FI124927B - Adenovirus vectors and methods and uses thereof - 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 in vitro cells and pharmaceutical compositions comprising said vectors. The present invention also relates to said vectors and a pharmaceutical composition for use in treating cancer in a subject. Additionally, the present invention relates to an in vitro method for producing cellular anti-CTLA4 monoclonal antibodies, and the use of oncolytic adenoviral vectors for in vitro production of anti-CTLA4 monoclonal antibodies, and oncolytic adenoviral vectors for use in the treatment of tumor-specific apoptosis and in.

Background of the Invention

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

Gene transfer technology has been the subject of much research in the last twenty years. 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 chemoprotective genes, and "suicide genes" and may be introduced into the cell utilizing modified viral vectors or non-viral methods, such as electroporation, gene terminals, lipid or polymers.

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, optimal vectors must remain active in the target tissues or cells. All these properties of viral vectors have been developed over the last decades, and retroviral and adenoviral vectors and adeno-associated viral vectors, for example, have been extensively studied in biomedicine.

To further improve local penetration of tumor penetration and antitumor activity, selectively oncolytic agents, e.g., conditionally replicating adenoviruses, have been formed. Are-lytic adenoviruses are a promising tool for the treatment of cancers and have proven to be very safe and somewhat effective in clinical trials. Are-lytic adenoviruses kill tumor cells because the virus replicates in the tumor cell, whereby the final stage of replication leads to the release of thousands of virions into the surrounding tumor tissues for efficient tumor penetration and vascular reinfection. Tumor cells allow viral replication, whereas normal cells are spared because modifications are made to the viral genome that prevent replication in non-tumor cells.

Replication can be limited to tumor tissue either by partial deletions in the E1 region of the adenovirus or by the use of tissue or tumor specific promoters (TSPs). Insertion of such a promoter may enhance the effects of the vectors on target cells, and the use of exogenous tissue or tumor-specific promoters is common in recombinant adeno-viral vectors.

Most clinical trials have been performed on early-generation ade-viruses based on adenovirus 5 (Ad5). The antitumor activity of oncolytic adenoviruses depends on their gene transfer ability. Unfortunately, most tumors poorly express the most important Ad5 receptor, which is why modifications have been introduced into the Ad5 capsid. For example, capsid modification with the serotype 3 protrusion has improved infectivity and efficacy in ovarian cancer (Kanerva A, et al., Clin Cancer Res 2002; 8: 275-80; Kanerva A, et al., Mol Ther 2002; 5: 695-704; Kanerva A, et al., Mol Ther 2003; 8: 449-58). Further, since the Ad vectors strand and the penton base unit are key mediators of cell entry, targeting of recombinant Ad vectors can be achieved through genetic modifications of these capsid proteins (Dmitry I., et al. 1998, Journal of Virology, 72, 9706-9713 ). Most of the currently used oncolytic viruses are severely attenuated in replication due to many deletions in the critical viral genes. These viruses have excellent safety data, but their antitumor efficacy has been limited.

However, clinical and preclinical results indicate that treatment with non-armed oncolytic viruses is not sufficiently immunostimulatory to result in longer-term anti-tumor therapeutic immune responses. In this regard, oncolytic viruses have been armed to make them more immunostimulatory. In addition, viral replication and expression of immunomodulatory proteins within the tumor enhance the immune system by inducing cytokine production and release of tumor antigens (Ries SJ, et al., Nat Med 2000; 6: 1128-33).

Arming oncolytic viruses combines the benefits of traditional gene transfer with the efficacy of replicating agents. One purpose of viral armamentation is to induce an immune response against cells that allow viral replication. As mentioned above, viral replication, although immunogenic, is normally not sufficient alone to induce effective anti-tumor immunity. To enhance the induction of therapeutic immunity, viruses are armed with stimulatory proteins, such as cytokines, to facilitate the delivery of tumor antigens to antigen presenting cells, such as dendritic cells, and their stimulation and / or maturation. The introduction of immunotherapeutic genes into tumor cells and furthermore the translation of their proteins results in activation of the immune response and more efficient destruction of the tumor cells. The most important immune cells in this regard are natural killer cells (NKs) and cytotoxic CD8 + T cells.

A key realization in cancer immunotherapy has been the realization that, due to mechanisms of avoidance of the tumor immune system, induction of an anti-tumor immune response is not sufficient to eliminate the disease. Instead, due to the immunosuppressive nature of advanced tumors, a reduction in inhibitory T cells is also required (Dranoff G., Nat Rev Cancer 2004; 4: 11-22; de Visser KE et al., Nat Rev Cancer 2006; 6: 24-37). . One of these regulatory key pathways comprises antigen 4 (CTLA-4, CD152) associated with cytotoxic T lymphocytes, which acts against B7 / CD28-mediated stimulation. The preclinical anti-tumor activity of anti-CTLA-4 antibodies has previously been demonstrated in several tumor models (Leach DR et al., Science 1996; 271: 1734-6; Kwon ED et al., Proc Natl Acad Sci USA 1999; 96: 15074 -9). Currently, two fully human monolonal antibodies (mAbs) to CTLA-4 are being developed clinically; IgG1 ipilimumab (formerly MDX-010) and IgG2 tremelimumab (formerly CP-675,206). Several published studies seek to validate the biological and clinical activity of ipilimumab and treme-lumumab in patients with melanoma and other cancers (Kirkvood JM et al., Clin Cancer Res 2010; 16: 1042-8;

Hodi FS et al., N Engl J Med. 2010; 363; 8: 711-23; Ribas A et al., Oncologist 2007; 12: 873-83). Although antitumor activity has been seen in many experiments, serious and even fatal side effects have also been reported. The side effects concern the exposure of normal tissues due to systemic administration, while the efficacy is determined by the reduction of immune suppression in the tumor. Thus, the potential for local production of CTLA-4 mAb is justified. CTLA-4 is an activation-inducible type I transmembrane protein belonging to the Ig superfamily, expressed by T lymphocytes as a covalent homodimer that acts as an inhibitory receptor for costimulatory molecules B7.1 (CD80) and B7.2 (CD86) (Rib86). et al., Oncologist 2007; 12: 873-83). Inhibition of CTLA-4 by mAbs results in increased production of interleukin-2 (IL-2) and interferon-gamma (IFN-γ) by lymphocytes; and increased production of Class I molecules of the Major Histocompatibility Complex (MHC) (Lee KM et al., Science 1998; 282: 2263-6; ParadisTJ et al., Cancer Immunol Immunother 2001; 50: 125-33).

