FI121508B - Adenovirus vectors and related methods and uses - Google Patents

Description

Adenovirus vectors and related methods and uses

Field of the Invention

The present invention relates to the fields of life sciences and medicine. In particular, the invention relates to cancer treatments. More particularly, the present invention relates to oncolytic adenoviral vectors and cells and pharmaceutical compositions comprising said vectors. The present invention also relates to the use of said vectors in the manufacture of a medicament for the treatment of cancer of a subject. In addition, the present invention relates to methods for producing GM-CSF in a cell and for increasing a tumor-specific immune response in a subject, and for using an oncolytic adenovirus vector of the invention to produce GM-CSF in a cell and boosting a tumor-specific immune response in a subject.

Background of the Invention

Cancer can be treated with surgery, hormone therapy, chemotherapy and / or radiation, but in many cases, cancers, which are often characterized by being advanced, cannot be cured by current treatments. Therefore, new approaches to cancer cells, such as gene therapy, are needed.

Over the past twenty years, much has been done on gene transfer technology 20. The purpose of gene therapy for cancer is to introduce a therapeutic gene into a tumor cell. These therapeutic genes introduced into the target cell may, for example, repair mutated genes, suppress active oncogenes, or create additional cellular properties. Suitable exogenous therapeutic genes include, but are not limited to, immunotherapeutic, anti-angiogenic and chemo-protective genes, and "suicide genes" and may be introduced into the cell utilizing modified viral vectors or non-viral methods including electroporation, gene stop and lipid or polymer terminus.

Requirements for optimal viral vectors include an effective ability to locate specific target cells and to express the viral genome in the target cells. In addition, the optimal vectors should remain active in the target tissues or cells. All these characteristics of viral vectors have been developed in recent decades, and retroviral and adenoviral vectors and adeno-associated viral vectors, for example, have been extensively studied in biomedicine.

Selective oncolytic agents, such as conditionally replicating adenoviruses, have been selectively constructed to further enhance local penetration of tumor penetration and antitumor activity. Oncolytic adenoviruses are a promising tool for the treatment of cancers. Oncolytic adenoviruses kill tumor cells by replication of the virus in the tumor cell, whereby the final step of replication leads to the release of thousands of virions into the surrounding tumor tissue for efficient penetration and vascular reinfection. Tumor cells allow viral replication, whereas normal cells are spared due to modifications in the viral genome that prevent replication in non-tumor cells.

In addition to replication-mediated cell killing, oncolytic adenoviruses may also be provided with a variety of therapeutic transgenes. In this approach, the benefits of traditional gene delivery are combined with the efficacy of replication competent agents. One of the aims of providing viruses is to induce an immune reaction against cells that allow viral replication. Although viral replication is immunogenic, it alone is not normally sufficient to induce effective anti-tumor immunity. To enhance the induction of therapeutic immunity, viruses can be provided with stimulatory proteins such as cytokines and by facilitating the delivery of tumor antigens into dendritic cells. Introduction of immunotherapeutic genes into tumor cells and, in addition, translation of proteins lead to activation of the immune response and efficient destruction of tumor cells. The most important immune immune cells in this regard are natural killer (NK) cells and cytotoxic CD8 + T cells.

Adenoviruses are medium-sized (90-100 nm), non-enveloped icosahedral viruses with approximately 36 kilobase pairs of double-stranded line-25 ara DNA in a protein capsid. The viral capsid has filamentous structures that are involved in attaching the virus to the target cell. First, the knob domain of the fiber protein binds to the target cell receptor [e.g., CD46 or coxsackievirus adenovirus receptor (CAR)], second, the virus interacts with the integrin molecule, and third, the virus is endocytosed to the target cell. Thereafter, the viral genome is transferred from endosomes to the nucleus and the replication machinery of the target cell is also utilized for viral purposes (Russell W. C. 2000, J General Virol 81,2573-2604).

The adenovirus genome has early (E1-E4), intermediate (IX and IVa2) and late (G1-L5) genes, which are transcribed sequentially. Early gene products affect the host cell defense mechanisms, the cell cycle and cell metabolism. Mid- and late-stage genes encode structural proteins of viruses to produce new virions (Wu and Nemerow, 2004, Trends Microbiol 12: 162-168; Russell WC 2000, J General Virol 81,2573-2604; Volpers C. and Kochanek S. 2004). , J Gene Med 6 suppl 1, S164-71; Kootstra NA and Verma IM 2003, Annu Rev Phar-5 macol Toxicol 43, 413-439).

More than 50 different serotypes of adenoviruses have been found in humans. The serotypes are classified into six subgroups A-F, and the different serotypes are known to be associated with various diseases such as respiratory, conjunctivitis and gastrointestinal inflammation. Adenovirus serotype 5 (Ad5) is known to cause respiratory-thymus disease and is the most common serotype studied in the field of gene therapy. E1 and / or E3 regions were deleted from the first Ad5 vectors, allowing the introduction of foreign DNA into the vectors (Danthinne and Imperiale 2000). In addition, deletions and further mutations in other regions have provided additional features for viral vectors. Thus, various modifications of adenoviruses have been proposed to provide potent anti-15 tumor effects.

For example, EP1377671 B1 (Cell Genesys, Inc.) and US2003 / 0104625 A1 (Cheng C. et al.) Describe an oncolytic adenoviral vector encoding an immunotherapeutic protein granulocyte macrophage colonization stimulating factor (GM-CSF). EP1767642 A1 (Chengdu Kanghong Biotechnologies Co., Ltd.) also shows that the effects of oncolytic adenoviral vectors enhance the human immune response.

However, more efficient and accurate gene transfer and better specificity of gene therapies and sufficient tumor-killing ability are needed. The safety information of the therapeutic vectors must also be excellent. The present invention provides a cancer treatment with these above-mentioned properties by utilizing both the oncolytic and immunotherapeutic properties of adenoviruses in a novel and inventive manner.

BRIEF DESCRIPTION OF THE INVENTION It is an object of the invention to provide new methods and means for providing the above-mentioned properties of adenoviruses and thus also for solving the problems of conventional cancer therapies. More particularly, the invention provides novel methods and means for gene therapy.

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

4

The present invention relates to an oncolytic adenoviral vector comprising an adenovirus serotype 5 (Ad5) nucleic acid backbone, a 24 bp deletion (D24) E1 Rb binding constant region 2, and a nucleic acid sequence encoding granulocytic coliform macrophage 5 (Ad5). -CSF) in place of the deleted gp19k / 6.7K in the E3 region.

The present invention further relates to an in vitro cell comprising the adenoviral vector of the invention.

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

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

Furthermore, the present invention relates to an in vitro method for producing GM-CSF in a cell, wherein the method a) delivers a vehicle comprising the oncolytic adenoviral vector of the invention to cell b) expressing the GM-CSF of the vector in question in the cell.

Furthermore, the present invention relates to an in vitro method for enhancing a tumor-specific immune response of a subject, comprising a) delivering a vehicle comprising the oncolytic adenovirus vector of the invention to a target cell or tissue b) expressing GM-CSF of said vector in cell c) adding cytotoxic T cells; or the number of natural killer cells in that target cell or tissue.

The present invention further relates to the use of the oncolytic adenoviral vector of the invention for the production of GM-CSF in a cell to provide tumor-specific immunity in a subject.

The present invention further relates to the use of the oncolytic adenoviral vector of the invention to enhance the tumor-specific immune response of the subject.

The present invention provides a means for treating poorly responsive cancers. In addition, there are few limitations regarding the types of tumors that can be treated compared to many other treatments. In fact, all solid tumors can be treated with the present invention. Mass-large and troublesome tumors can be cured by the present invention. Treatment can be given intravenously, intravitreally, intravenously, and in combination. The method of treatment may provide systemic efficacy despite topical injection. The treatment can also kill cells that are thought to be tumor-causing ('' cancer stem cells '').

In addition to allowing the vector to be delivered to the target region, the vector of the invention also ensures expression and persistence of the transgene. In addition, the immune response against the vector and the transgene is minimized.

