 months or more.

In one embodiment of the invention, the administration of oncolytic virus is conducted through an intratumoral, intra-arterial, intravenous, intrapleural, intravesicular, intracavitary or peritoneal injection, or an oral administration. Any combination of administrations is also possible. The approach can give systemic efficacy despite local injection. Adoptive cell therapeutic composition may be administered intravenously or intratumorally. In one embodiment the administration of the adoptive cell therapeutic composition and/or oncolytic viral vectors coding for an anti-interleukin 8 neutralizing antibody is conducted through an intratumoral, intraarterial, intravenous, intrapleural, intravesicular, intracavitary or peritoneal injection, or an oral administration. In a specific embodiment of the invention TILs or T cells are administered intravenously and viral vectors intratumorally and/or intravenously. Of note, virus is delivered to the tumor separately from administration of T-cells; virus is not used to modify the T-cell graft ex vivo. In essence, the virus modifies the tumor in such a way that the T-cell graft can work better.

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 1 x10⁸ viral particles (VP) to about 1 x10¹⁴ VP, specifically from about 5x10⁹ VP to about 1x10¹³ VP and more specifically from about 8x10⁹ VP to about 1 x10¹² VP. In one embodiment oncolytic adenoviral vectors coding for a bispecific monoclonal antibody are administered in an amount of 1 x10¹⁰- 1 x10¹⁴ virus particles. In another embodiment of the invention the dose is in the range of about 5x10¹⁰ - 5x10^{1 1} VP.

The amount of cells transferred will also depend on the patient, but typical amounts range from 1 x10⁹- 1 x10¹² cells per injection. The number of injections also varies but typical embodiments include 1 or 2 rounds of treatment several (e.g. 2-4) weeks apart.

Any other treatment or combination of treatments may be used in addition to the therapies of the present invention. In a specific embodiment the method or use of the invention further comprises administration of concurrent or sequential radiotherapy, monoclonal antibodies, chemotherapy or other anti-cancer drugs or interventions (including surgery) to a subject.

The terms "treat" or "increase", as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete treatment or increase. Rather, there are varying degrees of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the present inventive methods can provide any amount of increase in the efficacy of T-cell therapy or any degree of treatment or prevention of a disease.

The present invention is further described in the following examples, which are not intended to limit the scope of the invention.

EXPERIMENTAL SECTION

Materials and methods

Patients

All patients reported here participated in the Advanced Therapy Access Program (ATAP), which was a personalized therapy program (Hemminki, Oksanen et al. 2013). Patients who were treated in ATAP had solid tumors refractory to standard treatments and no major organ dysfunctions. A More detailed description of the exclusion criteria has been previously reported (Koski, Raki et al. 2012). Written informed consent was received from all of the patients before participation in the treatment program, and the studies performed on patient materials were positively evaluated by the Helsinki University Central Hospital Operative Ethics Committee (HUS 62/13/03/02/2013).

Oncolytic viruses used in patient treatments

Viruses that were used in the treatments have been previously published (Hemminki O, Diaconu I et al. 2012, Pesonen S, Diaconu I et al. 2012, Pesonen S, Nokisalmi P et al. 2010, Nokisalmi P, Pesonen S et al. 2010, Koski, Kangasniemi et al. 2010, Pesonen, Diaconu et al. 2012, Cerullo, Pesonen et al. 2010). All of the analyses concerned only the first treatments that patients received with oncolytic adenoviruses. Viruses were based on either Ad5, Ad3 or a modified Ad5/3 capsid, where the Ad5 knob had been switched to Ad3 knob (Koski, Kangasniemi et al. 2010). Some of the viruses were armed with transgene coding for GM-CSF or CD40 ligand (Cerullo, Pesonen et al. 2010, Pesonen, Diaconu et al. 2012).

Treatments and response evaluation

Imaging response before and after (typically at 3 months) virus treatments evaluated by computer tomography (CT) or positron emission tomography with CT (F18-FDG-PET-CT). Modified RECIST 1 .1 criteria (Kanerva, Nokisalmi et al. 2013) were used for assessment of CT results, and previously described PET criteria (Koski, Ahtinen et al. 2013) were used for the PET-CT imaging results. Responses were graded as progressive disease or progressive metabolic disease (PD/PMD), stable disease or stable metabolic disease (SD/SMD), minor response or minor metabolic response (MR/MMR) and complete response or complete metabolic response (CR/CMR).

