阅读说明：本技术 将腺病毒和化学治疗剂组合用于治疗癌症 (Combination of adenovirus and chemotherapeutic agent for treatment of cancer ) 是由 L·库尔克 T·兰基 S·佩索宁 E·哈维斯托 A·沃兰托 L·瓦西莱夫 于 2017-01-11 设计创作，主要内容包括：本发明涉及溶瘤腺病毒载体和化学治疗剂。更具体地,本发明涉及溶瘤腺病毒载体和化学治疗剂用于癌症治疗。如本文所述的联合疗法具有优异的安全性,并且具有有效的治疗活性。(The present invention relates to oncolytic adenoviral vectors and chemotherapeutic agents. More particularly, the invention relates to oncolytic adenoviral vectors and chemotherapeutic agents for cancer therapy. The combination therapy as described herein has excellent safety and has potent therapeutic activity.)

Use of an ONCOS-102 adenovirus (Ad5/3-d24-GMCSF) in combination with a chemotherapeutic agent in the manufacture of a preparation or kit for combination therapy for the treatment of human malignant mesothelioma, wherein the virus and the chemotherapeutic agent are administered in combination to a patient.

2. The use of claim 1, wherein the chemotherapeutic agent is selected from the group comprising: all-trans retinoic acid, azacitidine, azathioprine, bleomycin, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, erlotinib, etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib, mechlorethamine hydrochloride, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, temozolomide, teniposide, thioguanine, valrubicin, vinblastine, vincristine, vindesine, and vinorelbine.

3. The use of claim 1 or 2, wherein the chemotherapeutic agent is pemetrexed and cisplatin, or pemetrexed and carboplatin.

4. Use according to claim 3, wherein the molar ratio between pemetrexed and cisplatin is from 10:0.75 to 10: 3.

5. The use according to claim 4, wherein the molar ratio between pemetrexed and cisplatin is 10: 1.5.

6. Use according to claim 3, wherein the molar ratio between pemetrexed and carboplatin is comprised between 5:8 and 5: 2.

7. Use according to claim 6, wherein the molar ratio between pemetrexed and carboplatin is 5: 4.

8. The use of any one of claims 1 to 7, wherein the amount of virus is 5x10¹⁰To 5x10¹¹VP (viral particle).

9. The use of claim 8, wherein the virus is administered at 3x10¹¹VP/5 ml.

10. The use of any one of claims 1 to 9, wherein the chemotherapeutic agent and the virus are administered in effective amounts.

11. The use of any one of claims 1 to 10, wherein viral administration is by intratumoral (i.e., direct injection into the tumor), intraperitoneal, intrapleural, or intraluminal administration, and the chemotherapeutic agent is administered intravenously or intraperitoneally.

12. The use of any one of claims 1 to 11, wherein the virus is administered prior to and also during the administration period of the chemotherapeutic agent.

13. The use of any one of claims 1 to 11, wherein the chemotherapeutic agent is administered prior to and also during the administration period of the virus.

14. The use according to any one of claims 1 to 13, wherein the virus is administered to the patient from 1 to 10 times and the chemotherapeutic agent is administered from 1 to 6 times.

15. Use according to any one of claims 1 to 14, further comprising administering to the patient one, two, three or four or any combination thereof from the list of agents selected from the group consisting of cyclohexamide, folic acid, cyanocobalamin and dexamethasone.

16. Use according to any one of claims 1 to 15, further comprising the administration of cyclophosphamide.

17. The use according to any one of claims 1 to 16, further comprising administering a checkpoint inhibitor selected from the group consisting of anti-PD-1, anti-PD-L1 and anti-CTLA 4 antibodies.

18. Use according to any one of claims 1 to 17, wherein the malignant mesothelioma is malignant pleural mesothelioma.

Technical Field

The present invention relates to the field of medicine. In particular, the present invention relates to a novel strategy for treating human cancer using the ONCOS-102 adenovirus in combination with two chemotherapeutic agents. Also disclosed are methods of treating malignant mesothelioma using such a combination of a virus and a chemotherapeutic agent. In addition to the virus and both chemotherapeutic agents, other drugs may also be included in the treatment regimen.

Background

Malignant Mesothelioma (MM) is an aggressive and rare form of cancer that develops from the mesothelium. MM is mainly due to exposure to asbestos and exhibits a long latency, typically >30 years. Median survival time after diagnosis in mesothelioma patients is typically only 9-12 months. MM affects the pleura (lining of the lungs and internal chest wall) in 85.5% of MM cases, the peritoneum (lining of the abdominal cavity) in 13.2% of MM cases, the pericardium (sac surrounding the heart) in 0.5% of MM cases, and the tunica vaginalis (sac surrounding the testes) in 0.8% of MM cases. MM tumors often respond poorly to standard therapies and the incidence is increasing worldwide. The low incidence of MM has long limited the discovery of new drugs and new therapeutic modalities are therefore highly desirable.

Typically, cancer is treated with conventional treatment regimens such as, for example, surgery, hormonal therapy, chemotherapy, radiation therapy, and/or other therapies. However, in many cases, cancers that are often characterized by advanced stages cannot be cured with existing therapies.

Viral therapy is a relatively novel treatment that takes advantage of the natural ability of some viruses to kill the cells in which they proliferate and the ability to spread to neighboring cells (thereby amplifying the therapeutic effect of the initial input dose). In viral therapy, cancer cell transduction and viral replication are carefully controlled by genetic engineering of the viral genome to achieve effective and safe tumor eradication. Safe tumor eradication requires the introduction of various genetic modifications into the adenovirus genome, thereby limiting replication to only tumor cells and ultimately achieving selective eradication of the tumor without adverse effects on healthy tissue.

Specific deletions of the adenovirus' critical regulatory genes can be used to produce dysfunctional proteins or a lack of expression thereof, which results in a dependence on specific genetic characteristics present in the target cell. Partial deletion of E1A results in replication limitation in normal cells, but allows replication in target cells such as cancer cells. Conditionally replicating viruses characterized by a 24 base pair deletion in CR2 (constant region 2) have been generated, shown to be effective and selective in treating glioma and breast cancer xenografts (Fueyo et al, 2000; Heise et al, 2000). E1A, whose cancer specificity stems from dysfunction, is unable to release the E2F1 transcription factor, resulting in the need for free E2F 1. E2F1 is abundant in cancer cells, with the pRb pathway most frequently disrupted (Hanahan and Weinberg 2000).

Clinical and preclinical results indicate that treatment with unarmed oncolytic viruses is not immunostimulatory enough to result in a sustained anti-tumor therapeutic immune response. In this regard, oncolytic viruses have been armed to be more immunostimulatory. Viruses can be engineered to express highly immunogenic proteins, such as granulocyte-macrophage colony stimulating factor (GM-CSF). When immunogenic proteins are expressed within the tumor microenvironment, they are potent stimulators of specific and long-lasting anti-tumor immunity. Introduction of immunotherapeutic genes into tumor cells and further translation into proteins results in activation of the immune response and more efficient destruction of tumor cells. In this regard, the most relevant immune cells are Natural Killer (NK) cells and cytotoxic CD8+ T cells.

ONCOS-102(Ad 5/3-D24-GM-CSF; disclosed in WO 2010/072900) is a serotype 5 adenovirus that comprises a chimeric capsid for enhanced gene delivery to cancer cells and a 24bp deletion in the Rb binding site of the E1A region for localized replication of cancer cells. ONCOS-102 is equipped with granulocyte-macrophage colony stimulating factor (GM-CSF) to enhance immune stimulation. The safety and immunological activities of ONCOS-102 have been demonstrated in phase I clinical study (NCT 01598129). In this phase I study, local treatment of pleural mesothelioma with ONCOS-102 induced a systemic anti-tumor CD8+ T cell response, and CD8+ T cells infiltrated into the tumor of the last line refractory malignant pleural mesothelioma patient.

Koski et al (2010) disclose the treatment of a total of 21 patients with advanced solid tumors refractory to standard therapy with ONCOS-102. From these studies, ONCOS-102 appears to be safe in treating cancer patients. Signs of desirable efficacy were also seen.

Publication WO 99/59604 discloses a method for treating squamous cell carcinoma consisting of direct injection of adenovirus into the carcinoma and administration of the two chemotherapeutic agents cisplatin and 5-fluorouracil.

Siurala et al (2015) disclose capsid-modified oncolytic adenovirus CGTG-102 (identical to ONCOS-102) in combination with doxorubicin (with or without ifosfamide). This combination was found to be effective when the established soft tissue sarcoma tumor was tested in vivo in syrian hamsters with full immunocompetence.

Publication US 7393478B 2 discloses a method for inhibiting tumor growth in a subject comprising administering to the subject an effective amount of a micelle comprising cisplatin and a therapeutic agent. The therapeutic agent in this method may be, for example, GM-CSF.

In practice, the treatment of Malignant Mesothelioma (MM) has remained unchanged since 2003. The most commonly used first-line chemotherapy of MM is with the chemotherapeutic agents Pemetrexed (alimita), cisplatin (Platinol), carboplatin (Paraplatin), and combinations thereof. Despite the advances made in conventional cancer treatment regimens, human mesothelioma remains incurable and new treatment regimens are urgently needed.

Summary of The Invention

It is an object of the present invention to provide a novel combination of an oncolytic adenovirus and a chemotherapeutic agent, a novel combination therapy for treating cancer in a patient using an oncolytic adenovirus and a chemotherapeutic agent, and also to solve the problems associated with conventional cancer therapies.

One aspect of the present invention is an ONCOS-102 adenovirus for use in the treatment of human cancer, preferably for use in the treatment of human malignant mesothelioma, wherein administration of the virus to a human patient in need thereof is performed in combination with administration of two chemotherapeutic agents.

Another aspect of the invention is a method for treating human cancer, preferably human malignant mesothelioma, in a patient comprising the step of administering to said patient an ONCOS-102 adenovirus and two chemotherapeutic agents.

Yet another aspect of the invention is the use of the ONCOS-102 adenovirus in the treatment of human malignant mesothelioma, wherein the virus is administered to a patient in combination with two chemotherapeutic agents.

Brief Description of Drawings

The following drawings are included to further illustrate certain aspects and features of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments (including examples).

FIG. 1 treatment of human mesothelioma cells JL-1, MSTO-211H and H226 with pemetrexed and cisplatin or pemetrexed and carboplatin alone or in combination with ONCOS-102(10 VP/cell) or with ONCOS-102 alone in an in vitro efficacy study. a) Antitumor efficacy was measured by MTS cell viability assay. Cell viability was determined 72 hours post-treatment relative to untreated cells (mock). The amount of b) necrotic cells (PI) and c) early apoptotic cells (FITC-labeled annexin-V) 48 hours after treatment was analyzed by flow cytometry. Error bars, mean ± SEM: p <0.05, p <0.01, p < 0.001.

FIG. 2. combination therapy of human mesothelioma cells JL-1, MSTO-211H and H226 using pemetrexed and cisplatin or pemetrexed and carboplatin alone or in combination with ONCOS-102(10 VP/cell), or treatment with ONCOS-102 alone-immunogenic tumor cell death in vitro. a) Extracellular ATP was measured from the supernatant 48 hours (J1-1, MSTO-211H) and 72 hours (H226) after treatment with the ATP assay kit. b) Secretion of extracellular HMGB1 into the supernatant was measured 3 days after treatment using an ELISA assay. c) Calreticulin exposure on the outer cell surface of the tested human mesothelioma cells was measured 24 hours (H226, J1-1) and 48 hours (MSTO-211H) after treatment by flow cytometry. Error bars, mean ± SEM: p <0.05, p <0.01, p < 0.001.