The present invention solves the problem of therapeutic resistance to conventional therapies. Furthermore, the present invention provides means and methods for selective treatment without causing toxicity or damage to healthy tissues. The advantages of the present invention also have different and minor side effects compared to other treatments. Importantly, the treatment is synergistic with many other therapies, including chemotherapy and radiotherapy, and can thus be used in combination regimens.

The induction of an immune response against cells that allow replication of unstained viruses is normally not potent enough to induce the development of therapeutic tumor immunity. To overcome this weakness, the present invention provides a stock virus with a potent antitumor immunity. The present invention provides a cancer treatment in which tumor cells are destroyed by oncolysis by virions. In addition, the invention affects a variety of mechanisms that activate the human immune response, such as activation of natural killer cells (NKs) and dendritic cells (DCs).

Compared to prior art adenoviral agents, the present invention provides a simpler, more effective, less expensive, non-toxic and / or safer means of treating cancer. In addition, helper viruses are not needed.

The novel products of the invention provide further improvements in the treatment of cancer.

Brief description of the figures

Figure 1 schematically shows Ad5-D24-GMCSF. The virus has a 24 base pair deletion in the E1A constant region 2. gp19k and 6.7K in E3 has been replaced by human GM-CSF cDNA. ADP refers to the adenovirus death protein.

6

Figure 2 schematically shows the first step of cloning to generate a GM-CSF-carrying shuttle plasmid (pTHSN).

Figure 3 schematically shows the second step of cloning to generate a plasmid containing all adenovirus genes and a 24 base pair deletion in the 5 E1 region (mediating selective replication of cancer cells).

Figure 4 schematically depicts the third step of cloning to generate a plasmid containing all adenovirus genes except gp19k and 6.7K, which have been replaced by GM-CSF (pAd5D24.GM-CSF). Ad5-RGD-D24-GMCSF, Ad5 / 3-D24-GMCSF and Ad5-pK7-D24-GMCSF were similarly created. Figure 5 shows that GMCSF expression does not inhibit viral replication and cell killing effect. Figure 5a shows the results of an MTS assay showing the cell killing capacity of lung cancer (A549) with newly created Ad5-D24-GMCSF virus. Figure 5b shows the results of an MTS assay showing the killing of cancer-causing JIMT-1 cells ('' cancerous chicken-15 cells '') by Ad5-D24-GMCSF. Figure 5c shows the results of an MTS assay showing the killer activity of breast cancer cells (MDA-MB-436) with newly created Ad5-D24-GMCSF virus. Figure 5d shows the results of an MTS assay showing the killing efficiency of MDA-MB-436 with newly created Ad5-D24-GMCSF, Ad5-RGD-D24-GMCSF and Ad5 / 3-D24-GMCSF viruses.

Figure 6a shows the expression of human GMCSF associated with adenovirus. The A549 cell line was infected with Ad5D24 or Ad5D24-GMCSF, medium was harvested over time, and expression of GMCSF was analyzed by FAC-SARRAY.

Figure 6b shows that adenovirus-expressed GMCSF retains its biological activity in human lymphocytes. TF1 cells, which require human GMCSF to survive, were grown in the presence of recombinant human GMCSF (produced in E. coli, purchased from Sigma) or supernatant from cells infected with Ad5-D24 GMCSF.

Figures 7a and 7b show an in vitro analysis of transduction of a patient's tumor 30 to test for infectivity of the Ad5-based virus.

Figure 7c shows a prediction of tumor transduction by Ad5-D25-GMCSF by staining its receptor CAR (coxsackievirus-adenovirus receptor) in stored tumor samples.

Figure 8a shows the in vivo efficacy of Ad5-D24-GMCSF in Syrian-35 hamsters (capable of replicating human adenovirus) with pancreatic cancer tumors. Both Ad5D24 and Ad5-D24-GMCSF remove tumors within 16 days of treatment. The virus was given 1 x 109 virus particle pathways on days 0, 2 and 4.

Figure 8b shows that intravenous injection of Ad5-D24-GMCSF resulted in high levels of hGMCSF in serum of Syrian hamsters.

Animals treated with Ad5D24E3 or Ad5-D24-GMCSF were sampled on day 4 and serum levels of human GMCSF were analyzed by FACSARRAY.

Figure 8c shows that healing of FlapTI tumors with Ad5-D24-GMCSF (but not with Ad5D24) protected the Syrian hamsters from subsequent 10 FlapTI re-exposures. This indicates that Ad5-D24-GMCSF can induce a tumor-specific immune response. Animals previously treated with Ad5D24 or Ad5D24-GMCSF (Figure 8a) were re-exposed to the same tumor and tumor growth was measured over time.

Figure 8d shows that induction of a tumor-specific immune response by Ad5-D24-GMCSF, improvement of FlapTI tumors by Ad5-D24-GMCSF did not protect Syrian hamsters from Flak tumors. Animals previously treated with FlapTI tumors with Ad5D24 or Ad5D24-GMCSF (Figure 8a) were re-exposed to a different tumor and tumor growth was measured over time.

Figure 9a shows the potency of Ad5-D24-GMCSF in combination with metronomic oral cyclophosphamide. Computed tomography showed a 88% reduction in tumor at 71 days after initiation of treatment.

Figure 9b shows the efficacy of Ad5-D24-GMCSF treatment in an ovarian cancer patient treated with it. Computed tomography showed complete disappearance of all measurable tumors (indicated by arrow).

Figures 10a-d show that Ad5-D24-GMCSF elicited a T cell response to both tumor epitopes and adenovirus (present in tumor cells). T cells harvested from patients treated with Ad5-D24-GMCSF were analyzed by IFN-gamma ELISPOT in the context of stimulation with peptide blend from Adenovirus 5 and 30 survivor antigen survivors.

Figure 11 shows the induction of adenovirus hexon-specific T cells. Leukocytes collected from patients treated with Ad5-D24-GMCSF were stained with CD3, CD8 and hexone-specific tetramer antibodies and analyzed by flow cytometry before and after treatment. Floito increased hexon-specific cytotoxic T cells from 0.21% to 2.72%.

8

Figure 12 shows the reduction of circulating regulatory T cells in R73. CD4 positive, CD127 negative but Foxp3 rich PBCMs are considered to be effective regulatory T cells.

Detailed Description of the Invention

The adenoviral vector

In Ad5, as in other adenoviruses, the icosahedral capsid is composed of three major proteins: hexone (II), penton base protein (III), and protruding strand (IV), in addition to small proteins 10 and; VI, Vili, IX, lila and IVa2 (Russel W. C. 2000, J General Virol 81,2573-2604). Proteins VII, small peptide mu and terminal protein (TP) are linked to DNA. Protein V provides a structural link to the capsid via protein VI. A virus-encoded protease is required for processing some structural proteins.

The oncolytic adenoviral vector of the present invention is based on an adenovirus serotype 5 (Ad5) nucleic acid backbone, a 24 base pair deletion (D24) in the E1 Rb-binding constant region 2 (CR2), and a nucleic acid sequence coding for granulosa. factor (CM-CSF) in place of the deleted gp19k / 6.7K in the E3-20 region (Figure 1).

The Ad5 genome contains early (E1-4), intermediate (IX and IVa2) and late (G1-5) genes with inverted terminal repeats (LITR and RITR, respectively) containing DNA: n sequences required for replication. The genome also contains 25 packaging signals (Ψ) and MLP (major late promoter).

Transcription of the early stage gene E1A initiates a replication cycle, followed by expression of the E1B, E2A, E2B, E3 and E4 proteins. E1 proteins modulate cellular metabolism in a way that renders the cell more susceptible to viral replication. For example, they interfere with NF-κΒ, p53 and pRb proteins. E1A and E1B together inhibit apoptosis. The E2 (E2A and E2B) and E4 gene products mediate DNA replication, and the E4 products also affect viral RNA metabolism and inhibit host protein synthesis. The E3 gene products are responsible for defense against the host immune system, promote cell lysis and release of virus progeny (Russel W. C. 2000, J General 35 Virol 81,2573-2604).

9

The intermediate genes IX and IVa2 encode the small proteins of the viral capsid. MLP affects the expression of late-stage genes L1-5, which results in the production of viral structural components, encapsulation and maturation of viral particles (Russel W. C. 2000, J General Virol 81, 5, 2573-2604).