Serum IL-8 quantification Serum IL-8 was analyzed from venous blood samples after collection using standard laboratory techniques. The laboratory reference value of 62 ng/l was used as the cutoff to determine high and low baseline IL-8 levels. The IL-8 change status was assigned based on the changes in IL-8 in samples taken during 100 days after treatment with oncolytic adenovirus by comparing post-treatment values with baseline IL-8 levels. A decrease of at least 50% was required for decrease status and an increase of at least 100% was required for increase status. If no decrease or increase was observed, the patient was assigned to the "no change" group.

Tumor load and peripheral blood cell counts

Tumor load was assessed from pre-treatment CT and PET-CT images. Based on the metastases in different organs and size of the primary tumor a tumor load score (0-21 ) describing the overall tumor load was calculated according to previously described methodology (Taipale, Liikanen et al. 2016a). In this study tumor load score was available for 60 patients. The median of the total tumor load (5) score was determined as the cutoff value for high tumor load.

Peripheral blood cell counts were obtained in the laboratory of the treating hospital using standard protocols. Baseline blood samples were obtained from patients on the day of the treatment or one day before. Neutrophil count was obtained by subtracting the lymphocyte count from total leucocyte count. Neutrophil to lymphocyte ratio was determined by dividing the baseline neutrophil count by the lymphocyte count.

RNA microarrays

Gene expression in pre- and post-treatment tumor and liquid biopsy samples was analyzed using RNA microarrays and following computational methods as previously described (Taipale, Liikanen et al. 2016b). Expression data was normalized using sample specific normalization to account for differential gene expression in different sample types. Baseline measurements of serum IL-8 were not available for patients with RNA microarray data. The log2 expression values were compared at baseline to determine high and low baseline gene expression. Change in the expression value between pre- and post-treatment samples was calculated and patients with negative a change were grouped into decrease group whereas patients with a positive change were assigned to increase group.

Enzyme-linked ImmunoSpot (ELISPOT) assay

ELISPOT analysis was carried out using patient derived peripheral blood mononuclear cells (PBMCs) as described earlier (Cerullo, Pesonen et al. 2010). Stimulation of the PBMCs was done using the human adenovirus serotype 5 penton (HAdV-5; Prolmmune, Oxford, UK) to evaluate anti-viral immune response, and with the tumor-associated BIRC5 PONAB peptide Survivin (Prolmmune) to assess responses for a tumor-specific antigen. A total of 10 spot forming units were regarded to as the lower limit of detection for the baseline and difference between pre- and post-treatment samples. ELISPOT readout changes between -3 and +3 were labeled as "no change", while less than -3 was considered decrease and above 3 increase in anti-Ad5 or anti-survivin ELISPOT (Liikanen, Ahtiainen et al. 2013).

Ovarian tumor sample analyses

Samples were obtained from 5 patients with ovarian tumors. The local ethics committee positively evaluated the collection of samples, and the patients gave a written informed consent before sample collection. After collection, the samples were stored in growth medium on ice for transport. Tumor blocks were cut into small fragments with a knife and enzymatically digested overnight to obtain a tumor cell suspension. After digestion cell suspensions were filtered and incubated with ACK lysis buffer (Life Technologies, Carlsbad, CA), after which the cells were washed with growth media. The cells were then plated with or without adenovirus and anti- IL-8 antibody in 96-well plates and cell proliferation was measured after 7-13 days using MTS assay (CellTiter 96 AQueous One Solution Proliferation Assay, Promega, Fitchburg, Wl). Neutralizing anti-IL-8 antibody (R&D Systems, Minneapolis, MN) was applied in designated wells at concentration of 2 ug/ml.