FIG. 3 oncolytic efficacy, receptor expression profile and viral infectivity assay of ONCOS-102 measured in three mesothelioma cell lines (J1-1, MSTO-211H, H226). a) Oncolytic efficacy of ONCOS-102(0.1VP, 1VP, 10VP, 100VP and 1000 VP/cell) was measured by MTS cell viability assay 3 days after treatment initiation. Cell viability curves were determined relative to untreated cells (mock). b) Expression of CAR, DSG2 and CD46 on mesothelioma cell lines was measured by flow cytometry after specific antibody staining. c) Five treatment groups were tested for ONCOS-102 infectivity. The determination of the infectivity of ONCOS-102 is based on the visual quantification of infected cells after staining with viral hexon protein and the calculation of the final spot detected. For each 5 replicates, 5 non-overlapping fields of view images were obtained using the AMG EVO XL microscope. For infectivity comparisons, data are expressed as the average number of spots in 5 wells. Error bars, mean ± SEM: p <0.05, p <0.01, p < 0.001.

FIG. 4. analysis of the efficacy of ONCOS-102(1E +8 VP/mouse, intratumoral (i.t.)) in combination with pemetrexed (10mg/kg in 100. mu.l, Intraperitoneal (IP)), cisplatin (1.5mg/kg in 100. mu.l, IP) or carboplatin (8mg/kg in 100. mu.l, IP) in a human mesothelioma xenograft model of BALB/c nude mice (7 mice per group, 14 tumors per group) and comparison to each treatment mock or alone. a) Treatment of group 8 with ONCOS-102(i.t.) and chemotherapy (IP) according to the treatment protocol (Table 1), with subcutaneous H226 tumor bearing (6X 10)⁶Individual cells/tumor) of BABL/c nude mice. Animals were treated every 3 days for the entire 2 months. b) Survival status was calculated by the Kaplan-Meier test.c) Human GM-CSF produced by ONCOS-102 was analyzed from tumors, liver, and serum after painless sacrifice of animals using ELISA techniques. d) Adenovirus copies (E4 gene) were measured by qPCR from tumors, liver and serum of animals that died painlessly. Error bars, mean ± SEM: p<0.05，**p<0.01，***p<0.001。

FIG. 5 anti-tumor efficacy of ONCOS-102(1E +8 VP/mouse, i.t.), pemetrexed (10mg/kg in 100 μ l, IP) and cisplatin (1.5mg/kg in 100 μ l, IP), a combination of all three of the chemotherapeutic agents, or a combination of the chemotherapeutic agents in a human mesothelioma xenograft model of BALB/c nude mice (2 tumors per group of 2 mice per mouse). a) Treatment of 4 groups with ONCOS-102(i.t.) and chemotherapy (IP) for subcutaneous H226 tumor bearing (7X 10)⁶Individual cells/tumor) of BABL/c nude mice. Animals were treated every 3 days throughout the 54 days. b) Body weight was monitored and measured throughout the experiment. Body weight (g) was converted to a percentage value of body weight, with the body weight on day 0 set to 100%. c) Survival status was calculated by Kaplan-Meier test. Error bars, mean ± SEM: p<0.05，**p<0.01，***p<0.001。

FIG. 6. antitumor efficacy of ONCOS-102(2E +8 VP/mouse, i.t.), pemetrexed (6.7mg/kg in 100. mu.l, IP) and carboplatin (5.4mg/kg in 100. mu.l, IP), combinations of all three chemotherapeutic agents, or combinations of the chemotherapeutic agents in a human mesothelioma xenograft model of BALB/c nude mice (2 tumors per group of 2 mice, 2 tumors per mouse). a) Treatment of 4 groups with ONCOS-102(i.t.) and chemotherapy (IP) for subcutaneous H226 tumor bearing (7X 10)⁶Individual cells/tumor) in BALB/c nude mice. Animals were treated every 3 days for the entire 30 days. b) Body weight was monitored and measured throughout the experiment. Body weight (g) was converted to a percentage value of body weight, with the body weight on day 0 set to 100%. c) Survival status was calculated by Kaplan-Meier test. Error bars, mean ± SEM: p<0.05，**p<0.01，***p<0.001。

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Mesothelioma is a cancer that appears to be treatment-resistant to standard therapies, with an increasing number of lethal cases worldwide. The lungs of the chest and abdominal organs such as the stomach, intestines, liver and heart are encased in membranes such as the pleura, peritoneum and pericardium, respectively. The portion covering the surface of such a film is called the mesothelium. Tumors that develop from such mesothelium are called mesotheliomas. There are three major histological subtypes of malignant mesothelioma: epithelioid, sarcomatoid and bipolar subtypes. As used herein, "malignant mesothelioma" (MM) refers to mesothelioma affecting pleura, peritoneum, pericardium, or tunica vaginalis. To date, the most effective standard treatment for MM is the combination of pemetrexed and cisplatin, which results in a Response Rate (RR) of 41%. The combination of cisplatin and pemetrexed improves survival in mesothelioma patients compared to single-drug chemotherapy. However, MM remains a fatal disease with a median PFS/OS prognosis of 12 months from the start of treatment, and new treatment modalities are needed.

As used herein, the term "antiviral response" refers to a response of a cell to a viral infection, including, for example, interferon production, cytokine release, chemokine production, lymphokine production, or any combination thereof.

As used herein, the expressions "normal host cell" and "normal tissue" refer to a non-cancerous, uninfected cell or tissue having an intact antiviral response.

As used herein, the term "oncolytic agent" refers to an agent capable of inhibiting tumor cell growth and/or killing a tumor cell.

As used herein, the term "subject" refers to any living organism, including humans and animals, human and animal tissues, and human and animal cells.

As used herein, the term "patient" refers to any subject (preferably a human) having a disease, such as MM, that may benefit from treatment with a combination therapy as described herein.

Adenoviruses are non-enveloped viruses with an icosahedral capsid with a diameter of 70-90 nm. Their genome is a linear double-stranded DNA of between 25 and 45 kilobases in size, with Inverted Terminal Repeats (ITRs) at both ends, and a terminal protein attached to the 5' end.

The icosahedral capsid is formed from three major proteins, with hexon trimers being most abundant. Each of the 12 apices of the capsid also comprises a pentameric protein, the penton base (penton base) covalently attached to the fiber. The fiber is a trimeric protein, which protrudes from the base of the penton and is a rod-like structure with rounded heads. Other viral proteins IIIa, IVa2, VI, VIII and IX are also associated with the viral capsid. Protein VII, small peptide mu and Terminal Protein (TP) were associated with DNA. Protein V provides structural attachment to the capsid via protein VI.

As used herein, the term "capsid" refers to the protein shell of a virus, which includes the hexon protein, the fibrin, and the penton base protein.

All human adenoviruses share similarities in their fiber structure. Each having an N-terminal tail, a handle with a repeating sequence, and a C-terminal knob domain with a globular structure. The round-head domain is primarily responsible for binding to target cell receptors, and its globular structure presents a large surface for lateral and apical binding. The difference in length and bendability of the fiber proteins of adenoviruses from different subgroups is most pronounced.

The fibers are involved in the attachment of the virus to the target cells. First, the knob domain of fibrin binds to receptors of target cells, second, the virus interacts with integrin molecules, and third, the virus is endocytosed into target cells. Subsequently, the viral genome is transported from the endosome into the nucleus, and replication of the viral genome can begin.

As used herein, the "Ad 5/3 chimera" of the capsid refers to a chimera wherein the knob portion of the fiber is from Ad serotype 3 and the remainder of the fiber is from Ad serotype 5.

Adenoviruses rely on cellular mechanisms to replicate the viral genome. They can infect quiescent cells and induce them to enter a cell cycle S-phase like state, thereby enabling viral DNA replication. The adenovirus genome can be divided into immediate early (E1A), early (E1B, E2, E3, E4), mid (IX, Iva) and late (L1-L5) genes.

The E3 gene product is not essential for in vitro viral replication, but is dedicated to controlling various host immune responses. E3-gp19K inhibits transport of the major histocompatibility complex class I (MHC) from the Endoplasmic Reticulum (ER) to the plasma membrane, thereby preventing presentation of peptides by the MHC to T lymphocytes.

The adenovirus E1A protein was originally described as a pRb binding protein capable of inducing DNA replication in quiescent normal cells. One of the key functions of the E1A protein is to disrupt pRb-E2F interactions, thereby releasing the E2F transcription factor to activate transcription of E2F responsive promoters and genes they control, such as adenovirus E2A. Conserved region 2(CR2) in the E1A protein forms a strong interaction with the pocket binding domain of pRb, and CR1 mediates the actual disruption of E2F binding of pRb. Conditionally replicating viruses characterized by a 24 base pair deletion in CR2 have been generated, shown to be effective and selective in the treatment of glioma and breast cancer xenografts. Their cancer specificity stems from the inability of dysfunctional E1A to release the E2F1 transcription factor, resulting in the need for free E2F 1.

ONCOS-102 adenoviruses have been disclosed for a long time in the publication WO 2010/072900. ONCOS-102 is serotype 5 adenovirus (Ad5), showing the following modifications, which differ from the Ad5 genome:

deletion of 24 base pairs (bp) in constant region 2(CR2) of the E1A gene. The dysfunctional E1A protein is unable to bind to and release the E2F1 transcription factor from retinoblastoma protein (Rb), resulting in the need for free E2F1 for adenoviral gene transcription. Free E2F1 is abundant in cancer cells, with the pRb pathway most often disrupted. Therefore, a virus with this 24bp deletion in E1A is able to replicate efficiently in cancer cells. Transcription of the E1A gene into mRNA is under the control of the endogenous E1A promoter.

2. A deletion of 965bp was introduced in the early 3(E3) region encoding the 6.7K and gp19K proteins. These proteins are associated with the ability of adenoviruses to evade host immune control mechanisms, and their function is consumptive of adenovirus replication.

3. A transgene encoding the human granulocyte macrophage colony stimulating factor (GM-CSF) protein has been inserted in the E3 region, replacing 6.7K and gp 19K. Transcription of the GM-CSF gene into mRNA is controlled by the endogenous E3 promoter. In other words, 965 base pairs encoding viral genes gp19K and 6.7K have been deleted from the E3 region in the ONCOS-102 adenovirus and transgenic GM-CSF has been introduced to replace them.

4. Serotype 5 fibers are replaced by serotype 3 fibers allowing the virus to enter the cell through the serotype 3 receptor rather than the serotype 5 receptor CAR.

In the ONCOS-102 adenovirus, the native E1A promoter is present, i.e., it is not replaced by another promoter.

Briefly, in the ONCOS-102 adenovirus, GM-CSF is under the control of the endogenous viral E3, which results in the initiation of replication-related transgene expression at about 8 hours post-infection. The virus replicates in a tumor-selective manner, resulting in tumor-localized production of GM-CSF. Tumor specificity was achieved by a 24-bp deletion, which eliminated the Rb-binding site of E1A, and as demonstrated in previous reports, the virus selectively replicated in cells with defects in the p16-Rb pathway, including most, if not all, human cancers. The oncolytic potency of the ONCOS-102 adenovirus was shown to be more effective than the wild-type control virus.