Compared to the wild-type adenovirus genome, the adenoviral vector of the invention lacks 24 base pairs of CR2 in the E1 region, more specifically in the E1A region, and gp19k and 6.7K in the E3 region. In a preferred embodiment of the invention, in addition to the partial E1 and E3 regions, the on-10 colic adenoviral vector of the invention further comprises one or more regions selected from the group consisting of E2, E4, and late stage regions. In a preferred embodiment of the invention, the oncolytic adenoviral vector comprises the following regions: left ITR, partial E1, pIX, p1Va2, E2, VA1, VA2, L1, L2, L3, L4, partial E3, L5, E4 and right ITR. The regions may be in the vector in any order, but in a preferred embodiment of the invention the regions are in a sequential order in the 5'-3'-direction. Open reading frames (ORFs) may be on the same DNA strand or on different DNA strands. In a preferred embodiment of the invention, the E1 region comprises a virus compression signal.

As used herein, the term "'adenovirus serotype 5 (Ad5) nucleic acid backbone'" means an Ad5 genome or osagenome comprising one or more regions selected from the group consisting of partial E1, pIX, plVa2, E2 derived from Ad5. , VA1, VA2, L1, L2, L3, L4, partial E3, L5 and E4.

25 In this context, the term '' partial 'region' means an area that is missing a portion relative to the corresponding wild-type region. Partial E1 "refers to the E1 region containing D24, and" "partial E3" refers to the E3 region lacking gp19k / 6.7K.

In this context, the terms "VA1" and "VA2" refer to virus-associated RNAs 1 and 2, which are transcribed by the adenovirus but not translated. VA1 and VA2 have their share in the fight against cellular defense mechanisms.

As used herein, the term "viral packaging signal" refers to a portion of a viral DNA that is composed of a series of AT-rich sequences and controls the 35 capsidization process.

The 24 base pair deletion (D24) of E1 affects the CR2 region responsible for binding the Rb tumor suppressor / cell cycle regulatory protein and thus allows the initiation of the synthesis step (S), i.e. the DNA synthesis or replication step. The interaction of pRb and E1A requires eight amino acids 121-127 of the conserved region of E1A protein (Heise C. et al. 2000, Nature Med 6, 1134-1139), which are deleted in the present invention. The vector of the present invention comprises a deletion of nucleotides corresponding to amino acids 122-129 of the vector of Heise C. et al. (2000, Nature Med 6, 1134-1139). Viruses containing D24 are known to have a reduced ability to bypass the G1-S checkpoint and replicate efficiently only in cells where this interaction is not required, for example in tumor cells with a defective Rb-p16 pathway (Heise C. et al. 2000, Nature Med 6, 1134-1139; Fueyo J. et al. 2000, Oncogene 19, 2-12).

The E3 region is not essential for viral replication in vitro, but the E3-15 proteins play an important role in the regulation of the host immune response, that is, in the suppression of both native and specific immune responses. The gp19k / 6.7K deletion in E3 represents a deletion of 965 bp in the E3A region of the adenovirus. In the resulting adenovirus construct, both gp19k and 6.7K genes have been deleted (Kanerva A. et al. 2005, Gene Therapy 12, 87-2094). The gp19k gene product is known to bind and attract major histocompatibility complex I (MHC1) molecules in the mucosa and prevent cytotoxic T lymphocytes from recognizing infected cells. Because many tumors have an MHC1 deficiency, deletion of gp19k enhances viral tumor selectivity (virus is cleared more rapidly than wild-type virus from nor-25 normal cells, but there is no difference in tumor cells). 6.7K proteins are expressed on cell surfaces and participate in the down-regulation of TNF-like apoptosis-inducing ligand (TRAIL) receptor 2.

In the present invention, the GM-CSF transgene is inserted into the gp19k / 6.7k deleted E3 region under the E3 promoter. This restricts the expression of the transgene on tumor cells that allow viral replication and subsequent activation of the E3 promoter. The E3 promoter may be any exogenous or endogenous promoter known in the art, preferably an endogenous promoter. In a preferred embodiment of the invention, the nucleic acid sequence encoding GM-CSF is under the control of the viral E3 promoter 35.

GM-CSF participates in the immune response through a variety of mechanisms, including recruitment of natural killer cells (NKs) and stimulation of APCs (antigen presenting cells). The APC can then recruit, activate and target T cells to the tumor. The 5 nucleotide sequence encoding GM-CSF can be derived from any animal, e.g., human, monkey, rat, mouse, hamster, dog or cat, but preferably the GM-CSF is encoded by the human sequence. The nucleotide sequence encoding GM-CSF may be modified to enhance the effects of GM-CSF or unmodified, i.e. wild-type. In a preferred embodiment of the invention, the nucleic acid sequence encoding GM-CSF is wild-type.

The vector of the invention may also comprise modifications other than partial deletions of CR2 and E3 and insertion of the GM-CSF sequence as mentioned above. In a preferred embodiment of the invention, all other regions of the Ad5 vector are of the wild type. In another preferred embodiment of the invention, the E4 region is of the wild type. In a preferred embodiment of the invention, there is a wild type region upstream of the E1 region. '' Upstream 'means immediately before the E1 region in the direction of expression. The E1B region may also be modified in a vector of the invention.

Insertion of exogenous elements can amplify the effects of vectors on target cells. The use of exogenous tissue or tumor-specific promoters is common in recombinant adenoviral vectors and can also be utilized in the present invention. For example, viral replication may be restricted to target cells by, for example, promoters including but not limited to CEA, SLP, Cox-2, Midkine, hTERT, hTERT variants, E2F, E2F variants, CXCR4, SCCA2 and TTS. They are usually added to regulate the E1A region, but in addition or alternatively, other genes such as E1B or E4 can also be regulated. Exogenous insulators, i.e., inhibitors of non-specific enhancers (Enhancer), left ITR, native E1A-30 promoter, or chromatin proteins may also be incorporated into recombinant thiadenovirus vectors. Optionally, any additional components or modifications may be used in the vectors of the present invention, but are not required.

Most adults are exposed to the most widely used Adenovirus-35 serotype Ad5 and therefore the immune system is able to rapidly produce neutralizing antibodies (NAb) against it. In fact, the prevalence of anti-Ad5 NAb 12 can be up to 50%. It has been shown that NAb can be initiated against most immunogenic proteins of the adenovirus capsid and, on the other hand, it has been shown that even small changes in the Ad5 fiber protrusion can allow escape from the capsid-specific NAb. Thus, modification of the protrusion is important to maintain or increase gene transfer when using adenovirus contact in humans.

In addition, Ad5 is known to bind to a receptor called a CAR through the stroke portion of the fiber, and modifications of this portion or strand may improve access to the target cell and cause enhanced oncolysis in some cancers (Ranki T. et al. 2007, Int J Cancer 121, 165-174 ). Indeed, capsid-modified adenoviruses are preferred tools for improved gene transfer to cancer cells.

In one embodiment of the invention, the oncolytic adenovirus vector comprises a capsid modification. As used herein, "capsid" refers to a protein coat of viruk containing hexon, strand and penton base proteins. Any capsid modification, i.e., modification of the hexone, strand and / or pentone base proteins known in the art that enhances virus delivery to a tumor cell, can be utilized in the present invention. Modifications may be genetic and / or physical and include, but are not limited to, modifications that add ligands that recognize specific cellular receptors and / or inhibit binding of native receptors and that replace the adenovirus vector strand or caudal region with another adenovirus protuberance. specific molecules (e.g., FGF2) are added to adenoviruses. Thus, Kap linkage modifications include, but are not limited to, adding small peptide motif (s), pep-25 thid (s), chimerism (s) or mutation (s) to a strand (e.g., the protrusion, tail or stem portion). ), hexon and / or penton base. In a preferred embodiment of the invention, the capsid modification is Ad5 / 3 chimerism, insertion of an integrin binding region (RGD) and / or modification of the heparin sulfate binding polylysine into the strand.

30 In this context, "Ad5 / 3 chimerism" of the capsid refers to chimerism in which the protrusion of the strand is Ad serotype 3 and the end strand is Ad serotype 5.