One of the most important mechanisms used by tumors to evade anti-tumor immunity is mediated through regulatory T cells. Of the many approaches hitherto used to reduce T-Regs, anti-CTLA4 mAb is the only one whose safety and efficacy have been demonstrated in a large randomized study (Hodi FS et al., N Engl J Med. 2010; 363; 8: 711-23). . Although this experiment represents a breakthrough in tumor immunotherapy, it was preceded by a negative phase 3 study with another anti-CTLA4 mAb (Ribas A et al. J Clin Oncol ASCO suppl. 2008; 287). In addition, in all anti-CTLA4 mAb assays, serious immune-related adverse reactions (irAEs) have resulted in deaths, which raises concerns about the safety of this approach. Therefore, other approaches, such as the use of a gene therapy model, are needed as it could increase the local concentration to obtain increased efficacy while reducing the adverse effects associated with systemic exposure.

Combining expression of tumor-localized anti-CTLA4-scFv with methods to remove systemic T-Reg has been reported to have synergistic effects (Tuve S, et al. Cancer Res 2007; 67: 5929-39). Also, the authors did not detect autoimmune reactions in a mouse model when tumors continuously express anti-CTLA-4-scFv antibody and anti-CD25 antibodies are administered i.p. (Tuve S, et al. Cancer Res 2007; 67: 5929-39). In contrast, when anti-CTLA4 mAb is administered systemically with T-Reg cells, autoimmune reactions are observed (Sutmuller RP et al., J Exp Med 2001; 194: 823-32; Takahashi T et al. J Exp Med 2000; 192 : 303-10). One possible reason for the synergy is that removal of T-Reg cells reduces the number of regulatory cells, while anti-CTLA-4 reduces the activity of suppressive cells. In addition, anti-CTLA4 can also reduce suppression of antigen-presenting cells.

Adenoviruses are medium-sized (90-100 nm), non-enveloped icosahedral viruses with approximately 36,000 base pairs of double-stranded linear DNA in a protein capsid. The viral capsid has filamentous structures that are involved in attaching the virus to the target cell. First, the protruding domain of the fiber protein binds to the target cell receptor [e.g., CD46 or coxsackievivirus adenovirus receptor (CAR)], second, the virus interacts with the integrin molecule, and third, the virus is endocytosed to the target cell. Next, the viral genome is transported from endosomes to the nucleus, and the replication machinery of the target cell is also utilized for viral purposes (Russell W. C., J General Virol 2000; 81, 2573-2604).

The adenovirus genome contains 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 Nemerov, Trends Microbiol 2004; 12: 162-168; Russell WC, J General Virol 2000; 81, 2573-2604; Volpers C. and Kochanek S., J Gene Med 2004; 6 suppl 1, S164-71; Kootstra NA and Verma IM, Annu Rev Pharmacol Toxicol 2003; 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 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 foreign DNA to be introduced into the vectors (Danthinne X, Imperiale, MJ. Gene Therapy. 2000; 7: 1707-1714). In addition, deletions and further mutations in other regions have provided additional features for viral vectors. Thus, various modifications of the adenoviruses have been proposed to provide effective antitumor effects.

However, more efficient and accurate gene transfer and increased specificity of gene therapies and sufficient tumor-killing capacity are needed. The safety data of 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 tools for gene therapy.

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

The present invention relates to an oncolytic adenovirus vector comprising 1) a nucleic acid backbone of adenovirus serotype 5 (Ad5) comprising a capsid modification, preferably a capsid modification of the adenovirus serotype 3 (Ad3), (D5 / 3 capsidele), ) In the Rb binding lane 2 and 3 of E1) in place of the deleted gp19k / 6.7Kr1 region of the nucleic acid sequence encoding a CTLA-4 specific fully human monoclonal antibody (aCTLA-4 mAb).

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

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

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

The present invention also relates to a pharmaceutical composition of the invention for use in treating cancer in a subject.

The present invention also relates to a method of treating a subject's cancer, the method comprising administering a vector or pharmaceutical composition of the invention to a subject suffering from cancer, in particular a cancer that is poorly responsive to conventional chemotherapeutic and / or radiation therapies.

The present invention further relates to an in vitro method for producing a fully human monoclonal antibody to CTLA-4 in a cell comprising: a) delivering a vehicle comprising an oncolytic adenovirus vector of the invention to a cell; and b) expressing the CTLA-4 specific monoclonal antibody in the cell.

Additionally, the present invention relates to a method of enhancing a tumor-specific immune response of a subject, comprising delivering a vehicle comprising the oncolytic adenoviral vector of the invention to a target cell or tissue, expressing a fully human CTLA-4 specific monoclonal antibody in the cell, The amount of mAb production in tumors (but not in normal tissues) is due to tumor-specific replication of the viral genome.

The present invention further relates to the use of the oncytic adenovirus vector of the invention for the production of a fully human CTLA-4 specific monoclonal antibody in a cell in vitro.

The present invention further relates to an oncolytic adenoviral vector of the invention for the production of a fully human CTLA-4 specific monoclonal antibody in a cell.

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 further relates to an oncolytic adenoviral vector of the invention for enhancing the tumor-specific immune response of a subject.

The present invention further relates to an oncolytic adenoviral vector of the invention for use in enhancing a tumor-specific immune response and apoptosis in a subject.