The oncolytic adenovirus expressing GM-CSF can directly act on cancer cells through the oncolytic effect and induce anti-cancer immunity at the same time. GM-CSF is a potent inducer of systemic anti-tumor immunity associated with the recruitment and maturation of Antigen Presenting Cells (APCs), primarily dendritic cells, and the recruitment of innate immunity-armed cells. However, systemically elevated cytokine levels represent a risk of toxic side effects. In addition to the direct risk of side effects mediated by high serum concentrations of GM-CSF, there is an indirect risk caused by the recruitment of myeloid-derived suppressor cells (MDSCs). While the immunosuppressive effects of MDSCs are often potentially harmful to cancer patients, they may be particularly counterproductive in the case of cancer immunotherapy. Therefore, it is crucial to localize GM-CSF expression to the tumor site.

The ONCOS-102 adenovirus has shown good oncolytic potential in vitro and the production of functionally active human GM-CSF (Koski et al, 2010). The virus was shown to be effective in preventing the growth of invasive syngeneic pancreatic tumors in immunocompetent hamsters. Evidence of virus replication in tumors is shown by measuring virus copy number. The selectivity of replication was also demonstrated because there was no increase in viral copy number in the directly injected liver tissue. Local replication-related production of GM-CSF in tumors was demonstrated, while little leakage of GM-CSF into serum or liver was observed. It has also been shown that low doses of cyclophosphamide in combination with the ONCOS-102 adenovirus can enhance anti-tumor effects, while cyclophosphamide treatment alone does not result in a significant reduction in tumor growth.

Overall, treatment of patients with advanced cancer with the ONCOS-102 adenovirus appears safe, and has observed promising evidence of possible efficacy. Although the virus is present in serum for a long period after a single dose, multiple injections have the potential to improve tumor transduction and enhance anti-tumor immunity.

As used herein, "chemotherapy" refers to the use of a compound or drug in the treatment of disease, although the term chemotherapy is most commonly associated with cancer treatment. Cancer chemotherapeutic compounds include nearly 100 individual drugs.

The most common side effects of chemotherapeutic drugs are nausea and vomiting. Most individuals also suffer from bone marrow suppression, or suppression of bone marrow that produces red blood cells, white blood cells, and platelets. These and other side effects are also exacerbated by the suppression of the immune system with the associated risk of destruction and lack of production of leukocytes and opportunistic infections.

Other side effects common to a wide range of chemotherapeutic agents include: alopecia (baldness); loss of appetite; weight loss; taste change; stomatitis and esophagitis (inflammation and sores); constipation; diarrhea; fatigue; damage to the heart; a change in the nervous system; lung injury; reproductive tissue damage; liver damage; kidney and urinary system damage.

It has been shown that cancer cell death can be immunogenic or non-immunogenic. Immunogenic Cell Death (ICD) involves changes in cell surface structure that result in the release of pro-immunogenic factors. Subsequently, it attracts APC to take up tumor antigens, processes them, and finally elicits an anti-tumor immune response (specific anti-tumor T cells). The success of cancer treatment, whether using chemotherapy, oncolytic viruses, or a combination of both, depends on the induction of immunogenic tumor cell death and the induction of anti-tumor immune responses. Some chemotherapeutic agents and oncolytic adenoviruses are known to act as potent inducers of ICD and thus have a beneficial effect on the anti-cancer immune response, thereby contributing to anti-tumor activity. ICDs can be assessed by the presence of ICD biomarkers such as Calreticulin (CRT) in the outer plasma membrane, followed by extracellular release of high mobility histo-1 protein (HMGB1) and Adenosine Triphosphate (ATP).

As disclosed earlier, cancer treatment with the ONCOS-102 adenovirus, as well as therapy with chemotherapeutic agents, have shown some efficacy when used alone. The inventors wanted to investigate whether the combination of adenoviral gene therapy with other therapies (such as conventional therapies) could be more effective in the treatment of MM than either alone.

As used herein, "combination therapy" is directed to administering ONCOS-102 with a chemotherapeutic agent, preferably either pemetrexed and cisplatin or pemetrexed and carboplatin, to a patient in need thereof. In combination therapy, the virus as well as the chemotherapeutic agent may be administered in several doses over several days. Administration of the chemotherapeutic agent may begin before viral administration begins, and a dose of the chemotherapeutic agent may be further administered during administration of the viral dose. The treatment regimen may also include priming with the virus first, followed by administration of the chemotherapeutic agent, and then continuing with both the virus and the chemotherapeutic agent. In another embodiment, the combination therapy first comprises the chemotherapeutic agents pemetrexed and cisplatin, but during the combination therapy cisplatin is replaced with carboplatin. This may be necessary, for example, because of the excessive side effects that occur when cisplatin is used in therapy. Other drugs may also be administered concurrently with the combination therapy.

As used herein, "concurrently" refers to a drug or therapy that has been administered before, after, or concurrently with a combination therapy as described herein. While the duration of therapy may vary from a few minutes to several weeks. Typically, the therapy that is concurrent with the combination therapy described herein lasts for days or weeks.

As used herein, the term "priming" refers to the use of an apoptosis-inducing pretreatment with the ability of conventional cytotoxic chemotherapy. Tumor initiation with oncolytic viruses leads to immunogenic cancer cell death, which is associated with the presentation of calreticulin on the cell surface and the release of natural adjuvants, particularly high mobility histone B1(HMGB1), and ATP from within the dead cells, ultimately leading to DC stimulation and subsequent activation of the adaptive immune response. This virus-induced change in the tumor environment is essential in eliciting a meaningful anti-tumor immune response. Antigen presenting cells capture tumor antigens from dying tumor cells and process them for MHC class I and class II presentation, migrate to draining lymph nodes and stimulate antigen-specific B and T cells. For example, the ONCOS-102 adenovirus can result in the induction of cytotoxic tumor-specific CD8+ T cells. As used herein, the term "priming" also refers to the ability of a chemotherapeutic agent, when administered prior to administration of a virus, to cause death of such immunogenic cancer cells. On the other hand, priming a tumor with a chemotherapeutic agent also means enlarging the interstitial space with a pre-treatment that induces apoptosis.

As used herein, the term "effective amount" refers to an amount of a compound, therapeutic agent, virus, or drug that is capable of achieving the desired result. For example, an effective amount of a chemotherapeutic agent and/or an adenovirus is an amount sufficient to achieve a beneficial or desired clinical result (including a clinical result). An effective amount may be administered in one or more administrations. As described herein, an effective amount is an amount sufficient to ameliorate, stabilize, reverse, slow and/or delay the progression of, or cure, malignant mesothelioma. In the present invention, the effect of chemotherapeutic agents and the ONCOS-102 adenovirus can be monitored, for example, by monitoring the response of the tumor to the treatment. Monitoring of the tumor response can be carried out by any suitable method known in the art. It can be performed, for example, with methods of measuring cellular immune status such as those listed by Ranki et al (2014). For example, monitoring can be carried out by measuring the presence of tumor infiltrating lymphocytes in the tumor, TH1 type responses can also be monitored with microarrays, and IFN γ enzyme linked immunospot assay (ELISPOT) can also be used in the monitoring.

An effective amount is thus an amount that is capable of eliciting a desired effect or response in a tumor or that is desired. As understood in the art, an effective amount may vary depending on the patient's medical history as well as other factors such as the type and/or dosage of chemotherapeutic agent used, etc.

As used herein, the term "toxicity" refers to a toxic event associated with the administration of a chemotherapeutic agent. Such events include, but are not limited to, neutropenia, thrombocytopenia, toxic death, fatigue, anorexia, nausea, rash, infection, diarrhea, mucositis, and anemia. Toxicity effects can be monitored by any conventional method known in the art.

As used herein, the term "treatment period" refers to the period of time when combination therapy is administered. The treatment period may consist of several administrations of the ONCOS-102 and chemotherapeutic agent, wherein the administrations may be performed in multiple cycles. The treatment period may last for weeks or months. The treatment period can last for one year.

As used herein, the term "administration phase" refers to the period of time during which an adenovirus, chemotherapeutic agent, or other drug is administered to a patient. During the administration period, a single dose or several doses of the agent may be administered. The administration period may be minutes, hours, days, weeks or months. The administration period may consist of a cycle. For example, a three week (21 day) cycle may be used in the administration of the chemotherapeutic agent.

Platinum-based antineoplastic agents (informally referred to as platins) are chemotherapeutic agents for the treatment of cancer. They are coordination complexes of platinum (coordination complexes). Carboplatin and cisplatin are platinum-based antineoplastic agents.

Pemetrexed is chemically similar to folic acid and belongs to the class of chemotherapeutic drugs known as folic acid antimetabolites. Pemetrexed acts by inhibiting three enzymes used in purine and pyrimidine synthesis, Thymidylate Synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT). By inhibiting the formation of precursor purine and pyrimidine nucleotides, pemetrexed prevents the formation of DNA and RNA, which are required for the growth and survival of dividing normal and cancer cells.

Pemetrexed has been approved by the FDA for the treatment of unresectable MPM in combination with cisplatin (500 mg/m respectively)²And 75mg/m²). The large phase III EMPHACIS trial showed improved results in patients treated with cisplatin and pemetrexed compared to cisplatin alone. Median survival of patients in the combination group was also shown to be 12 months, compared to 9 months (p ═ 0.02) using cisplatin alone. Because of the high toxicity of cisplatin in many MM patients, carboplatin has been tested as a replacement. However, these first-line chemotherapeutic agents all show low efficacy against MM.

Therapeutic compositions are generally formulated for a particular route of administration. In the combination therapy described herein, pemetrexed is used in its effective concentration. For example, for MM treatment, pemetrexed may be administered at up to 500mg/m²Is administered. When a21 day cycle is used, pemetrexed may be administered, for example, as a10 minute intravenous (i.v.) infusion on day 1 of each 21 day cycle. For example, in the case of side effects, the dosage may be lower, such as 200mg/m²To 450mg/m²More preferably 250mg/m²To 375mg/m². Pemetrexed may also be administered intraperitoneally where appropriate.

In the combination therapy described herein, cisplatin is used at its effective concentration. In the treatment of MM, a typical dose of cisplatin is 75mg/m over 2 hours, starting approximately 30 minutes after the end of pemetrexed infusion²Intravenous infusion. The amount of cisplatin administered may be in the range of 30mg/m²To 75mg/m²To change between. For example, in the case of side effects, the dosage may be lower, such as 30mg/m²To 70mg/m²More preferably 35mg/m²To 60mg/m²Most preferably 37.5mg/m²To 56.25mg/m². Cisplatin may also be administered intraperitoneally where appropriate.

In combination therapy with the ONCOS-102 adenovirus, cisplatin and pemetrexed can be administered in a molar ratio of 0.75:10 to 3: 10. Most preferably in a ratio of 1.5: 10.

Herein, the textIn the combination therapy, carboplatin is used at an effective concentration. In the combination therapy of MM, a typical dose of carboplatin is 400mg/m for patients with normal renal function². Carboplatin may be infused intravenously over 30-60 minutes, starting about 30 minutes after the end of the infusion of pemetrexed, which has been infused over a period of about 10 minutes. However, the dose of carboplatin is generally according to the Calvert formula (in mg rather than mg/m)²Calculated) calculation: total dose (mg) ═ x (GFR +25) (target AUC), this formula considers the patient's Glomerular Filtration Rate (GFR) and the target area under the concentration versus time curve (AUC in mg/ml x min). Thus, the carboplatin dose can be adjusted and reduced, for example, due to side effects, especially due to reduced renal function.