In this context, the "RGD region" refers to the arginine-glycine-aspartic acid (RGD) motif, which is exposed on the surface of penton base unit 35 and interacts with cellular αν integrins that support adenovirus internalization. In a preferred embodiment of the invention, the capsid modification is an RGD-4C modification. "RGD-4C modification" refers to the insertion of the integrin-binding RGD-4C motif in the H1 loop of the filament region. 4C refers to the four cysteines that form sulfur bridges in RGD-4C. The construction of a recombinant 5 Ad5 filament gene encoding a strand containing a RGD-4C peptide is described in detail in, for example, Dmitriev I. et al. (1998, Journal of Virology, 72, 9706-9713).

In this context, '' heparan sulfate-binding polylysine modification '' means the addition of seven lysine sequences to the c-terminus of the fiber extension.

Expression cassettes are used to express transgenes at a target, for example a cell, using vectors. As used herein, the term "expression cassette" refers to a DNA vector or portion thereof comprising nucleotide sequences encoding cDNAs or genes or nucleotide sequences that direct and / or regulate expression of said cDNAs or genes. The same or different expression cassettes may be inserted into one or more vectors. The Ad5 vectors of the present invention may comprise either multiple expression cassettes or a single cassette. However, only one expression cassette is sufficient. In a preferred embodiment of the invention, the oncolytic adenoviral vector comprises at least one Express Express cassette. In one embodiment of the invention, the oncolytic adenovirus-20 vector comprises only one expression cassette.

The cell comprising the adenoviral vector of the invention may be any cell, for example a eukaryotic cell, a bacterial cell, an animal cell, a human cell, a mouse cell and so on. For example, the cell may be used to produce an adenoviral vector in vitro.

In a method of producing GM-CSF in a cell, a vehicle comprising the vector of the invention is transported to a cell, and in addition, the GM-CSF gene is expressed and the protein is paracrine translated and secreted. The '' transporter '' can be any viral vector, plasmid or other medium, such as a particle, capable of delivering the vector of the invention to a target cell. Any conventional method known in the art can be used to deliver a vector to a cell.

A tumor-specific immune response may be increased in a subject using the present invention. Cytotoxic T cells and / or natural killer cells are stimulated, produced and targeted as a consequence of GM-CSF expression. In a preferred embodiment of the invention, the amount of natural killer cells and / or cytotoxic T cells is increased in the target cell or tissue. Various immune response markers (e.g., inflammatory markers) may be defined to monitor or investigate the effects of the invention. The most common markers include, but are not limited to, proliferation of pro-inflammatory cytokines and tumor or adenovirus-specific cytotoxic T cells, recruitment and activation of antigen presenting cells, or enlargement of local lymph nodes. The levels of these markers can be assayed according to any conventional method known in the art, including, but not limited to, methods utilizing antibodies, probes, primers, and the like, for example, ELISPOT assay, tetramer assay, pentamer assay, and blood or tumor cell types.

Cancer

The recombinant Ad5 vectors of the invention are constructed to provide replication competence in cells defective in the Rb pathway, in particular the Rb-p16 pathway. These defective cells include all tumor cells in animals and humans (Sherr C.J. 1996, Science 274, 1672-1677). In a preferred embodiment of the invention, the vector is capable of selectively replicating in cells defective in the Rb pathway. In this context, '' errors in the Rb pathway '' refer to mutations and / or epigenetic changes in any of the genes or proteins of the pathway. Due to these errors, tumor cells overexpress E2F and thus binding of Rb to E1A CR2, which is normally required for efficient replication, is unnecessary.

Any cancer or tumor, including malignant and benign tumors, as well as primary tumors and metastases, may be the subject of gene therapy. In a particular embodiment of the invention, the cancer is any solid tumor. In one advantageous embodiment, the cancer is selected from the group consisting of nasopharyngeal cancer, synovial carcinoma, hepatocellular carcinoma, kidney cancer, connective-30 tissue cancer, melanoma, lung cancer, bowel cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, throat cancer, oral cancer, liver cancer, bone cancer , pancreatic cancer, placental cancer, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, von Hippel-Lindau's disease, zollinger-ellison syndrome, adrenal cancer, cancer of the gallbladder, cancer of the urinary tract, urinary tract cancer, , oligo-dendroglioma, neuroblastoma, meningioma, spinal tumor, bone cancer, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary location, carcinoid, gastrointestinal carcinoid, fibrosarcoma, breast cancer, Paget's taurus, cancer, esophageal cancer, gallbladder cancer, head cancer, eye cancer 5-day, neck cancer, kidney cancer, wilms tumor, liver cancer, Kaposi's sarcoma, prostate cancer, lung cancer, testicular cancer, Hodgkin's disease, non-Hodgkin's lymphoma, suicide, myeloma Mon, ovarian cancer, endocrine pancreas, glucagonoma, pancreatic cancer, paratyroidinen cancer, penile cancer, aivolisäkesyöpä, soft tissue sarcoma, 10 retinoblastoma, small intestine cancer, stomach cancer, thymic, thyroid, cancer, trofoblastisyöpä, hydatidiform mole, uterine cancer, endometrial cancer, vulval, ulkosynnytinsyöpä, akustikus neurinooma , mucosis fun-goides, insulinoma, carcinoid syndrome, somatostatinoma, gingival cancer, heart cancer, lip cancer, membrane cancer, oral cancer, nerve cancer, cortical cancer, corpus luteum cancer, peritoneal cancer, pharyngeal cancer, pharyngeal cancer, s.

Pharmaceutical composition

The pharmaceutical composition of the invention comprises at least one type of vector of the invention. In addition, the composition may comprise at least 20 two, three or four different vectors of the invention. In addition to the vector of the invention, the pharmaceutical composition may also comprise other vectors such as other adenoviral vectors, other therapeutically effective agents, other agents such as pharmaceutically acceptable carriers, buffers, excipients, adjuvants, antiseptics, fillers, stabilizers or thickeners, and / or that are normally found in similar products.

The pharmaceutical composition may be in any form suitable for administration, for example solid, semi-solid, or liquid. The formulation may be selected from the group consisting of, but not limited to solutions, emulsions, suspensions, tablets, pellets, and Kap-30.

In a preferred embodiment of the invention, the oncolytic adenoviral vector or pharmaceutical composition acts as an in situ cancer vaccine. In this context, "in situ cancer vaccine" means a cancer vaccine that both kills tumor cells and enhances the immune response against tumor cells. Virus-35 replication is a potent signal of danger to the immune system (necessary for a TH1-like response) and thus acts as a potent costimulatory phenomenon for GM-CSF-mediated maturation and activation of APCs and NK cell recruitment. Lysis of tumor cells also helps to present tumor fragments and epitones to APCs in addition to inflammation producing costimulation. Thus, an epitope-independent (i.e., non-HLA-restricted) response is produced in each tumor and therefore occurs in situ. The tumor-specific immune response is activated in the target cell and surrounding cells, for example in the target tissue.

The effective dose of the vectors depends at least on the subject to be treated, the type of tumor, the location of the tumor, and the stage of the tumor. For example, the dose may range from about 10e8 viral particles (VP) to about 10e14 VPs, preferably from about 5x10e9 VPs to about 10e13 VPs, and more preferably from about 8x10e9 VPs to about 10e12 VPs. In one particular embodiment of the invention, the dosage is about 5x10e10-5x10e11 VP.

The pharmaceutical compositions can be produced by any of the conventional processes known in the art, for example, utilizing one of the following: batch, batch feed and perfusion culture modes, column chromatography purification, CsCl gradient purification and low shear perfusion modes.

Allocation

The vector or pharmaceutical composition of the invention may be administered to any eukaryotic target selected from the group consisting of plants, living and humans. In a preferred embodiment of the invention, the subject is a human or animal. The animal may be selected from the group consisting of pet animals, domestic animals, and farm animals.

Any conventional method of administering a vector or composition to a subject can be used. The route of administration will depend on the formulation or form of the composition, the disease, the location of the tumors, the patient, the comorbidities, and other factors. In a preferred embodiment of the invention, administration is by intravenous, intramuscular, intravenous, intravenous, pleural, intracellular, intraperitoneal or peritoneal injection or oral administration.