For the TIL/TAN co-cultures, TILs and TANs were isolated from the cell suspension by selecting CD3- and CD15-positive cells, respectively. The selection was performed using magnetic microbeads and LS columns (Miltenyi Biotec, Bergisch Gladbach, Germany) according to reagent supplier's instructions. Following selection, co-cultures were plated at 1 :1 ratio for both cell types. Cells were stimulated by adding 5 ul of anti-CD3/anti-CD28 Dynabeads (Thermo Fisher Scientific, Waltham, MA) into the wells. Neutralizing anti-IL-8 antibody or control anti-lgG1 antibody (R&D Systems) were added to appropriate wells at the final concentration of 2 ug/ml. Recombinant human IL-8 (Peprotech, Rocky Hill, NJ) was used at 500 ng/ml.

IL-8 production was measured from the supernatants 24 hours after the start of incubation using IL-8 Flex Set (BD Biosciences, San Jose, CA) according to protocols supplied by the manufacturer. T cell proliferation and activation were analyzed by fluorescence activated cell sorting (FACS) using conjugated antibodies for CD3, CD4, CD8, CD25 and CD69 (eBioscience, San Diego, CA).

Construction of anti-IL8 coding oncolytic adenoviruses

Purified DNA from plasmid BAC-Ad5/3-A24 was electroporated into SW102 bacteria. Second, part of the sequence located in E3-Region was replaced by the sequence of a GalK/Amp selection cassette which was amplified from plasmid pT GalK-Amp using primers with a 5'-overhang of corresponding to the flanking position in E3-region. Purified PCR-product was electroporated into SW102 containing BAC-Ad5/3-A24 to generate BAC B-Ad5/3- A24-GalK/amp. Recombinants were identified by amp-selection and verified by EcoRV-digest.

Third, resulting BAC-Ad5/3-A24-GalK/amp was used for insertion of anti-IL-8. Genes were synthesized according sequences and inserted into pTHSN vector. Plasmids were used for PCR-amplification of cytokine sequences and additional flanking sequences from primers binding in the E3-region allowing efficient homologous recombination. Purified PCR-product was electroporated into SW102 bacteria containing B-Ad5/3A24-GalK/amp. Clones with successful exchange of the selection cassette by therapeutic transgenes were identified by DOG-selection and sequence was verified by EcoRV digest.

Statistical analysis

Statistical analysis was conducted using SPSS Statistics v23 (International Business Machines Corporation, Armonk, NY) , Microsoft Excel (Microsoft Corporation, Redmond, WA) and GraphPad Prism (GraphPad Software, La Jolla, CA). Differences between average IL-8 levels were tested with one-way ANOVA and Student's t-test. Log-rank test was utilized to compare overall survival between different IL-8 groups and subgroups defined by ELISPOT or NLR measurements. Differences in treatment responses between groups were analyzed with Fisher's exact test. Hazard ratios for IL-8 and patient characteristics were estimated using Cox proportional hazards regression model. P smaller than 0.05 were regarded to as statistically significant.

Results

Normal baseline IL-8 before treatment with oncolytic adenovirus correlates with longer overall survival

Baseline IL-8 levels were measured from pre-treatment peripheral blood samples of 103 patients (Table 1). Patients were divided into high and low baseline groups based on the "normal" laboratory reference range for serum IL-8 (62ng/l). Survival between high and low baseline groups was compared using the Kaplan-Meier method. We found significant differences in the survival of high and low baseline IL-8 patients (p<0.001 ) (Figure 1a). We also measured other inflammatory cytokines, including IL-6, IL-10, TNFa and GM-CSF, in baseline serum samples, but found no differences in overall survival when using reference values or even sample median as cutoff (Figure 2a-d). Despite a clear difference in survival, there was only a non-significant trend between baseline IL-8 and treatment response, although the number of patients imaged and pseudoprogression could have impacted the p-value. The proportions of patients displaying disease control were 53% and 33% in IL-8 low and high groups, respectively (p=0.182) (Figure 1 b).