In combination therapy with the ONCOS-102 adenovirus, the dose administered is typically about 500mg/m²Pemetrexed and carboplatin for about AUC 5. In a preferred embodiment, and where the patient's renal function is normal, about 500mg/m may be administered²Of pemetrexed and about 400mg/m²The carboplatin of (1). However, the amount administered may be at 250mg/m²And 500mg/m²Of pemetrexed and 200mg/m²And 400mg/m²May vary from carboplatin to carboplatin. In some cases, particularly when the chemotherapeutic agent exhibits toxic effects, about 375mg/m can be administered²Of pemetrexed and about 300mg/m²The carboplatin of (1). Thus, pemetrexed and carboplatin may be administered at a molar ratio of 5: 4. However, the ratio may vary between 5:8 and 5: 2.

It is an object of the present invention an ONCOS-102 adenovirus for use in the treatment of human malignant mesothelioma, wherein the virus is administered in combination with two chemotherapeutic agents to a patient in need thereof.

In the combination therapies described herein, the ONCOS-102 adenovirus can be administered in several doses and at different times than the chemotherapeutic agent, and the chemotherapeutic agent can also be administered in several doses.

Another object of the invention is an ONCOS-102 adenovirus for use in the treatment of human malignant mesothelioma, wherein the virus is administered to a patient in combination with two chemotherapeutic agents, and wherein the two chemotherapeutic agents are pemetrexed and cisplatin or pemetrexed and carboplatin.

Another object of the invention is an ONCOS-102 adenovirus for the treatment of human malignant mesothelioma, wherein said virus is administered in combination with pemetrexed and cisplatin to a patient in need thereof, and wherein the molar ratio between pemetrexed and cisplatin is from 10:0.75 to 10: 3.

Another object of the invention is an ONCOS-102 adenovirus for use in the treatment of human malignant mesothelioma, wherein the virus is administered in combination with pemetrexed and cisplatin to a patient in need thereof, and wherein the molar ratio between pemetrexed and cisplatin is 10: 1.5.

Another object of the invention is an ONCOS-102 adenovirus for use in the treatment of human malignant mesothelioma, wherein the virus is administered in combination with pemetrexed and carboplatin, wherein the molar ratio of pemetrexed to carboplatin is between 5:8 and 5: 2.

Another object of the invention is an ONCOS-102 adenovirus for use in the treatment of human malignant mesothelioma, wherein the virus is administered in combination with pemetrexed and carboplatin, wherein the molar ratio between pemetrexed and carboplatin is 5:4, to a patient in need thereof.

In a preferred embodiment of the present invention, various combinations of cyclohexamide, cyanocobalamine (cyanocobalamine), folic acid and dexamethasone may be used in addition to ONCOS-102 and pemetrexed and cisplatin or pemetrexed and carboplatin. More specifically, from the list of agents consisting of cyclohexamide, cyanocobalamin, folic acid and dexamethasone, one, two, three or four and any combination thereof may be used simultaneously with a combination therapy comprising ONCOS-102 and pemetrexed and cisplatin or ONCOS-102 and pemetrexed and carboplatin. As one example, the subject may be treated with one or more adjuncts that reduce or eliminate hypersensitivity reactions administered before, during, and after administration of the chemotherapeutic agents of the combination therapy, such as dexamethasone, folic acid, and vitamin B12 administered before, during, and after administration of the chemotherapeutic agents. In certain embodiments, the subject is treated by: orally administering 2-25mg dexamethasone one day, and one day after administration of the chemotherapeutic agent; orally administering 400 μ g per day of folic acid starting 7 days before administration of the chemotherapeutic agent, over at least one treatment period, and over a period lasting 21 days after the last administration of the chemotherapeutic agent; and 1000 μ g of vitamin B12 injected intramuscularly 1 week prior to the first administration of the chemotherapeutic agent during the treatment period.

The ONCOS-102 can be administered to a patient in need thereof using any conventional method. The route of administration depends on the formulation or form of the composition, the disease, the location of the tumor, the patient, complications, and other factors. In a preferred embodiment of the invention, administration is by intratumoral, intramuscular, intraarterial, intrapleural, intravesical, intracavity or intraperitoneal injection or oral administration. Preferably, administration is performed as intratumoral injection or intraperitoneal injection.

Personalized injections can be made for each patient based on the location and size of the tumor. For example, the virus may be injected (i.t.) in a volume of 0.5ml to 10 ml. Several, preferably up to five, different tumor sites can be injected. The volume of the intraperitoneal dose can vary between 200ml and 800 ml. Preferably, the administration volume is 500 ml.

The effective dose of the carrier will depend at least on the subject in need of treatment, the tumor type, the location of the tumor, and the stage of the tumor. The dosage may be, for example, from about 10⁸Individual Virus Particles (VP) to about 10¹⁴VP, preferably about 5X10⁹VP to about 10¹³VP, more preferably about 8X10⁹VP to about 10¹²And (3) a VP. In one embodiment of the invention, the dose is about 5x10¹⁰-5x10¹¹Within a range of one VP. Thus, the amount of ONCOS-102 adenovirus to be administered can be at 5x10¹⁰-5x10¹¹Within a range of one VP. Preferably, ONCOS 102 is at 3x10¹¹VP per 5ml was administered. On the other hand, in the case of expressed in plaque forming units, it can be administered by direct injection into the malignant mesothelioma tumor of the patient or intraperitoneally at about 10⁸-10¹²ONCOS-102 is administered at a dosage of each plaque forming unit.

An object of the present invention is an ONCOS-102 adenovirus for the treatment of malignant mesothelioma in humans, wherein said virus is administered in combination with two chemotherapeutic agents to a patient in need thereof, and wherein said virus isIn an amount of 5x10¹⁰To 5x10¹¹And (3) a VP.

Another object of the present invention is an ONCOS-102 adenovirus for the treatment of human malignant mesothelioma, wherein the virus is administered to a patient in combination with two chemotherapeutic agents, and wherein 3x10 is administered¹¹The virus was administered in an amount of 5ml per VP.

Another aspect of the invention is an ONCOS-102 adenovirus for use in the treatment of human malignant mesothelioma, wherein the virus is administered to a patient in combination with two chemotherapeutic agents, and wherein the chemotherapeutic agent and the virus are administered in effective amounts.

Another object of the present invention is an ONCOS-102 adenovirus for the treatment of human malignant mesothelioma, wherein the virus is administered in combination with two chemotherapeutic agents to a patient in need thereof, and wherein the virus is administered by intraperitoneal administration or by direct injection into the tumor, and the two chemotherapeutic agents are administered by intravenous or intraperitoneal route.

According to one aspect of the invention, the ONCOS-102 adenovirus is used for the treatment of human malignant mesothelioma, wherein the virus is administered in combination with two chemotherapeutic agents to a patient in need thereof, and wherein the virus is administered prior to, and during the period of administration of the chemotherapeutic agents.

According to another aspect of the invention, the ONCOS-102 adenovirus is used for the treatment of human malignant mesothelioma, wherein the two chemotherapeutic agents are administered before and during the administration phase of the virus.

According to a preferred embodiment, the agents of the combination therapy may be administered with a first priming, wherein the virus is administered 1 to 10 times over a period of 1 to 10 months, followed by administration of the chemotherapeutic agent, e.g. 1 to 4 weeks after the start of the virus administration. Administration can be carried out, for example, in the following order: priming with virus was performed by administering the virus 6 times to a subject in need over a period of 4 months. Typically, the time between doses is shorter at the beginning of the treatment session than when the treatment is being performed. Three weeks after the start of the administration of the virus, administration of chemotherapeutic agents, preferably pemetrexed and cisplatin or pemetrexed and carboplatin, may begin. Administration of the chemotherapeutic agent may be continued, for example, for a total of six administrations over a period of about three weeks. The number of applications may vary from one to six.

The oncolytic adenoviral vectors of the invention induce virion-mediated oncolysis of tumor cells and activate human immune responses against tumor cells. In a preferred embodiment of the invention, the method or use further comprises administering an agent capable of down-regulating regulatory T cells in the subject. By "an agent capable of down-regulating regulatory T cells" is meant an agent that reduces the amount of cells identified as T suppressors or regulatory T cells. These cells have been identified as consisting of one or more of the following immunophenotypic markers: CD4+, CD25+, FoxP3+, CD127-, and GITR +. Such agents that reduce T suppressor or regulatory T cells may be selected from anti-CD 25 antibodies or chemotherapeutic agents.

The immunomodulatory function of transgenic GM-CSF is the central mechanism of action of armed oncolytic adenoviruses, and moreover, adenoviruses themselves are strong activators of the immune system, which contributes significantly to the overall anti-tumor efficacy of the virus.

As used herein, "viral sensitizer" refers to an agent that can enhance the efficacy of an oncolytic virus. Agents suitable for such combination therapy or for use as viral sensitizers include, but are not limited to, all-trans retinoic acid, azacitidine, azathioprine, bleomycin, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, erlotinib, etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib, mechlorethamine hydrochloride, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, temozolomide, teniposide, thioguanine, valrubicin, vinblastine, vincristine, vindesine, and vinorelbine. Preferably, the virus sensitizer to be used is cyclophosphamide.

Checkpoint inhibitors such as anti-PD-1/PD-L1 or anti-CTLA 4 antibodies (such as Pembrolizumab, Nivolumab, or ipilimumab) are also suitable for use with combination therapy.

One aspect of the present invention is a method for treating malignant mesothelioma in a patient, comprising the step of administering to the patient an ONCOS-102 adenovirus and two chemotherapeutic agents. The VEGF-102 adenovirus and the two chemotherapeutic agents are administered in effective amounts.

Another aspect of the invention is a method of treating malignant mesothelioma in a patient, comprising the step of administering an ONCOS-102 adenovirus and two chemotherapeutic agents to the patient in an amount and for a time sufficient to kill malignant mesothelioma cells or prevent growth of malignant mesothelioma cells.

A preferred aspect of the present invention is a method for treating malignant mesothelioma in a patient, comprising the step of administering to said patient an ONCOS-102 adenovirus and two chemotherapeutic agents, wherein said chemotherapeutic agents are pemetrexed and cisplatin or pemetrexed and carboplatin.

Another preferred aspect of the invention is a method for treating malignant mesothelioma in a patient, comprising the step of administering to said patient an ONCOS-102 adenovirus and two chemotherapeutic agents, wherein the chemotherapeutic agents are administered first, then the administration phase of the virus is started, and the chemotherapeutic agents may be administered during the administration phase of the virus.

Another preferred aspect of the invention is a method for treating malignant mesothelioma in a patient, comprising the step of administering to said patient an ONCOS-102 adenovirus and two chemotherapeutic agents, wherein the virus is administered first, followed by the start of the chemotherapeutic agent day administration period, and the virus may also be administered during the chemotherapeutic agent administration period.

Another preferred aspect of the invention is a method for treating malignant mesothelioma in a patient, comprising the step of administering an ONCOS-102 adenovirus and two chemotherapeutic agents to said patient, wherein the ONCOS-102 adenovirus is administered to said patient 1 to 10 times and the chemotherapeutic agent is administered to the patient 1 to 6 times.