Even one administration of the oncolytic adenoviral vector of the invention can provide therapeutic effects. However, oncolytic adenoviral vectors or pharmaceutical compositions may also be administered several times during a course of treatment. For example, oncolytic adenoviral vectors or pharmaceutical compositions may be administered 1-10 times during the first two weeks, during the first four weeks, monthly or during the treatment period.

17

For example, they are given 3 to 7 times in the first two weeks, then in the fourth week and then monthly. They can also be given four times in the first two weeks, then in the fourth week and then monthly. The length of the treatment period can vary, for example from 2 to 12 months through 5 months or more.

To avoid neutralizing antibodies in a subject, the vectors of the invention may vary between treatments. For example, the subject is administered an oncolytic adenovirus vector having a different capsid filament protector compared to the prior treatment vector. In this context, the "" capsule filament protrusion "refers to the protrusion portion of the filament protein (Figure 1).

Gene therapy is effective on its own, but combining adenoviral gene therapy with another therapy, such as conventional therapy, may be more effective than either alone. For example, each agent of combination therapy may function independently in tumor tissue, adenoviral vectors may sensitize cells to chemotherapy or radiation therapy, and / or chemotherapeutic agents may enhance viral replication or affect target cell receptor status. The combination therapy agents may be administered simultaneously or sequentially.

In a preferred embodiment of the invention, the use further comprises administering concurrent radiation therapy to the subject. In another preferred embodiment of the invention, the use further comprises the administration of concurrent chemotherapy to the subject. As used herein, "" parallel "" refers to treatment given before, after or concurrently with the gene therapy of the invention. The period of co-treatment may vary from minutes to several weeks. Preferably, the concurrent treatment lasts for several hours.

Suitable agents for combination therapy include, but are not limited to, matrix All-trans retinoic acid, Azacitidine, Azathioprine, Bleomycin, Carboplatin, Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide, Cytarabine, Downo-rubicin, Docetaxel, Doxifluridine, Doxifluridine, Doxifluridine, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Imatinib, Mechlo-30 rethamine, Mercaptopurine, Methotrexate, Mitoxantrone, Oxaliplatin, Paclitaxel, Pemetrexed, Temozolomide, Teniposide, Tioguanine, Valrubicin, Vin.

18

In a preferred embodiment of the invention, the use further comprises administering to the subject verapamil or another calcium channel blocker. '' Calcium channel blocker '' refers to a class of drugs and natural substances that interfere with calcium channel conduction and can be selected from the group consisting of verapamil, dihydropyridines, gallopamil, diltiazem, mibefradiil, bepridil, flendirilene.

In a preferred embodiment of the invention, the use further comprises administering autophagy-inducing agents to the subject. Autophagy refers to a catabolic process in which the cell's own components are degraded through its lysosomal 10 machinery. "Autophagy-inducing agents" means substances capable of inducing autophagy, and may be selected from the group consisting of, but not limited to, mTOR inhibitors, PI3K inhibitors, lithium, tamoxifen, chloroquine, bafilomycin, temsirolimimus, temsirolimimus. In a particular embodiment of the invention, the method further comprises administering temozolomide to the subject. Temozolomide may be either oral or intravenous.

In one embodiment of the invention, the use further comprises administering chemotherapy or anti-CD20 treatment or other treatment that inhibits neutralizing antibodies. "Anti-CD20 treatment" refers to agents capable of killing 20 CD20 positive cells and which may be selected from the group consisting of rituximab and other anti-CD20 monoclonal antibodies. '' Neutralizing Antibody Treatment '' refers to agents that are capable of inhibiting the development of antiviral antibodies that are normally produced as a result of infection and which may be selected from the group consisting of various chemotherapeutic agents, immunomodulatory agents, corticoids, and other drugs. These agents may be selected from, but are not limited to, cyclophosphamide, cyclosporine, azathioprine, methylprenizolone, etoposide, CD40L, CTLA4Ig4, FK506 (tacrolimus), IL-12, IFN-gamma, interle , anti-CD8, anti-CD4 antibodies, myeloablation, and oral adenoviral proteins.

The oncolytic adenoviral vector of the invention initiates virion-mediated oncolysis of tumor cells and activates the human immune response against tumor cells. In a preferred embodiment of the invention, the use further comprises administering agents that are capable of downregulating T-regulatory cells in a subject. 'Substances capable of downregulation of regulatory T cells' refers to substances which reduce the number of cells identified as inhibitor T cells or regulatory T cells. These cells have been identified to contain one or more of the following immunophenotype markers: CD4 +, CD25 +, FoxP3 +, CD127- and GITR +. Such inhibitory T-5 cells or regulatory T-cell depressants may be selected from the group consisting of anti-CD25 antibodies or chemotherapeutic agents.

In a preferred embodiment of the invention, the use further comprises administering cyclophosphamide to the subject. Cyclophosphamide is a common chemotherapeutic agent also used in some autoimmune diseases. In the present invention, cyclophosphamide can be used to enhance viral replication and the effects of GM-CSF-initiated stimulation of NK cells and cytotoxic T cells to elicit an improved immune response against the tumor. It can be used as intravenous bolus doses or orally in small doses metronomically.

The following, for example, illustrate the present invention, but are not intended to be limiting in any way.

eXAMPLES

Example 1. Cloning of three D24-GM-CSF-type viruses - out of PCR lots of hGM-CSF 20 - Create Sunl / Munl sites = ^> 445 bp (as pORF-GM-CSF as template) - PCR product and pTHSN Sunl / Munl Digestion - Ligation of Adhesive Ends => - Pmel Linearized pShuttle-D24 + pTG3602 = ^> pAd5-D24

- Ad5-D24-GM-CSF

25 Homol. recomb: S / fl-linearized pAd5-D24 + Fspl-linearized pTHSN-GM-CSF => pAd5-D24-GM-CSF Pacl linearization and transfection => Ad5-D24-GM-CSF.

All cloning steps were confirmed by PCR and several restriction di gestions. The pTHSN-GMCSF shuttle plasmid was sequenced. Isolation of wild-type E1-30 was confirmed by PCR. The E1 region, transgene, and strand were screened for final virus by sequencing and PCR, and then introduced into a pure laboratory for production. To this end, viral DNA was extracted by overnight incubation with appropriate buffer solution and after PCR and sequencing was performed to analyze the integrity of the strand and GMCSF cDNA. All steps of viral production, including 35 transfections, were performed on A549 cells to avoid the risk of wild-type recombination as described previously (Kanerva A. et al. 2003, Mol Ther 8, 449-58; Baeurschmitz GJ et al. 2006, Mol Ther 14, 164 -74). GM-CSF is under the control of the E3 promoter, leading to the expression of replication-associated transgenes that begin approximately 8 h after infection. E3 is intact except for 5 except for the 6.7K / gp19K \ n deletion. Ad5 / 3-D24-GM-CSF and Ad5-RGDD24-GM-CSF were constructed identical except using a salvage plasmid with either the serotype 3 protrusion or the RGD-4C in the H5 loop of the Ad5 strand (Figures 2-4).

Example 2. In Vitro Analysis of D24-GM-CSF Type Viruses The in vitro potency of D24-GM-CSF type viruses was investigated in lung-10 cancer cells (A549), explant thymus cells derived from breast cancer stem cells (JIMT-1). and breast cancer cells (MDA-MB-436) using MTS cell death assays. The MTS assay is currently a standard method for studying cell viability in publications related to cancer gene therapy. Ad5Luc1 is a replication-defective virus and serves as a negative control. Ad5wt is a wild type Ad5 virus (Ad300wt strain) and was used as a positive control. Ad5-d24-E3 has an isogenic 24 base pair deletion in E1, but its E3 is intact. VP stands for virus particles.

Briefly, Ad5-D24-GMCSF had positive control-like oncolytic activity in vitro and therefore transgene production did not impair the oncolytic activity of the virus (Figures 5a-c). Similar data were obtained for Ad5 / 3-D24-GM-CSF and Ad5-RGD-D24-GM-CSF (Figure 5d).