The present invention provides a novel tool for the treatment of cancers that are poorly responsive to, or not amenable to, current therapies. 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. The present invention can help destroy tumors of greater mass and more complex tumors than previous techniques. The treatment can be administered intravenously, intravitreally, intravenously and in combination. The method of treatment can provide systemic efficacy despite local injection. The treatment can also kill cells that are thought to be tumor-causing ("cancer stem cells") (Eriksson M et al. Mol Ther2007; 15 (12): 2088-93).

Not only does the vector of the invention allow delivery of the vector to the target region, it also ensures expression and stability of the transgene. The present invention solves the problem of treatment resistance to conventional therapies. In addition, the present invention provides means and methods for selective treatment with less toxicity or damage to healthy tissues. The present invention also has the advantages of different and reduced side effects compared to other treatments. Importantly, the treatment is synergistic with many other therapies, including chemotherapy, small molecule inhibitors and radiation therapy, and can thus be used in combination regimens.

The induction of an immune response against cells that allow replication of unarmed adenoviruses is normally not potent enough to induce the development of therapeutic tumor immunity. To overcome this weakness, the present invention provides armed adeoviruses with a fully human CTLA-4 specific monoclonal antibody that enhances antitumor immunity by activating cytotoxic T cells and antigen presenting cells and reducing regulatory T- cells and other suppressive cells. Anti-CTLA-4 mAbs can also directly induce apoptosis of tumor cells that frequently express CTLA-4. Many of the above-described anti-tumor mechanisms mediated by CTLA-4 inhibitory antibodies are realized through the adenoviral vectors of the invention: (A): Anti-CTLA-4 mAbs can inhibit an immune response from a CTLA-4 molecule on activated T cells. suppressive signaling (Chambers CA et al., Annu Rev Immunol 2001; 19: 565-94). (B): An important T cell inhibitory subset (regulatory T cells, T-Reg) constitutively expresses CTLA-4 and can bind B7 molecules on the surface of dendritic cells, which are key antigen presenting cells (Paust S et al. ., Proc Natl Acad Sci USA 2004; 101: 10398-403). The subsequent addition of indolamine-2,3-dioxygenase and other suppressive networks results in induction of T cell tolerance in the microenvironment instead of cytotoxic action (Munn DH et al., J Clin Invest 2004; 114: 280-90). (C): T cells expressing CTLA-4 (including T-Reg cells) may also bind directly to activated T cells, since costimulatory B7 molecules are expressed on the surface of activated human T cells. CTLA-4-blocking mAbs interfere with this suppressive signaling and lead to local expansion of tumor-antigen-specific T cells (Paust S et al., Proc Natl Acad Sci USA 2004; 101: 10398-103). (D): CTLA-4 is expressed on the surface of many tumor cells (Contardi E et al., Int J Cancer 2005; 117: 538-50), presumably reflecting a direct immunosuppressive effect on B7 and DCs of cytotoxic T cells. Interestingly, CTLA-4 blocking mAbs can induce direct killing of tumor cells by initiating apoptosis (Ribas A et al., Oncologist 2007; 12: 873-83; Contardi E et al., Int J Cancer 2005; 117: 538 -50) and in vivo this could be further enhanced by antibody-dependent cellular cytotoxicity (Jinushi M et al. Proc Natl Acad Sci USA 2006; 103: 9190-5). (E): CTLA-4 expressed by the tumor may initiate increased inaminamine-2,3-dioxygenase in tumor-infiltrating DCs, and monoclonal antibodies specific for CTLA-4 could also block this effect (Ribas A et al., Oncologist 2007; 12: 873-83).

Thus, five different mechanisms render the adenoviruses of the invention comprising monoclonal anti-CTLA-4 effective anti-tumor approaches without systemic exposure-related side effects that have led to safety concerns (Hodi FS et al., N Engl J Med. 2010; 363; 8: 711-23; Sanderson K et al., J Clin Oncol 2005; 23: 741-50 In addition to these five transgene-mediated mechanisms, oncolytic replication of the virus enhances antitumor activity, given the potent immunostimulatory mediated by the adenovirus itself activity (Tuve S, et al. Vaccine. 2009; 27 (31): 4225-39), oncolysis increases the total immunological use of the approach. In Example 9 below (Figures 1, 11, 12), we describe an additional improvement in this regard: Addition of CpG groups adenovirus genome makes the virus even more immunostimulatory.

Compared to prior art adenoviral devices, the present invention provides a simpler, more efficient, less expensive, non-toxic and / or safer means for the treatment of cancer. In addition, helper viruses or co-administration of the recombinant molecule are not required.

The present invention provides a new generation of improved and highly effective adenoviruses with high infectivity, which retains good safety but prevents higher levels of efficacy. Importantly, the present invention describes oncolytic adenoviruses that produce immunological factors critical for the efficacy of oncolytic viruses.

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

Brief description of the figures

Figure 1 shows the formation of single and double targeted oncolytic adenoviruses armed with anti-CTLA-4. Schematic representation of oncolytic asenoviruses Ad5 / 3-A24aCTLA4 (single-targeted; others are double-targeted), Ad5 / 3-hTERT-A24aCTLA4, Ad5 / 3-hTERT-A24aCTLA4-CpG, Ad5 / 3-E2F-A24aCT -CpG, replication-defective Ad5 / 3-aCTLA4, as well as unarmed oncolytic Ad5 / 3-A24 (positive control) and replication-deficient Ad5 / 3-Luc1 (negative control).

Figures 2A-2D show an evaluation of the expression and function of the anti-CTLA4 monoclonal antibody produced by the virus.

Figure 2A: Serum-free supernatants from virus-infected A549 (lanes 1 and 2, left to right) or PC3-MM2 (lanes 3 and 4) analyzed by Western blot to specifically detect human Ig (both heavy and light chains). in native (upper row) or denaturing conditions (lower row). Lanes 1 and 3: Ad5 / 3-A24aCTLA4-infected cells; lanes 2 and 4: cells infected with Ad5 / 3-aCTLA4.