Another preferred aspect of the invention is a method for treating malignant mesothelioma in a patient, comprising the step of administering to said patient an ONCOS-102 adenovirus and two chemotherapeutic agents, wherein the administration is by direct injection into the malignant mesothelioma tumor of said patient or at 10⁸-10¹²Administration of the ONCOS-102 was performed by intraperitoneal injection at doses of each plaque forming unit.

Another preferred aspect of the invention is a method for treating malignant mesothelioma in a patient, comprising the step of administering to said patient an ONCOS-102 adenovirus and two chemotherapeutic agents, wherein the amount of ONCOS-102 adenovirus to be administered is at 5x10¹⁰-5x10¹¹VP is within the range.

Another preferred aspect of the invention is a method for treating malignant mesothelioma in a patient, comprising the step of administering to said patient an ONCOS-102 adenovirus and two chemotherapeutic agents, wherein at 250mg/m²To 500mg/m²Of pemetrexed and 30mg/m²To 75mg/m²Or at a dose of 250mg/m²To 500mg/m²Of pemetrexed and 200mg/m²To 400mg/m²Administering the chemotherapeutic agent at a dose of carboplatin.

Another aspect of the invention is the use of the ONCOS-102 adenovirus in the treatment of human malignant mesothelioma, wherein the virus and two chemotherapeutic agents are administered in combination to a patient.

Cyclophosphamide (CPO) may be concurrently administered to the patient during the period of the combination therapy. Cyclophosphamide is a common chemotherapeutic agent that has also been used in certain autoimmune disorders. Cyclophosphamide has been shown to enhance the efficacy of oncolytic viruses by several mechanisms. It inhibits innate anti-viral responses, slows the production of anti-oncolytic virus neutralizing antibodies, targets T-regs and affects tumor vasculature, and enhances oncolytic virus extravasation. Several preclinical studies have shown that cyclophosphamide can delay the immune clearance of oncolytic viruses, enhance the persistence of viral infections and prolong the therapeutic effect. In the present invention, cyclophosphamide is used as a virus sensitizer to enhance the effects of virus replication and GM-CSF-induced stimulation of NK and cytotoxic T cells (to enhance the anti-tumor immune response). It may be administered in a bolus intravenous dose or low dose oral rhythmic administration. Other suitable viral sensitizers that may be used in embodiments of the present invention include temozolomide and erlotinib.

To reduce the adjustmentSex T cells, patients will receive low dose CPO 1 to 3 days before each injection of ONCOS 102. CPO will be, for example, at 300mg/m²By intravenous bolus injection. The bolus injection may be between 100 and 600mg/m²To change between. The route of administration of the CPO may also be, for example, oral administration. Rhythmic chemotherapy may also be used.

In addition, folic acid can also be administered to the patient prior to the initiation of the combination therapy and during the combination therapy. Administration of the folate can begin at least five days prior to administration of the first dose of the chemotherapeutic agent of the combination therapy. For example, administration can begin 1-2 weeks before administration of the chemotherapeutic agent of the combination therapy begins. Folic acid may be administered daily (oral administration; PO) and may also continue to be administered during the combination therapy. Administration may continue for up to about three weeks after the last dose of chemotherapeutic agent. A typical dose of folic acid is 4mg (PO). A preferred aspect of the present invention is a method for treating mesothelioma in a patient in need thereof, comprising (a) the step of administering to said patient an ONCOS-102 adenovirus and two chemotherapeutic agents, and the step of administering to said patient folic acid.

Cyanocobalamin (vitamin B12) may be administered to the patient before the start of the combination therapy as well as during the combination therapy. Administration of cyanocobalamin is typically initiated 1-2 weeks before the start of administration of the chemotherapeutic agent of the combination therapy. Cyanocobalamin can be administered, for example, at 9 week intervals and also during the combination therapy by intramuscular injection (i.m.). Administration may continue for up to about three weeks after the last dose of chemotherapeutic agent. A typical amount of cyanocobalamin is 1000mcg (. mu.g). A preferred aspect of the present invention is a method for treating mesothelioma in a patient in need thereof, comprising (a) the step of administering an ONCOS-102 adenovirus and two chemotherapeutic agents to said patient and the step of administering cyanocobalamin to said patient.

Folic acid and cyanocobalamin are commonly used to reduce hematologic and gastrointestinal toxicity associated with therapy. These side effects may be associated with the administration of, for example, pemetrexed.

Dexamethasone can also be administered to patients undergoing combination therapy. Typically, dexamethasone is administered one day before, one day after, and one day after administration of the chemotherapeutic agent of the combination therapy. Dexamethasone can be administered at 4mg BD (i.e. twice a day) for 5 days and at a frequency of once every three weeks for up to six cycles. One object of the present invention is a method for treating mesothelioma in a patient in need thereof, comprising the steps of (a) administering to said patient ONCOS-102 adenovirus and two chemotherapeutic agents and (b) administering dexamethasone to the patient.

The combination therapy described herein may further comprise administering cyclophosphamide to the patient. One object of the present invention is a method for treating mesothelioma in a patient in need thereof, comprising (a) a step of administering to said patient an ONCOS-102 adenovirus and two chemotherapeutic agents, and further comprising administering cyclophosphamide to the patient prior to step (a).

Another aspect of the invention is a method for reducing tumor growth in a patient, wherein the method comprises administering to the patient an ONCOS-102 adenovirus and two chemotherapeutic agents under conditions wherein tumor growth in the patient is reduced.

The method can include identifying the patient as having a tumor prior to administering the combination therapy. Diagnosis of tumors can be performed by any conventional method. The patient may be identified as having a tumor, for example, using diagnostic imaging techniques. The method can further comprise measuring a reduction in tumor growth after administering the combination therapy to the patient. Tumor reduction can be studied by any conventional method. The reduction in tumor growth can be measured, for example, using diagnostic imaging techniques.

Another aspect of the invention is the use of combination therapy to inhibit tumor growth. The signs of inhibition of tumor growth may be, for example, reduction in tumor weight and reduction in tumor volume. In addition, combination therapy can be used to inhibit tumor spread.

It should also be noted that the pseudoprogression of tumors corresponding to treatment-related increase in lesion size can affect the results of the imaging follow-up aimed at revealing the effect of current combination therapy on tumor size. This effect is observed after combination chemotherapy and radiation therapy in about 30% of patients. Thus, when using combination therapy as described herein, spurious progression may be present in certain parts of the patient. For those patients, reduction in tumor size is not a suitable indicator of the effectiveness of the therapy.

Also disclosed are methods of reducing cancer cell growth comprising administering to a subject in need thereof an effective amount of an ONCOS-102 adenovirus and two chemotherapeutic agents.

Also disclosed are methods of inhibiting or killing a tumor or cancer cell in a human patient comprising treating the patient with an ONCOS-102 adenovirus and two chemotherapeutic agents, wherein the chemotherapeutic agents are pemetrexed and cisplatin or pemetrexed and carboplatin.

In addition, methods of killing a tumor or cancer cell are disclosed, comprising contacting the tumor or cancer cell with an ONCOS-102 adenovirus and two chemotherapeutic agents, wherein the chemotherapeutic agents are pemetrexed and cisplatin or pemetrexed and carboplatin.

Further, methods for treating malignant mesothelioma comprising administering to a subject in need thereof an effective amount of an ONCOS-102 adenovirus and an effective amount of two chemotherapeutic agents to provide a combination therapy having an enhanced therapeutic effect compared to the effect of administering the ONCOS adenovirus alone or the effect of administering the two chemotherapeutic agents without the virus are also disclosed.

In one embodiment of the invention, ONCOS-102 functions as an in situ cancer vaccine. As used herein, "in situ cancer vaccine" refers to a cancer vaccine that both kills tumor cells and increases the immune response against tumor cells. Viral replication is a strong danger signal to the immune system (required for TH 1-type response) and thus can serve as a powerful co-stimulatory phenomenon for GM-CSF-mediated APC maturation and activation and NK cell recruitment. Tumor cell lysis also contributes to the presentation of tumor fragments and epitopes to APCs, and in addition, produces co-stimulation through inflammation. Thus, an epitope-independent (i.e. non-HLA-restricted) response is generated in the context of each tumor, which thus occurs in situ. Tumor-specific immune responses are activated in the tumor environment when specific tumor antigens are released from dying cells after tumor cell lysis.

In a preferred embodiment of the invention, the method or use further comprises administering concurrent radiation therapy to the subject.

In a preferred embodiment of the invention, the method or use further comprises administering to the subject an autophagy inducing agent. Autophagy refers to a catabolic process involving degradation of cellular self-components by lysosomal mechanisms. An "autophagy inducing agent" refers to an agent capable of inducing autophagy and may be selected from, but is not limited to, mTOR inhibitors, PI3K inhibitors, lithium, tamoxifen, chloroquine, bafilomycin, temsirolimus, sirolimus, and temozolomide. In a particular embodiment of the invention, the method further comprises administering temozolomide to the subject. Temozolomide may be oral or intravenous temozolomide.

It is an object of the present invention to develop a novel therapeutically effective use of ONCOS-102 oncolytic adenoviruses and chemotherapeutic agents with improved safety and increased cancer efficacy compared to viral therapy alone or chemotherapy alone.

The current experimental results show that ONCOS-102, as a monotherapy, kills human mesothelioma cell lines in vitro and shows some anti-tumor activity in a xenograft model of treatment refractory H226 malignant pleural mesothelioma. In contrast, the current standard of care (SoC) chemotherapy regimen for malignant mesothelioma (pemetrexed + cisplatin or pemetrexed + carboplatin) showed no anti-tumor efficacy in this mesothelioma xenograft model. However, surprisingly, when ONCOS-102 was combined with the SoC chemotherapy regimen, a synergistic antitumor effect was observed. The data provides a powerful theoretical basis for the combined use of ONCOS-102 and SoC chemotherapy in the treatment of malignant mesothelioma.

Chemotherapy-when administered with ONCOS-102-increases the expression of ICD markers in current xenograft models. It has been shown earlier that ONCOS-102 produces functional human GM-CSF in hamsters, inducing tumor-specific immunity, suggesting that GMC-SF may also be involved in the recruitment and activation of Dendritic Cells (DC) in current xenograft models, leading to stimulation of T cells, and ultimately the development of anti-tumor immunity. In the human mesothelioma cancer model described in detail in the examples, the function of GM-CSF is lost by the immune system of immunodeficient mice. However, the utility of GM-CSF for immune system modulation against mesothelioma has been demonstrated in clinical studies (Ranki et al, 2014).

Previous clinical data have demonstrated that ONCOS-102 is able to elicit a tumor-specific immune response in malignant pleural mesothelioma patients refractory to chemotherapy, meaning the induction of cytotoxic tumor-specific CD8+ T cells (Ranki et al, 2014). However, priming with ONCOS-102 before the start of chemotherapy did not show any additional benefit in current immunodeficient animal models. This is not surprising, as the immune system has little effect in an immunodeficient mouse model.