To test whether Ad5D24-GMCSF is capable of expressing the transfer gene, the A549 cell line was infected at 1000 VP per cell and the culture medium was harvested over time. The concentration of GMCSF (Figure 6a) in the culture medium was measured at 25 FACSARRAY. In addition, we also analyzed whether the GMCSF expressed by the virus retained its biological function. To this end, the TF1 cell line, whose growth and viability are completely dependent on human GMCSF, was treated with growth medium previously collected from the A549 cell line infected with Ad5D24-GMCSF. The viability of TF1 was assessed over time by the MTS assay. The result of this experiment was that the GMCSF expressed by the virus was able to stimulate the growth of such a cell line, and no difference was found compared to the same cell line treated with recombinant human GMCSF (Sigma) (Figure 6b).

21

Example 3. Transduction Preprocessing Assay I. Infection of Tumor Cells with Ad5Luc1

To verify that the tumor could be infected with Ad5-based viruses, tissue biopsies were homogenized and infected with luciferase 5 encoding Ad5Luc1 according to the standard protocol for infection. The cell was briefly inoculated into a well washed twice with PBS, the virus was thawed and resuspended in a minimum of medium and slowly poured onto the cells. The infection continued for 30 minutes and then the cells were again washed with PBS and the appropriate amount of complete medium was added. Luciferase quantification was determined after 24 hours. Please note that only a very small amount of tissue was obtained and therefore the number of cells could not be counted nor the amount of virus normalized to the amount of tissue. For this reason, quantitative analyzes were not performed, but qualitative data showed successful gene transfer in patients 012 and C3 (Figures 7a-b). Background values of luciferase 15 (about 200 RLU) were subtracted.

II. Immunohistochemical staining of CAR

Available archived samples of patient tumors were collected and analyzed for CAR (adenovirus serotype 5 receptor) expression by immunohistochemistry. Cancer cells, cytoplasms (M3), jejunum, 20 adenocarcinoma (C-3), pancreatic carcinoma (H-7), carcinoma lobular infiltrates (R 8), ovarian cancer liver metastasis (012) and synovia-lisarkooman lung metastases (S23), the CAR adenovirus staining is shown in Figure 7c.

Example 4. In Vivo Assay of D24-GM-CSF Viruses in Animals The in vivo efficacy of Ad5-D24-GM-CSF was tested in immunocompetent Syrian hamsters that are semi-permissive to human adenovirus replication (mice are non-permissive). (Ying B. et al. 2009, Cancer Gene Ther doi: 10.1038 / cgt.2009.6.). 7 * 10 6 HapT1 pancreatic cancer cells were injected subcutaneously and 1 * 109 viral particles (VP) Ad5-D24-GM-CSF or 30 Ad5D24E3 (which does not express GM-CSF) were injected intradermally on days 0, 2 and 4. the same amount of growth medium was injected at the same times. Figure 8b shows that intravenous injections of Ad5-D24-GMCSF resulted in high levels of hGM-CSF in serum of Syrian hamsters. Human GM-CSF is known to be active in Syrian hamsters (Wow, Exp To-35 xicol Pathol 2006 vol. 57 (4) pp. 321-8). Interestingly, all animals were tumor-free by day 16 except the control group (Figure 8a). Tumor incidence was analyzed for a further two weeks to see if the tumor recurred. However, at day 32 these animals still showed no evidence of tumor, so the first part of the experiment was terminated and animals in the peer group were euthanized. The remaining treated animals were at this stage exposed to the same tumor by subcutaneous injection of 7 * 10 6 HapT1 cells to the right side of the torso, while to the left were various tumors (1 * 10 6 HaK tumors) to which the animals were naive. Tumor growth was measured over time and results are shown in Figures 8c-d. It was of interest that animals previously treated with Ad5D24GMCSF did not suffer at all from HapT1 tumors, whereas Hak tumors grew normally while animals treated with Ad5D24E3 both grew with HapT1 and HaK tumors (Figures 8c-d).

Briefly, the results show that Ad5-D24-GM-CSF has 15 antitumor activity in immunocompetent animals with tumors and is capable of producing tumor-specific immunity to the extent that it inhibits the growth of the same tumor later.

Example 5. In vivo Analysis of D24-GM-CSF Viruses in Human Patients 20 I. Patients

Patients with advanced and difficult to treat solid tumors were asked to participate in an experimental practice approved by the Government. The data of patients receiving Ad5-D24-GM-CSF are listed in Table 1.

25 II. Treatments with Adenovirus Vector Encoding GM-CSF a) Ad5-D24-GM-CSF, Ad5 / 3-D24-GM-CSF or Ad5-RGD-D24-GM-CSF Treatments

Ad5-D24-GM-CSF, Ad5 / 3-D24-GM-CSF, and Ad5-RGD-D24-GM-CSF were produced according to clinical grade and patient treatment was initiated. This' 'phase 0' experimental use program has been discussed by the Hyksos Board of Surgical Ethics. The program has also been discussed by FinOHTA (Health Methods Assessment Unit) and the Ethics Committee of the Finnish Medical Association. Legal issues have been reviewed by the Ministry of Social Affairs and Health, the National Board of Medicines, the Legal Committee of the Finnish Medical Association, 35 National Center for Legal Protection of Health and the Social Welfare Board of the Parliament. The treatments have been approved by the Gene Technology Board.

23

The virus was diluted with sterile saline at the time of administration under appropriate conditions. After the virus was administered, all patients were observed overnight in hospital and then as outpatient patients. Treatment side effects were recorded and scored according to CTCAE (Common Terminology Criteria for Ad-5 verse Events) version 3.0.

Blood samples were taken before treatment and then for analysis. Table 1 summarizes the serum viral load of patients treated with Ad5D24-GMCSF. Quantitative PCR (qPCR) was used in this assay (see Method descriptions in Part III).

Tables 2, 3 and 4 summarize all adverse reactions reported during and following treatment. All adverse reactions are scored according to CTCAE version 3.0 (Common Terminology Criteria for Adverse Events). It should be noted that all patients had Grade 1 and / or 2 flu-like symptoms, but only two Grade 3 symptoms were reported, one of 15 patients with ovarian and pre-existing cancer and one had Grade 3 hyponatraemia.

Table 5 shows the efficacy estimate according to the RECIST criteria (Theras-se P. et al. 2000, J Natl Cancer Inst 92, 205-16). Interestingly, two complete responses (CR) and five stable diseases (SD) were observed in the 14 patients analyzed, resulting in a 50% clinical benefit level.

III. Detection of virus in the blood

Serum samples were taken from patients treated with Ad5-D24-GM-CSF (see Example 5,) and conventional PCR was performed using primers and conditions as described in Takayama et al. 2007, J. Med. Virol. 79: 278-284. Briefly, total DNA was isolated by adding 3 μg of carrier DNA (polydeoxyadenylic acid; Roche, Mannheim, Germany) in 400 μΐιβη serum and using the QIAamp DNA mini kit. The isolated DNA was eluted in 60 of nuclease-free water and the DNA content was measured by spectrophotometry. PCR amplification was based on primers and probes targeted to the E1A region surrounding the 24 base pair deletion [forward primer 5'-TCCGGTTTCTATGCCAAACCT-3 '(SEQ ID NO: 1), reverse primer 5'-TCCTCCGGTGATAATGACA and probe whether 5'FaM-TGATCGATCCACCCAGTGA-3'Mgbnfq (SEQ ID NO: 3)]. In addition, a probe complementary to the sequence contained in the 24 base pair deletion region 35 was used to test samples for wild-type adenovirus infection [probe wt 5, vic-TACCTGCCACGAGGCT-3'mgbnfq (SEQ ID NO: 4).

Real-time PCR conditions for each 25 μΙ reaction were as follows: 2X LightCycler480 Probes Master Mix (Roche, Mannheim, Germany), 800 nM of both front and back primers, 200 nM of each probe and 250 ng of isolated DNA. PCR reactions were performed on LightCycler (Roche, Mannheim, Germany) under the following PCR conditions: 10 min at 95 ° C, 50 cycles: 10 sec at 95 ° C, 30 sec at 62 ° C and 20 sec at 72 ° C. And 10 min at 40 ° C. All samples were tested twice. Exogenous TaqMan internal positive control reagents (Ap-10 plied Biosystems) were used at the same PCR times to test the PCR inhibitors in each sample.