Figure 2B: Functional assay showing the activity of virus-produced monoclonal anti-CTLA4 antibody. Jurkat cells were incubated with ionomycin, phorbol-12-myristate-13-acetate (PMA), and recombinant human B7, resulting in CD28- and CTLA-4-positive cells resembling T cells stimulated by antigen presenting cells but inhibited by suppressor cells, a condition often found in advanced tumors. Supernatant from cells infected with Ad5 / 3-A24aCTLA4 or Ad5 / 3-aCTLA4 resulted in inhibition of B7 / CTLA-4-mediated Suppression. This is seen in the increased production of the T-cell activation mark, IL-2. The recombinant anti-CTLA4 monoclonal antibody was included as a positive control.

Figure 2C: Loss of function assay showing the affinity of recombinant CTLA-4 (rCTLA-4) for a virus-produced monoclonal anti-CTLA4 antibody. Jurkat cells were activated as above, but now rCTLA-4 was also added to bind B7, which prevents CTLA-4 activation. When aCTLA4-containing supernatant was added, rCTLA-4 was inhibited and B7 was able to bind CTLA-4, thereby decreasing IL-2 production. Columns, mean of three independent experiments; rods, SE *, P <0.05; ***, P <0.001.

Figure 2D: CTLA4 expression levels in A549, SKOV3-ip1, PC3-MM2 tumor cell lines and low-passage tumor explant UT-SCC8. All tumor cell lines were positive for CTLA4 in fluorescence-assisted cell sorting.

Figure 3 shows the cell killing efficiency of anti-CTLA4-armed adenoviruses and control vectors. Tumor cell lines (A) PC3-MM2, (B) SKOV3-ip1, (C) A549 and (D) low-growth tumor implant UT-SCC8 were infected. Ad5 / 3-A24aCTLA4 had similar oncolytic activity as the positive control virus Ad5 / 3-A24 in all cell lines. Also, replication-defective Ad5 / 3-aCTLA4 had antitumor activity because tumor cells express CTLA4. Columns, Triple Assay Mean; rods, SE. **, P <0.01; ***, P <0.001.

Figure 4 shows tumor growth in suppression and apoptosis in immunodeficient mice bearing human prostate cancer xenografts treated with oncolytic adenovirus expressing anti-CTLA-4 monoclonal antibody. PC3-MM2 tumors (n = 8 / group) were formed in nude mice in which human anti-CTLA-4 lacks immunological activity. Therefore, the model only measures oncolysis and proapoptotic effect of anti-CTLA-4. After seven days, tumors (5-8 mm in diameter) were treated intravenously with 1x108 viral particles on days 0, 2 and 4 (arrows). Mock mice were injected with media only.

Figure 4A: Ad5 / 3-A24aCTLA4 had the best antitumor activity in this highly aggressive model. The tumor size is plotted against the average original size. Score, average; rods, SE; **, P <0.01 (Student's t-test on day 7).

Figure 4B: On day 5, ice sections of tumors were stained for anti-CTLA4 expression (human IgG, upper row) or apoptosis (active caspase-3, lower row) by immunohistochemistry (brown). Pictures were taken at a magnification of * 40.

Figure 4C: At day 7, human IgG levels in tumors and plasma of Ad5 / 3-A24aCTLA4 or Ad5 / 3-aCTLA4 treated mice were measured. Ad5 / 3-Ä24aCTLA4-treated tumors showed 81-fold higher anti-CTLA4 mAb compared to Ad5 / 3-αCTLA4-treated tumors (p <0.05). In addition, 43.3-fold more anti-CTLA4 mAb was detected in tumors treated with Ad5 / 3-A24aCTLA4 than in the plasma of the same animals (p <0.05). The mean plasma concentration was 392.6 pg / g (SE 312.0), which is below the tolerated concentrations reported in humans treated with ipilimumab (561 pg / ml) and tremel-mumab, 450 pg / m, respectively (REFS ) (1 ml of plasma weighs about 1 g). In Ad5 / 3-Ä24aCTLA4 tumors, the mAb concentration was 16,977 pg / g and thus much higher than in plasma. Columns, average of triplicate determinations; rods, SE.

Figure 5 shows the increased production of IL-2 and IFN-γ mediated by Ad5 / 3-Ä24aCTLA4 in stimulated peripheral blood mononuclear cells (PBMCs) in cancer patients. PBMCs from cancer patients (patient I244, patient C261, patient M158, or patient X258) were stimulated with ionomycin, PMA, and recombinant human B7 to mimic tumor-induced immune suppression and treated with filtered supernatants from virus-infected PC2-MM3. IL-2 (A) and IFN-γ (B) are indicative of T cell activation and were measured by FACS array. Recombinant monoclonal anti-CTLA-4 antibody was included as a positive control. Columns, mean of three independent experiments; rods, SE. *, P <0.05; **, P <0.01; ***, P <0.001.

Figure 6 shows Ad5 / 3-A24aCTLA4-mediated IL-2 and IFN-γ production in peripheral blood mononuclear cells (PBMC) of healthy donors. PBMCs from two healthy donors were stimulated with ionomycin, PMA, and recombinant human B7 and treated with filtered supernatants from virus-infected PC3-MM2 cells. Concentrations of IL-2 (A) or IFN-γ (B) in the media were measured with a FACS array. Columns, mean of three independent experiments; bars, SE. *, P <0, .05; ***, P <0.001.

Figure 7 is a diagram of the anti-CTLA-4 functionality assays shown in the examples shown in Figures 2, 5 and 6. In vivo, dendritic cells present antigens via the Tissue Compatibility Complex (MHC) to the T cell receptor (TCR) on the surface of the T cell. Costimulation is required instead of tolerance to the immune response. This is mediated by the binding of B7.1 or B7.2 (on the surface of DCs) to CD28 by the T cell. T cell activation can be quantified by the production of IL-2 and IFN-γιη.

Figure 7A: In the absence of an intact immune system, T cell activation can be stimulated by in vitro incubation of PBMCs with phorbol-12-myristate-13-acetate (PMA) and ionomycin, leading to activation independent of TCR and CD28 signaling. .