The current data strongly suggest that ONCOS-102in combination with first-line chemotherapy can be used as an effective treatment against MM. Because of its safety profile, ONCOS-102 replicates only locally in the tumor and exhibits a high tropism for mesothelioma cells. No major side effects were reported during the animal studies. Combination therapy shows a synergistic effect, being the most effective therapy for mesothelioma and therefore also more effective than standard first-line chemotherapy. In addition, the combination of ONCOS-102 with chemotherapy is a powerful tool that overcomes the major hurdles of the immunosuppressive microenvironment in the tumor microenvironment due to the induction of enhanced induction of immunogenic tumor cell death and ultimately induction of anti-cancer immune responses. The inventors have shown that combination therapy results in an increase in ICD, suggesting an enhanced activity of tumor immunogenic cell death. In addition, the combination therapy according to the invention improves the infectivity of ONCOS-102in mesothelioma cells. GM-CSF function is lost in this animal model, but it is known from previous studies that this cytokine plays an important role by directly recruiting APC and natural killer cells as well as by activating APC and maturing it at the tumor site.

In addition to enabling the delivery of the vector to the target site, the adenoviral vector of the invention also ensures the expression and persistence of the transgene. In addition, the immune response to the vector as well as the transgene is also minimized.

The present invention addresses the problems associated with treatment resistance of conventional therapies. In addition, the present invention provides tools and methods for selective treatment with lower toxicity or damage in healthy tissue. Advantageous aspects of the invention also include different and reduced side effects compared to other therapeutic agents. Importantly, the method is synergistic with many other forms of therapy, including radiation therapy, and is therefore suitable for use in combination regimens.

The present invention enables cancer treatment in which tumor cells are destroyed by virosomal-induced oncolysis. In addition, various different mechanisms of recruiting activation of the human immune response, including activation of natural killer cells (NK) and Dendritic Cells (DC), are useful for the therapeutic uses of the present invention.

Thus, the present application describes strategies and provides methods and means to effectively recruit the host's immune system against malignant cells and to provide both direct oncolytic and chemotherapeutic activity in malignant cells while maintaining excellent safety records.

Aspects of the present invention relate to novel methods and means for achieving efficient and accurate gene transfer in cancer therapy with increased specificity and sufficient tumor killing capacity.

The unexpected efficacy of the inventive therapy with oncolytic adenovirus in combination with the use of chemotherapeutic agents provided a significant improvement in therapeutic efficacy (compared to the current standard of care (SoC) chemotherapy regimen for malignant mesothelioma (pemetrexed + cisplatin or pemetrexed + carboplatin)), as demonstrated in vivo studies.

In summary, the data presented provide a strong theoretical basis for the use of ONCOS-102in combination with first-line chemotherapy for the treatment of malignant mesothelioma.

Examples

The following examples are given for the purpose of illustrating various embodiments of the invention only and are not meant to limit the invention. Those skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and attain the ends and advantages mentioned, as well as those objects, and advantages inherent therein. Variations thereof and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined by the scope of the claims.

Construction of ONCOS-102

The construction and characterization of a chimeric oncolytic adenovirus encoding human GM-CSF (ONCOS-102) has been previously described (WO2010/072900 and Koski et al). Briefly, Ad5/3-D24-GMCSF was generated and amplified using standard adenovirus preparation techniques. A pAdEasy-1 derived plasmid containing chimeric 5/3 fibers, pAdEasy5/3, was generated by homologous recombination of the Ad5/3luc1 viral genome with the BstXI-digested 8.9kb fragment of pAdEasy-1 in E.coli (Escherichia coli). Subsequently, the shuttle vector containing the 24bp deletion in E1A (pShuttled D24) was linearized with PmeI and recombined with pAdEasy5/3 to give pAd 5/3-D24. For insertion of the human GMCSF gene into the E3 region, the DNA fragment from the Ad5 genome was inserted into pGEM5Zf⁺(Promega, Madison, Wis.) to generate the E3 cloning vector pTHSN. Further digestion of pTHSN with SunI/MunI resulted in a deletion of 965bp in the E3 region (6.7K and gp19K deleted). A432 bp complementary DNA (Invitrogen, Carlsbad, CA) encoding human GM-CSF was amplified with primers characteristic of the specific restriction sites SunI/MunI flanking the gene, and then inserted into SunI/MunI digested pTHSN. pAd5/3-D24-GMCSF was produced by homologous recombination between FspI-linearized pTHSN-GMCSF and SrfI-linearized pAd5/3-D24 in E.coli. The Ad5/3-D24-GMCSF viral genome was released by PacI digestion and transfected into A549 cells for amplification and rescue. All stages of cloning were confirmed by PCR and multiple restriction digestions. The shuttle plasmid pTHSN-GM-CSF was sequenced. The absence of wild type E1 was confirmed by PCR. The final virus was examined for the E1 region, transgene, and fiber by sequencing and PCR. Viral production was performed on a549 cells according to cGMP principles of Oncos Therapeutics (Helsinki, Finland) to avoid the risk of wild-type recombination. The formula of the virus stock solution buffer solution is 10mmol/l Trizma alkali, 75mmol/l NaCl, 5% (wt/vol) sucrose, 1mmol/l MgCl, 10mmol/l L (+) histidine, 0.5% (vol/vol) EtOH, 0.02% Tween and 100 mu mol/l EDTA; as diluent, 0.9% (wt/vol) NaCl solution (b.braun, Melsungen, Germany) was used. ONCOS-102 was produced by Biovian (Turku, Finland) in GLP and stored at-80 ℃ until use. For use, the ONCOS-102 was thawed immediately prior to administration and stored on ice until use. After thawing, ONCOS-102 were diluted in a laminar flow box to obtain the desired concentration, syringes were prepared and stored on ice until use.

Cell lines

Human epithelial-like mesothelioma cell line JL-1(ACC 596, purchased from DSMZ) was cultured in Darbeck's modified eagle's medium (DMEM,10567-014, Gibco) supplemented with 20% heat-inactivated fetal bovine serum (FBS,16000-044, Gibco), 2mM L-glutamine, 1% penicillin and streptomycin (15140-122, Gibco). Human malignant biphasic mesothelioma MSTO-211H (ACC390, purchased from DSMZ) and human epithelial mesothelioma NCI-H226(H226, CRL-5826, purchased from ATCC) were cultured in RPMI 1640(A10491-01, Gibco) supplemented with 10% heat-inactivated FBS (16000-. All cell lines were incubated at 37 ℃ and 5% CO₂The incubation was performed.

Chemotherapeutic agents

Pemetrexed disodium (sc-219564), cisplatin (sc-200896), and carboplatin (sc-202093) were purchased from Santa Cruz Biotechnology and reconstituted in sterile water prior to use.

10mg of pemetrexed (pemetrexed) was suspended in 1ml of sterile water to obtain a10 mg/ml solution. This solution was used as stock solution and stored at room temperature. After addition of the solution, the material was shaken for 1 to 5 minutes to give a homogeneous solution. Stock solutions were further diluted in 0.9% NaCl and 10 mg/kg/mouse (in 100. mu.l) was used.

150mg of cisplatin (cis) was suspended in 150ml of sterile water to give a 1mg/ml solution. This solution was used as stock solution and stored at room temperature. After the addition of the solution, the material was shaken for 1 to 5 minutes to give a homogeneous solution. Stock solutions were further diluted in 0.9% NaCl and 1.5 mg/kg/mouse (in 100. mu.l) was used.

25mg of carboplatin (car) was suspended in 10ml of 0.9% NaCl to give a 2.5mg/ml solution. This solution was used as stock solution and stored at room temperature. After addition of the solution, the material was shaken for 1 to 5 minutes to give a homogeneous solution. Stock was further diluted and 8 mg/kg/mouse (in 100. mu.l) was used.

Cell viability-in vitro tumor cell killing assay

Mesothelioma cells were plated at 1.0x10 per well⁴Individual cells were seeded in 96-well plates. After overnight incubation, cells were infected with ONCOS-102 with a viral particle/cell ratio of 10 (VP/cell). The virus and chemotherapeutic agent were diluted in medium containing 5% FBS. Pemetrexed, cisplatin and carboplatin were tested at the following suboptimal, previously selected concentrations, respectively: 0.625mg/ml, 0.0026mg/ml, 0.0625mg/ml (H226 cells); 0.625mg/ml, 0.0006mg/ml, 0.00196mg/ml (Jl-1 cells); 0.083mg/ml, 0.0026mg/ml, 0.0625mg/ml (MSTO-211H cells). Eight different therapeutic combinations were evaluated: ONCOS-102 alone, Pemetrexed + cisplatin, Pemetrexed + Carboplatin, ONCOS-102+ Pemetrexed + cisplatin, ONCOS-102+ Pemetrexed + Carboplatin, ONCOS-102+ Pemetrexed + cisplatin (priming: Virus administration first, chemotherapy addition 24 hours post infection), ONCOS-102+ Pemetrexed + Carboplatin (priming: Virus administration first, chemotherapy addition 24 hours post infection), and mock (growth medium only). After 3 days, cell viability was determined by MTS according to the manufacturer's instructions (G3582, Promega).

Analysis of apoptotic and necrotic cells in vitro

Mesothelioma cells were plated at 5x10⁵Individual cells/well were seeded onto 6-well plates. Cells were infected with 10 VP/cell ONCOS-102 supplemented with chemotherapeutic agents according to the treatment protocol described above. After 48 hours, the amount of apoptotic and necrotic cells was measured by flow cytometry (4830-01-K, Trevigen Inc.) using the TACS Annexin V-FITC kit according to the manufacturer's instructions.

Immunogenicity of in vitro tumor cell death

The CRT is exposed. Cell lines at 5X10⁵Individual cells/well were seeded in duplicate onto 6-well plates. The cells were infected with 10 VP/cell ONCOS-102 and/or chemotherapeutic agents according to the treatment combinations described above. After 24 hours (H226, J1-1) and 48 hours (MSTO-211H), cells were harvested and stained with a 1:1000 dilution of rabbit polyclonal anti-calreticulin antibody (ab2907, Abcam) for 40 minutes at 4 ℃ followed by a 1:100 dilution of Alexa-Fluor 488 secondary antibody (A21206, Invitrogen) and analyzed by flow cytometry.

HMGB-1 is released. Cell lines were plated in triplicate on cell culture plates and infected with 10 VP/cell ONCOS-102 and/or chemotherapeutic agents according to the therapeutic combinations described above. After 72 hours, the supernatant was collected and HMGB-1 was measured with the Elisa kit according to the manufacturer's instructions (ST51011, IBL International).

And (4) ATP release. Cell lines were plated in triplicate onto cell culture plates and processed as described above. Supernatants were collected after 48 hours (Jl-1, MSTO-211H) and 72 hours (H226) and analyzed with an ATP assay kit for luminescence analysis according to the manufacturer's protocol (A22066, Invitrogen).

In vitro viral infectivity-immunocytochemistry assay (ICC)

Mesothelioma cell line was replicated in 5 replicates at 6x10⁵Individual cells/well were seeded in 24-well plates and treated with the 8 different treatment combinations described above. After 24 hours, the supernatant was aspirated and the cells were fixed by incubating them with ice-cold methanol for 15 minutes (322415-100ML, Sigma-Aldrich). The determination of the infectivity of ONCOS-102 is based on the visual quantification of viral hexon protein in infected cells. Briefly, cells were stained with a 1:2000 diluted mouse anti-hexon antibody (NB600-413, Novus Biologicals) at room temperature for 1 hour in the dark followed by a 1:500 diluted biotin-SP conjugated secondary antibody (115-. Subsequently Extravidin-peroxidase was added at 1:200 and incubated for 30 min at room temperature in the dark (E2886-1ML, Sigma-Aldrich). Finally, infected cells were visualized by adding the stain DAB for up to 5 minutes (D3939-1SET, Sigma-Aldrich). For each 5 replicates (wells), 5 images of non-overlapping fields of view were acquired using the AMG EVO XL microscope. Infectivity data are expressed as the average number of spots in 5 wells.