IL-8 levels in patients with different tumor types, tumor load and pre-treatment leucocyte counts

Different tumor types had slightly diverging average IL-8 levels (Figure

3a), but the differences were not considered significant. When patients were grouped based on the calculated tumor load score, which reflected the overall tumor burden of the patient, average IL-8 concentrations seemed to be higher, although not significantly, in patients with high tumor load score (Figure 3b). Since IL-8 is known to function as a chemoattractant for immune cells, and especially neutrophils, we analyzed the pre-treatment cell counts of two major leucocyte subsets, neutrophils and lymphocytes, in 86 patients with available IL-8 data. We then grouped the patients into high and low lymphocyte and neutrophil count groups, and compared baseline IL-8 levels between these groups (Figure 3c). Disparity in IL-8 levels was larger between the neutrophil groups in comparison to the lymphocyte groups, although the difference was not significant between patients with low and high neutrophil counts (p=0.085).

Post-treatment decrease in IL-8 independently predicts improved overall survival

We measured changes in IL-8 levels, following the first treatment with oncolytic adenovirus. Patients were categorized into increase, decrease and no- change groups based on post-treatment IL-8 levels (Figure 4). The minimal requirements for increase and decrease were a 100% growth and a 50% decline from baseline IL-8 levels during adenoviral immunotherapy, respectively. We analyzed survival between these three groups and found patients with post- treatment IL-8 decrease to have significantly longer overall survival (p<0.001 ) (Figure 5a).

Imaging responses after treatment were also compared between IL-8 change groups (Figure 5b). Interestingly, this comparison showed an almost twofold difference in disease control rate and between IL-8 decrease and the other two groups. However, partly due to the small sample size, the difference remained insignificant despite pooling of the increase and no-change groups (Fisher's exact test p=0.066). In additional analyses taking into account both baseline IL-8 status and IL-8 change, decrease in IL-8 seemed to correlate with a higher disease control rate. The effect was similar in both high and low baseline groups (Figure 5c). To further validate the survival effect of both baseline IL-8 and IL-8 change, we constructed a multivariate proportional hazards model with clinical variables of the patients (Table 2). In this analysis, normal baseline IL-8 and post- treatment IL-8 decrease were associated with significantly lower hazard ratios for tumor related mortality (HR 0.502, p=0.010 and HR 0.270, p=0.001 , respectively), which supports their role as independent prognostic factors for adenoviral immunotherapy. Impressively, IL-8 decrease was the strongest prognostic factor in this analysis, even stronger than the general condition or tumor type of the patients.

Treatment characteristics do not explain the observed changes in IL-8 levels

To investigate whether the characteristics of the treatment influence the post-treatment IL-8 change, we correlated IL-8 changes with treatment virus type (capsid), the arming device (transgene) or concomitant "virus sensitizing" treatment (Figure 6). In these comparisons, we observed no significant differences between patients who received virus with Ad5 capsid (Cerullo, Pesonen et al. 2010) or chimeric Ad5/3 capsid (Koski, Kangasniemi et al. 2010) (Figure 6a) or virus coding for no transgenes or coding the immunostimulatory granulocyte-macrophage colony-stimulating factor (GM-CSF) (Figure 6b). Additionally, IL-8 changes were not impacted by concomitant cyclophosphamide which was used with the aim of reducing regulatory T-cells (Cerullo, Diaconu et al. 201 1 ) (Figure 6c).

IL8 and IL-8 receptor RNA expression in tumor samples

To quantify IL8 at the tumor site, we measured RNA expression from pre- and post-treatment tumor or ascites/pleural fluid samples from an additional cohort of 15 patients treated with oncolytic adenoviruses (Table 3). Together with IL-8, we analyzed expression of the two IL-8 receptors CXCR1 and CXCR2 (Figure 7a). Variation in pre-treatment expression levels and pre-post changes was remarkably larger for IL-8 compared to its receptors, and thus IL-8 was focused on in the following analyses.

For the survival analysis, patients were grouped based on the baseline tumor-level expression of IL-8 mRNA and pre-post treatment change in expression (Figure 7b-c). IL-8 expression change was not correlated with overall survival, but we observed an interesting, albeit not statistically significant (p=0.058) in this small patient cohort, trend for longer survival in patients with low pre-treatment IL-8 expression in the collected samples.