A standard regression curve was generated using DNA isolated from serial dilutions of Ad5 / 3-D24-Cox2L (1 x 10 8 to 10 vp / ml) in normal human serum. The detection and quantification limits for the assay were 500 vp / ml serum.

Positive samples were confirmed by real-time PCR using LightCycler480 SYBR Green I Master Mix (Roche, Mannheim, Germany) and adenovirus-specific and GM-CSF sequence-specific primers [forward primer 5'-AAACACCACCCTCCTTACCTG-3 '(reverse primer 5') 5'-TCATTCATCTCAGCAGCAGTG-3 '(SEQ ID NO: 6)].

IV. Killing of differentiated tumor cells

Computer tomographs of a patient with ovarian cancer and a patient with mesothelioma (see Table 1) before and after treatment are shown in Figures 9a-b.

25 V. Haematological effects of treatment

Leukocytes, erythrocytes, Hb, platelets, bilirubin, INR, ALT, AST, ALP, creatinine, K, Na CRP, CA19-9, GT, The levels of D-dimers of fibrin and CEA were examined (Tables 3-4).

VI. Immune response to virus 30 a) IL-6. IL-10. TNF-g and IL-8

One potential disadvantage of adenoviral gene therapy is its early toxicity due to its viral components, which may be immunogenic and lead to sepsis-like shock and even death (Brunetti-Pierri et al., Gene Ther 2004; Raper et al. 2002). For this reason, it is very important to monitor for signs of a possible cytokinesis storm that can subsequently lead to organ failure. To this end, blood samples were taken from patients shortly after treatment and at the indicated time point, and the proinflammatory cytokines were analyzed by FAC-SARRAY as described in Cerullo V. et al. (2007, Mol Ther 15, 378–85). No significant changes were observed, indicating no early congenital toxicity.

5 b) Initiation of svtotoxic T cells against tumors and specific immunity against tumor epitopes

Oncolytic cell death gives the immune system the ability to recognize and kill tumor cells. This is potentially useful for tumor destruction and can facilitate treatments. The adenovirus is eliminated from the body within a relatively short time after administration; for this reason, it becomes extremely important to stimulate the immune system to recognize a particular tumor antigen so that treatment can lead to a lasting beneficial effect on the patient. In addition, in the presence of antibodies, the virus is neutralized so that it may lose its ability to infect metastases. Tumor-initiated effector T or NK cells can circulate freely and eventually kill metastasis far from the injected tumor. To demonstrate that adenovirus expressing GMCSF is capable of conferring adenovirus or tumor specific immunity, PBMCs collected from treated patients were analyzed by INF-gamma ELISPOT. ELISPOT was made by a blind company as an external blind and was not provided with information on treatments received by patients (Proimmune). Figures 10a-d show the results of this analysis. It is clear that when T cells were stimulated with a peptide pool derived from either a tumor antigen (survivin) or an adenovirus (penton), these cells were activated to produce INF gamma (INF gamma is a specific activating marker for T cell stimulation) in the same patients. Figure 11 shows the induction of Adenovirus Hexon specific T cells.

Leukocytes collected from patients treated with Ad5-D24-GMCSF were stained with CD3, CD8, and hexone-specific tetramer antibodies and analyzed by flow cytometry before and after treatment. The treatment increased the amount of hexone-specific cytotoxic T cells from 0.21% to 2.72%.

30 c) Decrease in Requlatory T cells

Previous data have shown that metronomic administration of cyslophosphamide reduces regulatory T cells (T-Regs) in experimental animals.

This information was utilized in patients receiving cyclophosphamide metronomically before and after treatment, and T-reg analysis was performed on PBMCs collected from these patients. The example shown in Figure 12 is one example from R73 patient and shows a reduction in circulating T-regs. Total PBMCs were collected from patients and frozen in appropriate media. During analysis, all samples were thawed and stained with CD and CD127 antibodies, followed by permeabilization and staining for Foxp3 transcription factor. Cells positive for CD4 and negative for CD127 but rich in Foxp3 were considered to be effective regulatory T cells (Figure 12).

Table 1. The table shows the presence of Ad5-D24-GMCSF in the serum of treated patients

Before Days after treatment ___ treatment_______ I I 1 I 2 | 3-7 | 8-12 | 13-20 | 21 ^ 0

Patient primary tumor virus Dose Viral load in the serum (VP / ml) (code) ___ (kok.VP) ________ C3__Tyhjäsuolisyöpä 8x10 * 0__0__500__500 __-__-__ 0 for M3 Hepatocellular __syöpä__1x101 ° __0__0__4896__0__0__0__0 012__Munasarjasyöpä 3,6x101u 0__0__0__0__0__0__0 014__Munasarjasyöpä 1x10 "0__0__0__500__0__0__0 G15__Mahalaukkusyöpä 1x10" 0__0__565__500__0__0__0 K18 Non-small cell 0 __keuhkosyöpä__2x1011 ___ 500 __, __ 0__0__0__856 T19 medullary thyroid __rauhassyöpä__2χ1θ "__ 0__765__500__500__0__0__0 U89__Munuaissyöpä_ 2x10" 0__0 __-__-__-__-__ 0 S100__Leiomyosarkooma 2x10 "0__500 __-__ 500 __-__-__- S108, synovial sarcoma __kooma__2x10" __ 0_______ M 50__Mesoteliooma_ 2,5x10 "0__0 __-__ 500__ -__ 0__0 R8__Breast Cancer_ 3x10 '' 0__500 __-__ 500 __-__ 0__0 M 32__Mesothelioma_ 3x10 "0__0__0 __-__-__ 0__0 X49 Cervical Eating-3x10" 0 4290 - - 37975 6706 1211 ____ "________ I52__M -__-__ 500 C58__Paksusuolen cancer 4x10 "0__1978 __-__ 4236 878 __-__- R73__Breast Cancer 4x10" 0_______ 088__Ovarian Cancer 4x10 "0_______ 1 27

Table 2. The table summarizes the adverse reactions reported by patients treated with Ad5-D24-GMCSF __Patients ___

Reported symptoms__Stage 1__Stage 2__Stage 3__Stage 4__Dose range

Fever 3_____P_ __1____K_ __6__4____S_

Pain at the injection site_____P_ __1____K_ ______S_

Muscle Pain 1_____P_ _____K_ __1__1____S_ Headache__1 ____ P_ __1____K_ __1_____S_

Väsymys__1 ____ P

1_____K_ __2__3____S_

Dyspnea_____P_ __1____K_ __2_____S_

Diarrhea_____P_ _____K_ __1_____S_

Hypotensio_____P

1 _____K_ __1_____S_

Kuvotus_____P

2 _____K_ __3_____S_

Vomiting_____P_ _____K_ __3_____S_

Dizziness_____P_ _____K_ __2_____S_

Cough 1_____P_ 1__1____K_ __3__3____S_

Vilu P_ 4_____K_ _ | 1 | | S ~ 5 P (low dose) = Cohort 1 dose interval 8x109 <D <3.6x101 ° K (mean dose) = Cohort 2 dose interval 1x1011 <D <2.5x1011 S (high dose = Cohort 3 dose interval 3x1011 <D <4x1011 28

Table 3. Haematological side effects after administration of Ad5-D24-GMCSF

Effect Early Toxicity (1-7 days) __ Late Toxicity (> 7 days) Dosage Grade 1 Grade 2 Grade 3 Grade 4 Grade 1 Grade 2 Grade 3 Grade 4__ Range

Hypo- 1_________P_ nat- 1_____1__1____K_

remia__6___1______S

Hypoka- 1_____2_____P_

leads to 2 1 K

__1_________S_

Anemia 1_________P_ 1_________K_ __2__4_____3____S_

Trom -______ 1____P_ bosyto -___ _ _K_

Penny 1__ _S

Leuco -_________ P_

Pen 1K

_ | | I | | | | | | S

5 P (low dose) = Cohort 1 dose interval 8x109 <D <3.6x101 ° K (mean dose) = Cohort 2 dose interval 1x1011 <D <2.5x1011 S (high dose = Cohort 3 dose interval 3x1011 <D <4x1011

Table 4. Liver Enzymes after Ad5-D24-GMCSF Administration 10___

Early Toxicity (1-7 days) __ Late Toxicity (> 7 days) _ An-

Stage I Stage II Stage III Stage IV Stage I Stage II Stage III Stage IV nos- __________ area ALT_________P_

Patients * \ 1K

_ 3 1 ~ --S ~ AST_________P_

Patients 1K

__1__1____1__1____s

Hyperbil -_________ P_ rubinemia_________K_

Patients 2 1 S

number ____ | _ | _ | ____ P (low dose) = Cohort 1 dose interval 8x109 <D <3.6x101 ° K (mean dose) = Cohort 2 dose interval 1x1011 <D <2.5x1011 S (high dose = Cohort 3 dose interval 3x1011 <D <4x1011 29

Table 5. Efficacy of Ad5-D24-GMCSF Treatment in Patients Treated with Different Ad5-D24-GMCSF Doses. The analysis was performed according to RECIST criteria. CR = full response, PR = partial response, SD = stable disease, PD = progressive disease, - = not available.