Expression of CAR, CD46 and DSG 2in mesothelioma cell lines

Human adenovirus infects cells after attachment to coxsackie virus B and Ad receptor (CAR), CD46 or desmoglein 2 (DSG-2). Mouse monoclonal anti-CAR antibody for mesothelioma cells (1. mu.g/1X 10)⁶Individual cells) (sc-56892FITC, Santa Cruz Biotech) were labeled at 4 ℃ for 40 minutes, or with miceMonoclonal anti-CD 46 antibody (1. mu.g/1X 10)⁶Individual cell) (ab789, Abcam) or mouse monoclonal anti-DSG 2 antibody (1. mu.g/1X 10)⁶Individual cells) (ab14415, Abcam) were labeled at 4 ℃ for 40 minutes, followed by flow cytometry measurements with a 1:2000 diluted Alexa-Fluor 488 secondary antibody (a21206, Abcam).

Demonstration of dose level and route

The immunomodulatory function of transgenic GM-CSF is the central mechanism of the action of ONCOS-102. In addition, adenoviruses are themselves strong activators of the innate immune system. BALB/c nude mice are immunodeficient and therefore can only be studied for replication-dependent oncolytic function of the virus, thereby losing the main mechanism of action (immune-mediated effect). Dose 5E +7 VP/tumor i.t. (1E +8 VP/mouse) was selected based on the results of two experimental studies in which doses 2E + 8/mouse and 1E + 8/mouse were tested (fig. 5 and 6). Suboptimal viral doses (non-curative) were chosen to allow evaluation of the potential additive and/or synergistic combination effects of ONCOS-102 and chemotherapy. The route of injection was selected based on the route used in the clinical trial (i.e. intratumoral).

The dose and route of administration of the chemotherapeutic agent is selected based on technical feasibility.

Animal selection, randomization, group assignment, housing, feeding and water and environmental adaptation

Animals were randomized into groups. Groups of animals were kept in separate cages and study cards were attached to the cages. These cards are labeled with study number, treatment group, individual animal ID, and their source and date of arrival. Individual animals in each cage were marked with ear punctures for identification. Only one cage was treated at a time to avoid confusion of animals between cages.

Animals were housed in a facility with a biosafety level of 2(BSL2) in an environment with monitored humidity and temperature. Animals were housed in individually ventilated plastic cages with filter lids (GreenLine, Scanbur).

Animals were fed standard pelleted feed provided ad libitum. Water was supplied ad libitum during acclimation and study.

Animals were acclimated for 7 days prior to the start of the experimental part. All animals remained in good health throughout the acclimation period.

Clinical observations

All animals were observed daily for clinical signs, morbidity or mortality during the acclimation and administration periods, and for an additional 60 minutes after each treatment. During the experiment, mice were killed as the tumor size reached the maximum allowed (1.5cm in any diameter). The health of the animals was followed daily and no signs of discomfort due to the tumor were seen. Mice were sacrificed with carbon dioxide on day 63, which was the end point of the experiment. In addition, dislocation of cervical vertebrae is also performed.

The body weight of the animals was tracked throughout the experiment and slight weight loss was observed in the following groups: pep + cis (10% reduction on day 48, 11% reduction on day 60), ONCOS-102 elicited + pep + cis (9% reduction on day 48, 12% reduction on day 60) (FIG. 5B), pep + car (6% reduction on day 60) (FIG. 6B). In the group of ONCOS-102 alone or ONCOS-102in combination with pemetrexed and cisplatin (single case), few animals were sacrificed due to health problems (> 30% weight loss and skin problems (> rash)). These adverse events may be treatment independent, as both mock treated animals also suffered from skin problems (rashes). The veterinarian is informed of these issues for further investigation, but the cause of these events remains unclear. In addition, necropsy revealed some changes in the morphology of the intestines (jejunum and ileum) and stomach of a few animals (pep + cis; pep + car).

Analysis of tumor size progression

Tumor size was measured and recorded starting 8 days after cell injection. In fig. 4A, the first day of treatment and the first day of tumor size measurement are indicated as day 0. Tumors were measured every three days.

Record the longest and shortest diameters and use 0.52x length x (width)²The formula (iv) calculates the tumor volume. The progression of tumor size is expressed as a percentage of the first measurement point (arbitrarily set to 100%) on day 0.

Data compilation and statistical analysis

Body weight was measured every 3 days throughout the experiment. The percent body weight change from baseline was calculated for each animal.

Tumor volume (table 2): absolute tumor volume is the endpoint of tumor volume analysis. Absolute tumor volumes were assessed on day 21 and day 48, respectively. Analysis of variance of duplicate measurements was used as an analytical method for tumor volume, since two tumors were measured for each mouse. The model contains the group effect and baseline values as covariates (as fixed effects). Pairwise comparisons between groups were adjusted using Tukey's method. Synergy calculations were performed by using FTV (fractional tumor volume) method.

Reasons for the selection of time points: day 21 is the last day that all animals were still alive, and on day 48, no more than one animal per group died.

Human mesothelioma xenograft model

Animal experiments were approved by the University of Helsinki animal Committee and the provincial government of Southern Finland. 6-8 week old mice were obtained from Scanbur (Karlslunde, DK) and isolated for 1 week prior to the start of the experiment. Mice were anesthetized with isoflurane furan (Baxter) and 50. mu.l of NCI-H226 cells were injected into both flanks (6E + 06/flank). Tumors were allowed to grow for 8 days prior to treatment. The virus was administered every 6 days. One group received only ONCOS-102, two groups received ONCOS-102 and chemotherapy (pemetrexed + cisplatin or pemetrexed + carboplatin) once every 6 days, while the other two groups received ONCOS-102 priming alternating over a 3 day cycle followed by combination therapy of chemotherapy (pemetrexed + cisplatin or pemetrexed + carboplatin) and ONCOS-102. Mock animals were treated with 0.9% saline. ONCOS-102 was diluted into 0.9% saline and treated at 5X10 per tumor⁷Dose of VP was injected intratumorally (two tumors per animal). Injections were performed in a fan-shaped pattern to ensure even distribution throughout the tumor. Pemetrexed, cisplatin, and carboplatin were diluted in 0.9% NaCl and administered intraperitoneally at doses of 10mg/kg, 1.5mg/kg, and 8mg/kg, respectively. The injection volume of each chemotherapeutic agent was 100 μ l.

Tumor size was measured with calipers every 3 days in two dimensions starting on the first treatment day. The longest and shortest diameters were recorded and used 0.52x length x (width)²The formula (iv) calculates the tumor volume. Swelling and swelling treating medicineThe progression of tumor size was expressed as a percentage of the first measurement point (arbitrarily set to 100%) on day 0 (fig. 4A). All animals were observed daily for clinical signs, morbidity or mortality during acclimation and administration, and for an additional 30 minutes after each treatment. During the experiment, mice were killed when the tumor size reached the maximum diameter allowed (1.5 cm).

Human GM-CSF ELISA

Total proteins from harvested BALB/c nude mouse tissue samples (tumor and liver) were extracted using tissue extraction reagent I (FNN0071, Invitrogen) supplemented with protease inhibitor cocktail (P-2714, Sigma-Aldrich) according to the manufacturer's instructions. Protein extracts and previously collected sera were analyzed for human GM-CSF concentration using ELISA (ab100529, Abcam) according to the manufacturer's instructions.

Quantitative real-time PCR

qPCR for adenovirus E4 copy number was performed according to the protocol previously described (primer FW: 5'-GGA GTG CGC CGA GAC AAC-3', primer RV: 5'-ACT ACG TCC GGC GTT CCA T-3', probe E4:5 '- (6FAM) -TGG CAT GAC ACT ACG ACC AAC ACG ATC T- (TAMRA) -3') (Koski et al 2010). Total DNA was extracted from BALB/c nude mouse samples (tumor, liver, Blood) using the QIAamp DNA Blood Mini kit (51106, Qiagen) according to the manufacturer's protocol. Subsequently, the isolated DNA was analyzed for adenovirus E4 copy number and normalized against murine β -actin (liver, blood) and human β -actin (tumor) (primer FW: 5'-CGA GCG GTT CCG ATG C-3', primer RV: 5'-TGG ATG CCA CAG GAT TCC AT-3', probe murine β -actin: 5 '- (6FAM) -AGG CTC TTT TCC AGC CTT CCT TCT TGG- (TAMRA) -3'; (primer FW: 5'-CAG CAG ATG TGG ATC AGC AAG-3', primer RV: 5'-CTA GAA GCA TTT GCG GTG GAC-3', probe human β -actin: 5 '- (6FAM) -AGG AGT ATG ACG CCG GCC CCT C- (TAMRA) -3'), using a LighCyryqPCR instrument (LighCycler 480, roche) to analyze the sample.

Statistical analysis

Statistical significance was analyzed using one-way anova and Tukey multiple comparison test and unparameterized Mann-Whitney test. Survival curves and statistical analysis were performed using the Kaplan-Meier test. All statistical analyses, calculations and tests were performed using GraphPad Prism 5(GraphPad Software, San Diego, USA). Results are expressed as mean ± SEM. All p values are bilateral or unilateral, and are considered statistically significant when < 0.05.

Example 1 in vitro antitumor Activity of the combination of ONCOS-102 with first line chemotherapy on mesothelioma

Oncolytic potency of ONCOS-102 was tested in three mesothelioma cell lines in vitro (fig. 1A). JL-1 (epithelial-like mesothelioma), MSTO-211H (malignant biphasic) and H226 (epithelial morphology) cells appear to be relatively resistant to oncolytic effects, as 10 VP/cell (sub-optimal dose) killed 18%, 24% and 11% of the cells in 3 days, respectively. Compared to MSTO-211H cells (pemetrexed + cisplatin or pemetrexed + carboplatin), JL-1 and H226 cell lines are more resistant to chemotherapy-mediated cytotoxicity. Incubation with chemotherapeutic agents only killed 10% of JL-1 and 11-12% of H226 cells within 3 days. In contrast, on day 3 of co-culture with pemetrexed + cisplatin and pemetrexed + carboplatin, 63% and 73% of MSTO-211H cells were killed, respectively. The combination of ONCOS-102 with chemotherapeutic agents significantly increased cytotoxicity in H226 and JL-1 cells compared to the results observed in monotherapy (viral or chemotherapy). However, when chemotherapy was combined with ONCOS-102, no increased cytotoxicity was observed in MSTO-211H cells. Overall, H226 and JL-1 cells were more resistant to oncolytic effects (ONCOS-102 alone) and cytotoxic effects of chemotherapeutic agents than MSTO-211H cells, which were particularly sensitive to chemotherapy.

Consistent with the cell viability results, the number of apoptotic H226 and JL-1 cells was generally lower in all treatment groups, but the combination treatment slightly increased the number of apoptotic cells compared to the monotherapy. In contrast to H226 and JL-1 cells, the number of apoptotic MSTO-211H cells was significantly higher in chemotherapy alone and in cells treated with chemotherapy + ONCOS-102 (FIG. 1B).