Baseline IL-8 status improves the prognostic value of anti-tumor T cell ELISPOT activity and neutrophil-to-lymphocyte ratio We evaluated the effects of baseline IL-8 in the context of anti-tumor T cell activity, as measured by anti-survivin ELISPOT, and pre-treatment neutrophil- to-lymphocyte ratio. In this analysis, we studied the correlation of anti-tumor T cell activity with overall survival separately for normal and high IL-8 patient groups (Figure 8a-b). For patients with high baseline IL-8, T cell activity did not influence survival considerably, whereas in patients with normal IL-8 T cell activity seemed to be associated with improved overall survival, although the difference was only borderline significant in this small patient group (n=23, p=0.052).

We have previously found that neutrophil-to-lymphocyte ratio (NLR) significantly predicts survival in patients treated with oncolytic adenovirus (Taipale, Liikanen et al. 2016a). In order to verify that IL-8 is not merely reflecting the neutrophil/lymphocyte balance in these patients, we combined baseline IL-8 and NLR status in a survival analysis (Figure 8c). Here we found a significant improvement in the prognostic value of both factors when used in a combination approach (p<0.001 ).

IL-8 blockade does not impair the oncolytic activity of adenovirus in human ovarian tumor cell suspensions

To study the feasibility of the combination of oncolytic adenovirus and IL-8 blocking antibody, we tested the cell killing abilities of the combination in an MTS assay using cell suspensions from human ovarian tumors samples (Figures 9- 14). Samples were obtained fresh from the operating room and processed immediately, which means that analyses were performed on a highly relevant human clinical substrate. In these experiments, the anti-IL-8 antibody alone did not increase the cell killing. Importantly, anti-IL-8 antibody did not blunt the oncolytic functionality of the unarmed Ad5/3-d24 virus. Instead, the combination treatment showed increased cell killing (Figures 9a, 12a), which can indicate that some tumors are susceptible to the combination of adenovirus and IL-8 antibody even in this ex vivo study where the complete immune system is not present.

IL-8 blockade together with adenovirus influences T cell proliferation and CD8 activation in TIL/TAN co-cultures extracted from patients undergoing surgery for expected ovarian cancer

Human ovarian tumor samples were processed to extract tumor infiltrating lymphocytes (TILs) and tumor associated neutrophils (TANs) for further analyses on the immune effects of adenovirus and anti-IL-8 antibody combination. The amount of T cell proliferation varied considerably, when TILs were grown in the presence of TANs (Figure 15), in some instances resulting in increased and in others in decreased number of T cells. Although anti-IL-8 treatment seemed to reduce the number of T cells in general, a combination treatment with adenovirus was able to restore the T cell levels to same as in the mock group. A similar pattern can be observed in helper T cell (Figure 16) and cytotoxic T cell (CTL) activation (Figure 17), although in some instances it was the adenovirus that seemed to reduce helper T cells, which was reversed by anti-IL-8 treatment (Figure 16c-f). Interestingly, it can be noted that recombinant IL-8 interfered with CTL activation even in the presence of adenovirus (Figure 17a,f). The different histology types of the tumors probably contribute to the variation seen in these results. Overall, T cell proliferation and activation of helper T cells seemed to be markedly worse in the more malignant tumor samples (patients OVCA2, OVCA5 and OVCA7).

IL-8 concentrations were also measured from different cell cultures after 24h incubation (Figures 18-19). TAN cultures seemed to have higher levels of IL-8 compared to TIL cultures. The antibody used in the experiments was able to efficiently block IL-8. IL-8 concentration was unchanged (Figure 18) or even reduced (Figure 19) after incubation with adenovirus. This suggests that oncolytic Ad5/3-d24 adenovirus is not likely to cause counterproductive IL-8 increase when used together with IL-8-blocking antibodies. Overall, based on these findings it seems that oncolytic adenovirus and anti-IL-8 treatment do not have critically interfering effects and in some situations they are able to counteract each other's negative functions when used as a combination treatment.

Novel oncolytic adenovirus constructs coding for a neutralizing IL-8 antibodies

Based on the previous results, we constructed oncolytic adenoviruses coding for neutralizing antibodies for against human IL-8 (Figure 20). The first virus, referred to as TILT-801 (SEQ ID NO:1 ), codes for a fully human antibody against human IL-8. This antibody is based on the lgG2 subtype. Similarly, the second virus, TILT-802 (SEQ ID NO:2), is also armed with a fully human IL-8 antibody, but this antibody is of the lgG1 subtype. Both viruses have the same Ad5/3-d24 backbone (Kanerva, Zinn et al. 2003). The cloning procedure of the viruses involves two steps of homologous recombination using vector plasmids with the desired gene segments (Figure 21 ).