Figure 7B: As a negative feedback control mechanism in T cell activation, expression of CTLA-4 is also increased.

Figure 7C: CTLA-4 has a 10 0-fold higher affinity than CD28 for B7, leading to T cell uptake.

Figure 7D: Inhibition of CTLA-4 signaling by anti-CTLA-4 antibodies inhibits inhibition leading to T cell activation.

Figure 7E: When soluble recombinant human CTLA-4 is added, the anti-CTLA-4 mAb is captured, leaving B7 free to bind to CTLA-4 for T cell capture.

Figure 7F: If recombinant CTLA-4 is added to the culture medium in the absence of anti-CTLA4 mAbs, it will capture B7, leaving the T cell in an activated state.

Figure 8 shows specific anti-CTLA4 mAb activity by Ad5 / 3-A24aCTLA4 in cancer patient-stimulated peripheral blood mononuclear cells (PBMC). PBMCs of cancer patients (patient I244, patient C261, patient M158, or patient X258) were stimulated with ionomycin, PMA, and recombinant human B7 and recombinant human CTLA-4; and treated with filtered supernatants from virus-infected PC3-MM2 cells. Concentrations of IL-2 (A) or IFN-γ (B) in the media were measured with a FACS array. Columns, mean of three independent experiments; bars, SE. *, P <0.05.

Figure 9 shows the specific anti-CTLA4 mAb activity mediated by Ad5 / 3-A24aCTLA4 in healthy donor-stimulated peripheral blood mononuclear cells (PBMC). PBMCs were incubated with ionomycin, PMA, and recombinant human B7, and treated with filtered supernatants from virus-infected PC3-MM2 cells. Concentrations of IL-2 (A) or IFN-γ (B) in the media were measured with a FACS array. Columns, mean of three independent experiments.

Figure 10 shows the utility of a replication-capable model in increasing the expression of anti-CTLA4 mAb compared to replication-defective virus. PC3-MM2 cells were inoculated at 20,000 cells per well and infected with 10 viral particles of the respective viruses. At 24, 48 and 72 hours after infection, supernatants were harvested and analyzed by ELISA for human IgG. A threefold increase was observed with the oncolytic virus Ad5 / 3-A24aCTLA4 compared to non-replicating Ad5 / 3-aCTLA4. Columns, average of four determinations; rods, SE. ***, P <0.001.

Figure 11 demonstrates the effect of an oncolytic adenovirus containing TLR-9 (toll-like receptor 9) stimulating CpG molecules in a xenograft mouse model of lung cancer. Nude mice (5 mice per group, two tumors per mouse) were implanted with A549 cells and treated as follows: saline (black triangle), CpG-rich oncolytic adenovirus (Ad5-A24CpG, white circle), oncolytic adenovirus (Ad5-A24CpG), black square), oncolytic adenovirus + recombinant CpG oligos (white square). The CpG-rich virus was most effective in transmitting anti-tumor immunity, and was even more effective than the oncolytic adenovirus when co-administered with the recombinant CpG molecule.

Figure 12 shows the results of an MTS cell killing assay performed on spleen cells taken from treated mice at 72 hours (same mice as in Figure 11). The number of A549 cells still alive at the indicated time point is reported.

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; VI, Vili, IX, lila and IVa2 (Russel W. C., J General Virol 2000; 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 comprising a capsid modification such as an adenovirus serotype 3 (Ad3) protuberance (Ad5 / 3 capsid chimerism) with a 24 bp deletion (D24) E1 region: and a nucleic acid sequence encoding a fully human monoclonal antibody specific for CTLA-4 (anti-CTLA4 mAb or aCT-LA4) in place of deleted gp19k / 6.7K.

In a preferred embodiment of the invention, the adenoviral vector is based on human adenovirus.

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 the packaging signal (Ψ) and MLP (major late promoter).

Transcription of the early stage gene E1A initiates a replication cycle, followed by expression of E1B, E2A, E2B, E3 and E4 proteins. E1 proteins modulate cell 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. E3 gene products are responsible for defense against the host immune system, promote cell lysis and release of virus progeny (Russel W. C., J General Virol 2000; 81,2573-2604).

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., J General Virol 2000; 81, 2573-2604).

Compared to the wild-type adenovirus genome, the adenoviral vector of the invention lacks 24 bp of the E1 region, more specifically the E1A region, CR2, and the gp19k and 6.7K E3 region, and comprises capsid modifications in the virus strand. In some embodiments, compared to the wild-type adenovirus genome, the adenoviral vector of the invention further comprises an hTERT promoter or an E2F promoter in the E1 region, particularly upstream of the E1A region, and lacks the gp19k and 6.7K E3 region. In some embodiments, the invention also includes an adenovirus backbone enriched in TLR-9 binding CpG islands inserted into the E3 region (Figure 1).

In a preferred embodiment of the invention, in addition to the modified / partial E1 and E3 regions, the oncolytic 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, p1x, 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, p1x, p1Va2, E2, VA1 derived from Ad5 , VA2, L1, L2, L3, L4, partial E3, L5 and E4 In a preferred embodiment, the vector of the invention comprises the backbone of Ad5 together with part of Ad3 (e.g., part of the capsid structure). which is missing some element compared to the corresponding wild type region. For example, "partial E3" refers to the E3 region lacking gp19k / 6.7K. In this context, "VA1" and "VA2" refer to virus-associated RNAs 1 and 2, which are transcribed by the adenovirus but not translated. VA1 and VA2 In this context, the term "viral packaging signal" refers to a portion of the viral DNA that is made up of a series of AT-rich sequences and directs the process of encapsidation. The 24 base pair deletion of E1 (D24) affects the CR2 domain responsible for binding the tumor suppressor / cell cycle regulatory protein Rb and thus allows the induction of the synthesis of the (S) phase, i.e., DNA synthesis or replication. The interaction of pRb and E1A requires eight amino acids, 121-127 (Heise C. et al. 2000, Nature Med 6, 1134-1139), of the E1A conserved region, which have been deleted in the present invention. The vector of the present invention comprises a deletion of nucleic acids corresponding to amino acids 122-129 from the vector of Heise C. et al (2000, Nature Med 6, 1134-1139). Viruses with D24 are known to have a reduced ability to bypass the G1-S checkpoint and to replicate effectively only in cells where this interaction is not necessary, e.g., tumor cells deficient in the 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 proteins play an important role in the regulation of the host immune response, that is, in the suppression of both natural and specific immune responses. The gp19k / 6.7K-6e deletion in E3 represents a deletion of 965 bp in the E3A region of the adenovirus. In the resulting adenovirus construct, both the gp19k and the 6.7K gene have been deleted (Kanerva A. et al., Gene Therapy 2005; 12, 87-94). The product of the gp19k gene is known to bind and isolate major histocompatibility complex I (MHC1) molecules in the mucosa and prevent cytotoxic T lymphocytes from recognizing infected cells. Because many tumors lack MHC1, deletion of gp19k increases viral tumor selectivity (virus is cleared more rapidly than wild-type virus from normal cells, but there is no difference in tumor cells). 6.7K proteins are expressed on cell surfaces and are involved in the downregulation of TNF-like apoptosis-inducing ligand (TRAIL) receptor 2.