Example 2 combination of ONCOS-102 and chemotherapy enhances immunogenic cell death and viral replication of mesothelioma cell lines

Immunogenic cell death markers, such as calreticulin a exposure to the cell surface and extracellular release of ATP and HMGB1 were measured from mesothelioma cell cultures after exposure to onocos-102, chemotherapeutic agents, or a combination of both (fig. 2). The most immunogenic of Jl-1 and H226 mesotheliomas tumor cell death was induced by treatment with ONCOS-102+ chemotherapy, as the highest amount of CRT exposure and extracellular release of HMGB1 and ATP was measured in these groups. In turn, the simultaneous administration of ONCOS-102 and chemotherapy in MSTO-211H cells was as immunogenic as chemotherapy alone, consistent with observations from cell viability assays, confirming the high sensitivity of these cells to chemotherapy.

The effect of chemotherapy on in vitro replication of ONCOS-102 was also evaluated. H226 and Jl-1 cells infected with ONCOS-102 and pretreated (primed) with chemotherapeutic agents (pemetrexed + cisplatin or pemetrexed + carboplatin) showed a significant increase in the number of infected cells relative to control cells (p <0.001 vs ONCOS-102) (fig. 3C). On the other hand, pretreatment of MSTO-211H cells with chemotherapy did not increase the number of infected cells. In contrast, concurrent administration of chemotherapy to three mesothelioma cell lines with ONCOS-102 resulted in inhibition or limitation of viral replication in vitro.

Example 3 ONCOS-102 shows a high tropism for mesothelioma cells

ONCOS-102 is a chimeric oncolytic adenovirus in which fiber knob 5 is replaced by a serotype knob 3 domain. Positive cells were screened for CD46, DSG2(Ad3), and CAR (Ad5) receptors (fig. 3B). MSTO-211H, H226 and JL-1 expressed high levels of CD46 (98%, 96% and 98%, respectively), DSG2 (95%, 7% and 64%, respectively) on their surface. CAR receptors are expressed by two of the three mesotheliomas, H226 (88%) and J11 (15%).

Example 4 ONCOS-102in combination with Pemetrexed and cisplatin or Pemetrexed and Carboplatin exhibit increased anti-tumor efficacy against MM in a human xenograft mesothelioma model

In view of the observed synergy of ONCOS-102 with pemetrexed and cisplatin or pemetrexed and carboplatin in vitro, it was next assessed whether there was also enhanced antitumor activity in an in vivo animal model. Thus, the human mesothelioma H226 cell line was implanted subcutaneously into BALB/c nude mice and tumors were treated according to the treatment protocol shown in table 1.

Tumor growth (volume) was calculated at the indicated time points and reported as a function of time (fig. 4A). H226 mesothelioma xenograft tumors appear to be refractory to standard chemotherapeutic agents (pemetrexed + cisplatin, pemetrexed + carboplatin) because none of the treatments significantly reduced tumor growth. Chemotherapy alone is the least effective treatment modality for mesothelioma, in part because of the suboptimal doses used. One animal treated with ONCOS-102+ pemetrexed + cisplatin showed complete tumor regression by day 21 (both tumors). In addition, one animal treated with ONCOS-102-primed + pemetrexed + cisplatin showed complete regression of both tumors by day 45. ONCOS-102 alone was able to control tumor growth to some extent (p-0.074 mimics control of ONCOS-102 on day 48). The most effective treatment modality for pleural mesothelioma was to prime with ONCOS-102 plus pemetrexed and cisplatin (this treatment showed 97% initial tumor size vs 473% (mock), 563% (pemetrexed + cisplatin) and 672% (pemetrexed and carboplatin) on day 60). In addition, in all combination regimens (ONCOS-102+ chemotherapy), we observed the most significant antitumor activity, in particular compared to chemotherapy alone, virus alone and mock (initial tumor size: 97% (virus challenge + pemetrexed + cisplatin), 138% (virus + pemetrexed + cisplatin), 151% (virus challenge + pemetrexed + carboplatin) and 163% (virus + pemetrexed + carboplatin) compared to day 60 206% (virus alone), 473% (mock), 563% (pemetrexed + cisplatin) and 672% (pemetrexed + cisplatin) in all combinations tested, ONCOS-102 and chemotherapeutic showed strong synergistic antitumor effect on day 48 (R ═ 2.19ONCOS-102+ pemetrexed + cisplatin; R ═ 1.7ONCOS-102+ pemetrexed + carboplatin; R ═ 2.5ONCOS-102 + pemetrexed + R ═ 1.41 ═ ONCOS-102+ carboplatin) . On day 21, the combination of ONCOS-102 with pemetrexed + cisplatin showed a synergistic effect on both regimens (R ═ 1.8ONCOS-102+ pemetrexed + cisplatin; R ═ 1.32ONCOS-102 elicitation + pemetrexed + cisplatin), while the combination with pemetrexed + carboplatin showed an additive effect (R ═ 1.0 and 0.93 for the combination group).

Table 1 the antitumor activity of ONCOS-102in combination with chemotherapeutic agents (pemetrexed and cisplatin or pemetrexed and carboplatin) in mesothelioma was evaluated in eight groups (a-H). Different treatment regimens, chemotherapy and virus concentrations were tested in a human mesothelioma xenograft model of BALB/c nude mice over a2 month period.

Table 2 discloses mesothelioma treatment of BALB/c nude mice with ONCOS-102in combination with pemetrexed + cisplatin or pemetrexed + carboplatin.

TABLE 2 combination therapy of mesothelioma with ONCOS-102 and pemetrexed + cisplatin or pemetrexed + carboplatin in BALB/c nude mice. Treatment synergy was assessed using FTV calculations. FTV (mean tumor volume (%) experiment)/(mean tumor volume control (%)). X (mean FTV (%) for chemotherapy) x (mean FTV (%) for ONCOS-102). (expected FTV divided by observed FTV). A ratio (R) of >1 indicates a synergistic effect, and a ratio of <1 indicates less than an additive effect.

Example 5 local replication of ONCOS-102in tumor tissue and production of human GM-CSF

Adenovirus E4 copy number and GM-CSF levels were quantified in murine organs (tumor, liver) and serum by qPCR and ELISA. ONCOS-102 was present only in tumors (local), while no viral particles were detected in serum or liver in all tested groups (fig. 4D). ONCOS-102 is producing GM-CSF protein, the highest concentration of which was detected in tumors in 5 tested groups (see FIG. 4C).

The present invention relates to the following embodiments:

an ONCOS-102 adenovirus for use in the treatment of human malignant mesothelioma, wherein the virus is administered to a patient in combination with two chemotherapeutic agents.

2. The ONCOS-102 adenovirus of embodiment 1 for use in treating malignant mesothelioma in a human, wherein the two chemotherapeutic agents are pemetrexed and cisplatin or pemetrexed and carboplatin.

3. The ONCOS-102 adenovirus of embodiment 2, wherein the molar ratio between pemetrexed and cisplatin is from 10:0.75 to 10:3, is used to treat human malignant mesothelioma.

4. The ONCOS-102 adenovirus of embodiment 3, wherein the molar ratio between pemetrexed and cisplatin is 10:1.5, is used to treat human malignant mesothelioma.

5. The ONCOS-102 adenovirus of embodiment 2, wherein the molar ratio between pemetrexed and carboplatin is between 5:8 and 5:2, is used to treat human malignant mesothelioma.

6. The ONCOS-102 adenovirus of embodiment 5, wherein the molar ratio between pemetrexed and carboplatin is 5:4, is used to treat human malignant mesothelioma.

7. The ONCOS-102 adenovirus according to any one of embodiments 1 to 6, wherein the amount of the virus is 5x10, for use in the treatment of human malignant mesothelioma¹⁰To 5x10¹¹ VP。

8. The ONCOS-102 adenovirus of embodiment 7 for use in the treatment of human malignant mesothelioma, wherein the virus is identified as 3x10¹¹VP/5 ml.

9. The ONCOS-102 adenovirus of any one of embodiments 1-8, wherein the chemotherapeutic agent and the virus are administered in effective amounts, for use in the treatment of human malignant mesothelioma.

10. The ONCOS-102 adenovirus of any one of embodiments 1-9, wherein the virus is administered intraperitoneally or by direct injection into a tumor, and the two chemotherapeutic agents are administered intravenously or intraperitoneally, for the treatment of human malignant mesothelioma.

11. The ONCOS-102 adenovirus according to any one of embodiments 1 to 10 for use in the treatment of human malignant mesothelioma, wherein the virus is administered prior to and also during the administration period of the two chemotherapeutic agents.

12. The ONCOS-102 adenovirus according to any one of embodiments 1 to 10 for use in the treatment of human malignant mesothelioma, wherein the two chemotherapeutic agents are administered before and also during the administration phase of the virus.

13. A method for treating malignant mesothelioma in a patient, comprising the step of administering to the patient an ONCOS-102 adenovirus and two chemotherapeutic agents.

14. The method of embodiment 13, wherein the virus and chemotherapeutic agent are administered in effective amounts.

15. The method of embodiment 13 or 14, wherein the chemotherapeutic agent is pemetrexed and cisplatin or pemetrexed and carboplatin.

16. The method of any one of embodiments 13-15, wherein the chemotherapeutic agent is administered first prior to beginning the administration phase of the virus, and is also administered during the administration phase of the virus.

17. The method according to any one of embodiments 13-15, wherein the virus is administered first before the administration phase of the chemotherapeutic agent is initiated, and the virus is also administered during the administration phase of the chemotherapeutic agent.

18. The method according to any one of embodiments 13-17, wherein the ONCOS-102 adenovirus is administered to the subject 1 to 10 times and the chemotherapeutic agent 1 to 6 times.

19. The method of any one of embodiments 13-18,wherein the administration of ONCOS-102 is 10⁸-10¹²The dosage of each plaque forming unit is administered either by direct injection into the malignant mesothelioma tumor of the patient or by intraperitoneal injection.

20. The method according to any one of embodiments 13-19, wherein the amount of the ONCOS-102 adenovirus to be administered is at 5x10¹⁰-5x10¹¹VP is within the range.

21. The method of any one of embodiments 13-20, further comprising administering cyclophosphamide prior to the step of administering the ONCOS-102 adenovirus and the two chemotherapeutic agents to the patient.

22. The method according to any one of embodiments 13-21, further comprising administering folic acid to the patient.

23. The method of any one of embodiments 13-22, further comprising administering cyanocobalamin to the patient.

24. The method of any one of embodiments 13-23, further comprising administering dexamethasone to the patient.

Use of an ONCOS-102 adenovirus in the treatment of human malignant mesothelioma, wherein the virus and two chemotherapeutic agents are administered in combination to a patient.

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Sequence listing

<110> Targovax Oy

<120> use of adenovirus in combination with chemotherapeutic agents for the treatment of cancer

<130> B5587PC

<160> 9

<170> BiSSAP 1.3

<210> 1

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide

<400> 1

ggagtgcgcc gagacaac 18

<210> 2

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide

<400> 2

actacgtccg gcgttccat 19

<210> 3

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide

<400> 3

tggcatgaca ctacgaccaa cacgatct 28

<210> 4

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide

<400> 4

cgagcggttc cgatgc 16

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide

<400> 5

tggatgccac aggattccat 20

<210> 6

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide

<400> 6

aggctctttt ccagccttcc ttcttgg 27

<210> 7

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide

<400> 7

cagcagatgt ggatcagcaa g 21

<210> 8

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide

<400> 8

ctagaagcat ttgcggtgga c 21

<210> 9

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> oligonucleotide

<400> 9

aggagtatga cgccggcccc tc 22