Hypothetical results from studies with the new constructs demonstrate oncolytic potency and stimulation of anti-tumor immune responses In future studies TILT-801 and TILT-802 are expected to demonstrate similar or increased oncolytic potency compared to the backbone virus in tumor cell lines that are indifferent or dependent on IL-8 activity, respectively (Figure 22). These novel viruses are also more likely to cause tumor growth inhibition in nude mice carrying human cancer xenografts that are sensitive to IL-8 blockade (Figure 23). Most importantly, studies with human tumor infiltrating lymphocytes and tumor associated neutrophils are expected to show increased cytotoxic T cell activation (Figure 24). This effect is achieved by reducing the immunosuppressive activity of the TANs through IL-8 blockade, which allows the formation of a more potent antitumor immune response following adenovirus infection and oncolysis.

Table 1. Patient characteristics.

Sex, no. of patients (total N=103)

Male 45 Female 58

Age (years)

Median 58 Range 5-77

WHO performance status (0-5), no. of patients

0 1 1

1 48

2 34

3 10

Tumor type, no. of patients

Ovarian cancer 18

Colorectal cancer 14

Sarcoma 1 1

Pancreatic 8

Prostate cancer 7

Breast cancer 7

Melanoma 6

Lung cancer 6

Head and neck cancer 5

Mesothelioma 4

Cholangiocarcinoma 4

Gastric cancer 3

Neuroblastoma 2

Anal cancer 1

Esophagus cancer 1 Hepatocellular carcinoma

Neuroendocrine carcinoma

Thyroid cancer

Urinary bladder cancer

Endometrial cancer

Cervical cancer

Previous treatments, no. of patients

Surgery

Chemotherapy

Radiotherapy

Immunotherapy

Virus used for treatment, no. of patients

ICOVIR-7 23

Ad5-d24-RGD 9

Ad5-d24-GMCSF 18

Ad5/3-cox2L-d24 18

Ad5-RGD-d24-GMCSF 7

Ad5/3-d24-GMCSF 28

Table 2. Multivariate analysis for prognostic value of baseline IL-8 and IL-8 change.

Hazard ratio (HR) for cancer mortality (n=98)

P value HR (95% CI)

Age 0.395 0.992 (0.974-1.010)

Sex (female/male) 0.804 1.073 (0.616-1.868)

Tumor type 0.360

(vs. Panc/Chol/HCC) CRC/Gastric 0.521 1.269 (0.612-2.631)

Melanoma 0.372 0.614 (0.210-1.793)

Lung 0.819 1.140 (0.371-3.503)

Gynecological 0.241 0.620 (0.278-1.379)

Other 0.286 0.674 (0.327-1.390)

WHO (low/high) 0.002 0.467 (0.288-0.755)

Baseline IL-8 (low/high) 0.010 0.502 (0.297-0.847)

IL-8 change 0.004

(vs. No change) Increase 0.060 0.576 (0.324-1.024)

Decrease 0.001 0.270 (0.122-0.596)

Table 3. Patients included in tumor gene expression analyses.

F=Female; M=Male. A= Ascites fluid; B= Biopsy; P=Pleural fluid. MMR=Minor metabolic response; SMD=Stable metabolic disease; PMD^: =Progressive metabolic disease; N/A=Not available.

Table 4. Sequence listing.

SEQ ID NO: Name:

1 Plasmid TILT-801

2 Plasmid TILT-802

3 LIT

4 Δ24

5 5/3 knob modification

6 Transgene IL-8 V light

7 Transgene IL-8 V heavy

8 Transgene IL-8 V light with leader sequence

9 Transgene IL-8 V heavy with leader sequence

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Claims

1 . An oncolytic viral vector comprising a nucleic acid sequence encoding an anti-interleukin 8 (IL-8) neutralizing antibody.

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

3. The oncolytic vector according to claim 2, wherein a backbone of the oncolytic adenoviral vector is an adenovirus serotype 5 (Ad5) or serotype 3 (Ad3) nucleic acid backbone.