5 ______RECIST____Longness (days)

Virus dose Number of patients (VP total) CR PR SD PD - S> 300 300 <S> 200 200 <S> 100 S <100

8x109 VP__1__ 1 -I

1x101 ° VP__1 1!

3.6x101 ° VP 1__1 -I

1x1011 VP__2__1__ 1 1 1 2x1011 VP 4 14 1 2 1 2.5x1011 yp 1 1 1 3x1011 VP 5 1 3 11 2 3.6x1011 yp -i 4x1011 VP | 3 I I 1 I 1 | l 1 | _ 3

Claims (37)

1. Oncolytic adenovirus vector, characterized in that it comprises a nucleic acid main chain of adenovirus serotype 5 (Ad5), a deletion 5 of 24 base pairs (D24) E1 within the stable region 2 that binds Rb and a nucleic acid sequence encoding granulocyte macrophage macrophages. (GM-CSF) instead of gp19k / 6.7K within the E3 range. An oncolytic adenovirus vector according to claim 1, characterized in that it further comprises one or more regions selected from a group consisting of E2 and E4 and late phase regions. An oncolytic adenovirus vector according to claim 1 or 2, characterized in that it comprises the following regions: left ITR, partial E1, pIX, plVa2, E2, VA1, VA2, L1, L2, L3, L4, partial E3, L5, E4 and right IOA. An oncolytic adenovirus vector according to claim 3, characterized in that the regions are in a consecutive order in the 5'-3 'direction. An oncolytic adenovirus vector according to any of the preceding patent claims, characterized by the wild-type region located upstream of the E1 region. An oncolytic adenovirus vector according to any of the preceding patent claims, characterized by the E1 region comprising the packing signal of the virus. An oncolytic adenovirus vector according to any of the preceding patent claims, characterized by the nucleic acid sequence encoding An oncolytic adenovirus vector according to any of the foregoing patent claims, characterized by the nucleic acid sequence encoding GM-CSF being wild-type. An oncolytic adenovirus vector according to any of the foregoing patent claims, characterized in that the E4 region is of a wild type. An oncolytic adenovirus vector according to any of the preceding patent claims, characterized in that it comprises a capsid modification. 11. Oncolytic adenovirus vector according to claim 10, characterized in that the capsid modification is Ad5 / 3 chimerism, insertion of the integrin binding region (RGD) and / or modification of heparin sulfate binding polylysine in the strand. An oncolytic adenovirus vector according to claim 10 or 11, characterized by the capsid modification being RGD-4C modification. An oncolytic adenovirus vector according to any of the preceding patent claims, characterized in that it comprises at least one expression cassette. An oncolytic adenovirus vector according to any of the preceding patent claims, characterized in that it comprises only one expression cassette. 15. Oncolytic adenovirus vector according to any of the preceding palo tent claims, characterized by the vector being capable of selectively replicating in cells with defects on the Rb pathway. 16. In vitro cell, characterized in that it comprises an adenovirus vector according to any of claims 1-15. Pharmaceutical composition, characterized in that it comprises an adenovirus vector according to any of claims 1-15. An oncolytic adenovirus vector or pharmaceutical composition according to any of the preceding claims, characterized in that it functions as an in situ cancer vaccine. Use of an oncolytic adenovirus vector according to any of the preceding claims for the manufacture of a medicament for treating cancer of an object. Use according to claim 19, characterized in that the cancer is selected from a group comprising nasopharyngeal cancer, synovial cancer, hepatocellular cancer, kidney cancer, connective tissue cancer, melanoma, lung cancer, colorectal cancer, colon cancer, colorectal cancer, brain cancer throat cancer, oral cancer, liver cancer, pancreatic cancer, chorion carcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, von Hippel-Lindau disease, Zollinger-Ellison's syndrome, adrenal cancer, anal cancer, gallbladder cancer, urinary bladder cancer, oligo-dendroglioma, neuroblastoma, meningioma, spinal cord tumor, bone cancer, osteo-condom, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoma, gastrointestinal carcinoma, fibrosarcoma, breast cancer, pancreatic cancer, uterine cancer , head cancer, eye cancer, neck cancer, kidney cancer, Wilms tumor, Kaposi's sarcoma, prostate cancer, testicular cancer is, Hodgkin's disease, non-Hodgkin's lymphoma, skin cancer, meiotelioma, multiple myeloma, ovarian cancer, endocrine pancreatic cancer, glucagonomic, pancreatic cancer, parathyroid cancer, penile cancer, brain cancer, fibromyalgic sarcoma, retinoblastoma thyroid cancer, trophoblast cancer, mola hydatidosa, life-maternal cancer, endometrial cancer, osteoarthritis, vulvar cancer, acoustic neuroma, mucosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gum cancer, heart cancer, gland cancer, diaphragm cancer ovarian cancer, peritoneal cancer, pharyngeal cancer, pleural cancer, salivary gland, tongue and tonsill cancer. Use according to claim 19 or 20, characterized in that the object is human or animal. Use according to any of claims 19-21, characterized in that the administration is by intratumoral, intramuscular, intra-arterial, intravenous, intrapleural, intravesical, intracavitary or peritoneal injection or orally. Use according to any one of claims 19-22, characterized in that oncolytic adenovirus vectors or pharmaceutical compositions are given several times during a treatment period. Use according to any one of claims 19-23, characterized in that the object is given an oncolytic adenovirus vector with a different capsid strand projection from a vector used in a previous treatment. Use according to any of claims 19-24, characterized in that the object is further given radiation treatment in parallel. GM-CSF is under the virus E3 promoter. Use according to any of claims 19-24, characterized in that the object is further given chemotherapy in parallel. Use according to any of claims 19-24, characterized in that the object is further given verapamil or another calcium channel broker in parallel. Use according to any of claims 19-24, characterized in that the object is further given autophagy inducing substances in parallel. Use according to any of claims 19-24, characterized in that the object is further given temozolomide in parallel. Use according to any one of claims 19-24, characterized in that the object is further given chemotherapy or anti-CD20 treatment or another blocking treatment of neutralizing antibody pairs. Use according to any one of claims 19-24, characterized in that the object is further provided with parallel substances capable of pigeon-regulating regulatory T cells. Use according to any of claims 19-24, characterized in that the object is further given cyclophosphamide. 33. An in vitro method for producing GM-CSF in a cell, characterized in that: a) introducing into a cell a carrier comprising an oncolytic adeno-virus vector according to any of claims 1-15, 10b ) expresses GM-CSF of said vector in the cell. 34. An in vitro method for increasing tumor-specific immune response in an object, characterized in that: a) introducing into a cell a carrier comprising an oncolytic adeno-virus vector according to any of claims 1-15, b) expressing GM-CSF of the vector in the cell and c) the amount of cytotoxic T cells and / or natural killer cells in said target cell or tissue increases. An oncolytic adenovirus vector according to any of the preceding claims for producing GM-CSF in a cell to elicit tumor-specific immunity in an object. An oncolytic adenovirus vector according to any of the preceding claims to increase the tumor-specific immunity of an object. 37. Oncolytic adenovirus vector according to claim 36, characterized in that the amount of cytotoxic T cells and / or natural killer cells is increased in said object cell or tissue.