In the present invention, the cDNA encoding the CTLA4 mAb is inserted into the gp19k / 6.7 / f deleted E3 region under the E3 promoter. This restricts the expression of the transgene on tumor cells, which allow replication of the virus 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 the anti-CTLA4 mAb is under the control of the viral E3 promoter. gp 19k-de \ eet \ o is particularly useful for expression of CD40L because it can increase MF1Cl presentation of tumor epitopes in tumors that retain this ability. In this context, stimulation of APC and T cells by anti-CTLA mAb may result in optimal benefits.

The anti-CTLA4 mAb acts through a variety of mechanisms such as activation of cytotoxic T cells, reduction of regulatory T cells (T-Regs), and cytotoxic T cells and antigen presenting cells by CTLA-4-expressing tumor cells ( inhibition of direct immunosuppression of dendritic cells). In addition to the mechanism mediated by these 5 transgenes, described in detail above, the oncolytic replication of the virus enhances the anti-tumor effect. The nucleotide sequence encoding the CTLA4 mAb can be from any animal, such as a human, monkey, rat, mouse, dog or cat depending on the subject being treated, but preferably the CTLA4 mAb is fully encoded by a human sequence. The nucleotide sequence encoding the CTLA4 mAb may be modified to enhance its effects, or unmodified, i.e., wild type. In a preferred embodiment of the invention, the nucleic acid sequence encoding the CTLA4 mAb is unmodified.

Addition of exogenous elements may increase the effects of vectors on target cells. The use of exogenous tissue or tumor specific promoters is common in recombinant adenoviral vectors and may also be utilized in the present invention. In some embodiments, the adenoviruses of the invention comprise a hTERT or hTERT variant or E2F for controlling the E1A region, preferably: from. hTERT directs the vector to cells expressing telomerase, while E2F directs the vector to cells lacking the Rb / p16 pathway. Such deficiencies result in high levels of free E2F expression and high activity of the E2F promoter. However, viral replication may be restricted to target cells by any other suitable promoter, including but not limited to CEA, SLP, Cox-2, Midkine, CXCR4, SCCA2 and TTS. They are usually added to control the E1A region, but in addition or alternatively other genes such as E1B or E4 can also be regulated. Exogenous insulators, i.e., inhibitory elements against non-specific enhancers, left ITR, native E1A promoter or chromatin proteins, may also be incorporated into recombinant adenoviral vectors. Any additional component or modification may optionally be used, but are not necessary in the vectors of the present invention.

In other embodiments of the invention, the adenoviral vectors of the invention exhibit a TLR-9 binding CpG-rich DNA region in the adenovirus backbone (Figures 1, 11, 12).

The oncolytic adenoviral vector of the invention comprises a Kap linkage modification. Most adults are exposed to the most widely used ade-novovirus serotype Ad5 and therefore the immune system is able to rapidly produce neutralizing antibodies (NAb) against it. In fact, the incidence of anti-Ad5 NAb can be up to 50%. It has been shown that NAb can be induced against most immunogenic proteins of the adenovirus capsid, and on the other hand it has been shown that even small changes in the Ad5 filament protrusion may allow escape from capsid-specific NAb (Särkioja M, et al., Gene Ther. 2008; 15 (12)). : 921-9). Thus, modification of the protuberance 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 a fiber protrusion, and modifications of this protrusion or fiber may improve target cell entry and result in enhanced oncolysis in many or some cancers (Ranki T. et al., Int J Cancer 2007; 121, 165- 174). Indeed, capsid-modified adenoviruses are preferred tools for improved gene transfer to cancer cells. As used herein, "capsid" means a viral protein coat containing hexon, filament and penton base proteins. Any capsid modulation, i.e., modification of the hexon, filament and / or penton base proteins known in the art to enhance virus delivery Modifications may be genetic and / or physical modifications and include, but are not limited to, modifications that add ligands that recognize specific cellular receptors and / or inhibit native receptors for binding, and substitute for adenoviral or non-adenoviral vector. adenovirus protrusion (chimerism) and specific molecules (e.g., fibroblast growth factor 2, FGF2) are added to adeno viruses. Thus, the capsid modifications include, but are not limited to, small peptide motif (s), peptide (s), chimerism (s) or mutation (s). to increase n thread (e.g., protrusion, tail or shaft), hexone and / or penton base unit. 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 a heparin sulfate binding polylysine into a strand. In a particular embodiment of the invention, the capsid modification is Ad5 / 3-Kime. In this context, "Ad5 / 3 chimerism" of the capsid means chimerism in which the protrusion of the strand is Ad serotype 3 and the end strand is Ad serotype 5.