4. The oncolytic vector according to claim 2 or 3, wherein said nucleic acid sequence encoding an anti-interleukin 8 (IL-8) neutralizing antibody is in the place of a deleted nucleic acid sequence in the E3 region of said oncolytic adenoviral vector.

5. The oncolytic vector according to claim 4, wherein the deletion of a nucleic acid sequence in the E3 region is a deletion of viral gp19k

6.7k reading frames.

6. The oncolytic vector according to any one of claims 2-5, wherein the vector comprises a 24 bp deletion (Δ24) in the adenoviral E1 sequence of said oncolytic adenoviral vector.

7. The oncolytic vector according to any one of claims 2-6, wherein the vector comprises an Ad5/3 fiber knob.

8. A pharmaceutical composition comprising an oncolytic vector according to any one of claims 1 -7 and at least one of the following: physiologically acceptable carriers, buffers, excipients, adjuvants, additives, antiseptics, filling, stabilising and/or thickening agents.

9. An oncolytic vector according to any one of claims 1 -7 or a pharmaceutical composition according to claim 8 for use in the treatment of cancer.

10. The oncolytic vector or pharmaceutical composition for use in treatment of cancer according to claim 9, wherein 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, pheo-chromocytoma, 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.

1 1 . The oncolytic vector or pharmaceutical composition for use in treatment of cancer according to claim 9 or 10 together with an adoptive cell therapeutic composition.

12. The oncolytic vector or pharmaceutical composition for use in treatment of cancer according to claim 1 1 for increasing the efficacy of adoptive cell therapy in a subject.

13. The oncolytic vector or pharmaceutical composition for use in treatment of cancer according to any one of claims 9-12 together with radiotherapy, monoclonal antibodies, chemotherapy, small molecular inhibitors, hormonal therapy or other anti-cancer drugs or interventions to a subject

14. An anti-interleukin 8 (IL-8) neutralizing antibody and an oncolytic adenoviral vector for use in the treatment of cancer.

15. A method of detecting and analysing whether a subject suffering from a cancer is responsive or non-responsive to the treatment with an oncolytic viral vector comprising the steps of:

- providing a biological sample taken from the subject;

- measuring the level of interleukin 8 (IL-8) in said sample; and

- selecting the subject for the cancer therapy with said oncolytic viral vector, wherein the selection is based on the level or a change of the level of interleukin 8 (IL-8) in said sample.

16. The method according to claim 15, wherein said biological sample is a blood or tumor sample.

17. The method according to claim 15 or 16, wherein a cutoff value between 50-80 ng IL-8/I determines high and low status groups and subjects in the low status group are selected for the cancer therapy.

18. A method of monitoring efficacy of a cancer therapy with an oncolytic viral vector comprising the steps of:

- providing a biological sample, preferably a blood or tumor sample, taken from a patient treated with an oncolytic viral vector; and - measuring the level of interleukin 8 (IL-8) in said sample.

19. The method according to claim 18, wherein a change in the level of interleukin 8 (IL-8) compared to an initial level before the treatment is indicative of the efficacy of the cancer therapy.

20. A method of treating cancer in a subject, wherein a

pharmaceutically effective amount of an oncolytic vector according to any one of claims 1 -7 or a pharmaceutical composition according to claim 8 is administered to a subject.

21 . Use of an oncolytic vector according to any one of claims 1 -7 for the manufacture of a medicament for the treatment of cancer.

22. A method of treating cancer in a subject, wherein a

pharmaceutically effective amount of an anti-interleukin 8 (IL-8) neutralizing antibody and an oncolytic adenoviral vector is administered to a subject.

23. The method according to claim 22, wherein the administration(s) of an anti-interleukin 8 (IL-8) neutralizing antibody and an oncolytic adenoviral vector to a subject is(are) conducted simultaneously or consecutively, in any order.

24. The method according to claim 20, 22 or 23, further comprising administration of concurrent or sequential radiotherapy, monoclonal antibodies, chemotherapy, adoptive T-cell therapy, small molecular inhibitors, hormonal therapy or other anti-cancer drugs or interventions to a subject.