The vector of the invention may also contain other modifications, such as modifications of the E1B region. In this context, "RGD" refers to the arginine-glycine-aspartic acid (RGD) motif revealed on the surface of the penton base unit and interacts with cellular α-ν-β 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 heterologous integrin-binding RGD-4C motif into 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 Ad5 filament gene encoding a strand containing a RGD-4C peptide is described in detail, for example, in an article by Dmitry I. et al. (Journal of Virology 1998; 72, 9706-9713). In this context, "heparan sulfate binding polylysine modification" means the addition of seven cycles of lysine to the C-terminus of a filament.

Expression cassettes are used to express transgenes in a subject, e.g., a cell, using vectors. As used herein, the term "expression cassette" means 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 expression cassette, however, only one expression cassette is sufficient In one preferred embodiment of the invention, the oncolytic adenoviral vector comprises at least one expression cassette, In one preferred embodiment, the oncolytic adenoviral 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. The cell may be an in vitro, ex vivo or in vivo cell. For example, the cell may be used to produce an adenoviral vector in vitro, ex vivo or in vivo, or the cell may be a target, e.g., a tumor cell infected with the adenoviral vector.

In a method for producing CTLA4 mAbs in a cell, a vehicle comprising the vector of the invention is delivered to a cell, and the CTLA4 mAb gene is expressed and the protein is paracrine translated and secreted. A "vehicle" can be any viral vector, plasmid or other medium, such as a particle, capable of delivering a 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.

The present invention can enhance the tumor-specific immune response in a subject. Activation of cytotoxic T cells, reduction of regulatory T cells (T-Regs), and direct inhibition of immunosuppression of CTLA-4-expressing tumor cells and dendritic cells results from the expression of CTLA4 mAb.

Various immune response parameters may be determined to monitor or investigate the effects of the invention. The most common signs include, but are not limited to, changes in tumor or adenovirus specific cytotoxic T cells in blood or tumors. Recruitment and activation of antigen presenting cells can be studied in tumors or lymphoid tissue. In addition, subgroups of regulatory cells (e.g., regulatory T cells) can be examined for number or activity. Serum cytokine profiles can illuminate the Th1 / Th2 environment, which is also important for immunity versus tolerance. 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, e.g., ELISPOT assay, tetramer assay, pentamer assay, intracellular cytokine staining, antibody assay. analysis of different cell types of blood or tumors.

Cancer

The recombinant Ad5 / 3 vectors of the invention are engineered to be capable of replication in cells lacking the Rb pathway, especially the Rb-p16 pathway. These deficient 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 deficient in the Rb pathway. As used herein, "deficiencies in Rb pathways" refers to mutations and / or epigenetic changes in any of the genes or proteins of the pathway. Due to these deficiencies, E2F is overexpressed by tumor cells and thus binding of Rb by E1A-CR2, which is normally required to release E2F required for efficient replication, is redundant.

In addition, some of the oncolytic adenoviral vectors of the invention have been engineered to be capable of replication in cells expressing human telomerase reverse transcriptase (hTERT), a catalytic subdomain of human telomerase. These include more than 85% of human tumors that are found to increase expression of the hTERT gene and its promoter, while most adult somatic cells lack or transiently express very low levels of enzymes. (Shay and Bacchetti 1997, Eur J Cancer 33: 787-791). Such Rb-p16 pathway-deficient / hTERT-promoter combinations can target any type of cancer or tumor, including both malignant and benign tumors, and may target primary tumors and metastases. E2F transcription factors regulate the expression of a diverse set of genes involved in key growth-controlling events in cells (Johnson and Schneider-Broussard 1998, Role of E2F in Cell Cycle Control and Cancer, Front Biosci 1998 Apr 27; 3: d447-8; Muller and; Helin 2000, The E2F transcription factors: key regulators of cell proliferation, Biochim Biophys Acta 2000 Feb 14; 1470 (1): M1-12).

In non-cycling normal cells, E2F is trapped in pRb / E2F complexes and thus little free E2F is freely available. The pRb pathway has been cleaved in almost all human cancers, leading to free E2F in most cancers, indicating its importance in controlling physiological growth. The pathway can be cut by mutation in any of a number of different molecules. However, a common feature is the resulting activation of the E2F promoter to provide increased E2F levels. E2F binds the promoter of many target genes, but autoactivation is also important for its function. Therefore, cells inactive for p16 / Rb exhibit high levels of E2F, which are further amplified through binding of E2F to their promoter (Hanahan and Weinberg 2000, Cell 7; 100 (1): 57-70; Johnson et al. ., 2002, Cancer Cell 1 (4): 325-37).

However, if E1A of the adenovirus is controlled by the E2F promoter (such as, for example, US 2008118470 A1), there is a risk of self-replicating a bad loop unless a D24 deletion of E1A / Rb is used concurrently. Specifically, low levels of E2F present even in normal cells would bind to the E2F promoter, leading to E1A expression leading to further release of E2F from the pRb / E2F complexes, leading to increased activation of the E2F promoter and thus greater E1F activation. . Thus, in the absence of the D24 deletion, which interrupts the binding of E1A to pRb, the E2F promoter results in oncolytic adenoviruses, which can also replicate in normal cells, which may have safety effects.

Non-methylated double-stranded DNA can stimulate TLR9, an endo-somatic receptor that combines an innate and acquired immune response. Addition of CpG-rich regions to the adenovirus backbone enhances the adenovirus's ability to stimulate TLR9 in antigen presenting cells, thereby increasing T-cell stimulation and maturation as well as NK activation (Nayak S, Herzog RW. Gene Ther. 2010 Mar; 17 (3): 295-304 ).

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, synovia-alkaline cancer, hepatocellular cancer, kidney cancer, connective 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, prolactoma, T-cell leukemia / lymphoma, neuroma, von Hippel-Lindau disease, zollin-ger-ellison syndrome, adrenal cancer, anal cancer, gallbladder cancer, urinary tract , neuroblastoma, meningioma, spinal neoplasm, osteochondromoma, chondrosarcoma, Evving's sarcoma, cancer of unknown primary location, carcinoid, gastrointestinal carcinoid, fibrosarcoma, breast cancer, Paget's disease, cervical cancer, cancer of the esophagus, esophageal cancer, head, neck cancer, kidney cancer, Wilms tumor, Kaposi's sarcoma, prostate cancer, testicular cancer, Hodgkin's disease, non-Hodgkin's lymphoma, skin cancer, mesothelioma, multiple myeloma, ovarian cancer, endocrine pancreatic cancer, endocrine pancreatic cancer, , soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymic carcinoma, thyroid cancer, trofoblastisyöpä, hydatidiform mole, uterine cancer, endometrial cancer, vulval, ulkosynnytinsyöpä, kuuloher-mokasvain, mucosis fungoides, insulinoma, carcinoid syndrome, somatostatin-tinooma, iensyöpä, heart cancer, lip cancer, the membrane of cancer , oral cancer, hormone cancer, palatine cancer, parathyroid cancer, peritoneal cancer, pharyngeal cancer, pleural cancer, salivary gland cancer, tongue cancer and tonsillectomy.

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 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 such as those described in US2010166799 A1, other therapeutically effective agents, other agents such as pharmaceutically acceptable carriers, buffers, adjuvants, adjuvants, antiseptics, and / or any components normally present 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 capsules.

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. Viral replication is a potent sign of danger to the immune system (necessary for TH1-type cytotoxic T cell response) and thus acts as a potent co-stimulatory factor. and NK cell recruitment.

A critical finding in the field of tumor immunology is that the induction of an anti-tumor immune response is not sufficient for a therapeutic effect. Instead, it is also critical to reduce tumor-mediated immune suppression. Tumor cell lysis also helps to present tumor fragments and epitopes to APCs and inflammation produces more costimulation. Thus, an epitope-independent (i.e., non-HLA-restricted) response is generated with each tumor and thus occurs in situ. The tumor-specific immune response is activated in the target cell, subsequently allowing anti-tumor effects to occur at the entire target level, e.g., distant metastases.

The effective dose of vectors depends on many factors, such as the subject to be treated, the type of tumor, the location of the tumor, and the category of the tumor. For example, the dose may vary from about 108 viral particles (VP) to about 1014 VP, preferably from about 5x109 VP to about 1013 VP, and more preferably from about 8x109 VP to about 1012VP. In one particular embodiment of the invention, the dosage is about 5x101 ° -5x1011 VP.

The pharmaceutical compositions can be produced by any of the conventional methods known in the art, for example, using one of the following: batch, batch feed and perfusion cultivation modes, column chromatography gradient purification, CsCl gradient purification and perfusion modes in low-throughput solids.

Allocation

The vector or pharmaceutical composition of the invention may be administered to any eukaryotic subject 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, intra-arterial, intravenous, pleural, intracellular, intrathecal or peritoneal injection or oral administration.

Even one administration of the oncolytic adenoviral vector of the invention can provide therapeutic effects. However, in a preferred embodiment of the invention, the oncolytic adenoviral vectors or pharmaceutical compositions are administered several times during a treatment period. Oncolytic adenoviral vectors or pharmaceutical compositions may be administered, for example, 1 to 10 times during the first two weeks, during the first four weeks, monthly or during a treatment period. In one embodiment of the invention, they are administered 3 to 7 times in the first two weeks, then in the fourth week and then monthly. In one particular embodiment of the invention, they are administered 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 or longer.

In addition, administration of the oncolytic adenoviral vectors of the invention may advantageously be combined with administration of other oncolytic adenoviral vectors, such as those described in US201066799 A1. The administration may be simultaneous or sequential.

To avoid neutralizing antibodies in a subject, the vectors of the invention may vary between treatments. In a preferred embodiment of the invention, the subject is provided with an oncolytic adenoviral vector having a different capsular filament protrusion compared to the prior treatment vector. In this context, the "capsular filament protrusion" refers to the protrusion portion of the filament protein (Figure 1a).

The gene therapy of the invention is effective on its own, but combining ade-noviral gene therapy with another treatment, such as conventional therapy, may be more effective than either alone. For example, each agent of the combination therapy may function autonomously in tumor tissue, adenoviral vectors may sensitize cells to chemotherapy or radiation therapy, and / or chemotherapeutic agents may enhance viral replication levels or affect target cell receptor status. Alternatively, the combination may modify the immune system of the subject so as to be beneficial to the efficacy of the treatment. For example, chemotherapy could be used to reduce suppressive cells, such as regulatory T cells. Alternatively, chemotherapy may be used after treatment with an oncolytic virus to enhance the immunological response by killing the tumor cells and the consequent release of epitopes and / or viruses. Chemotherapy can also sensitize tumor cells to on-lytic viruses and vice versa. The combination therapy agents may be administered simultaneously or sequentially. In a preferred embodiment of the present invention, patients are simultaneously receiving cyclophosphamide to enhance the immunological effect of the treatment.

In a preferred embodiment of the invention, the method or use further comprises administering concurrent radiation therapy to the subject. In another preferred embodiment of the invention, the method or use further comprises the administration of concurrent chemotherapy to the subject. In another preferred embodiment, the method or use further comprises administering to the subject other oncolytic adenovirus or vaccinia virus vectors. The vectors may be administered simultaneously or sequentially. As used herein, "concurrent" refers to treatment given before, after or concurrently with the gene therapy of the invention. The period of concurrent treatment may vary from minutes to several weeks. Preferably, the concurrent treatment lasts several hours. In one embodiment, cyclophosphamide is administered both intravenously.

Suitable agents for combination therapy include, but are not limited to, all-trans-retinoic acid, azacitidine, azathioprine, bleomycin, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, dau-norubicin, docetaxel, doxiflubidine, , fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib, mecorothamine, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, temozolomide, teniposide, thioguanine, valrubicin, vinblastine, vincristine

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

In a preferred embodiment of the invention, the method or use further comprises administering agents to induce autophagy. Autophagy refers to a catabolic process in which the cellular components are broken down by lysosomal machinery. "Autophagy-inducing agents" means substances which are 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, temozirolimus, temozirolimus, and temsirolimus. further comprises administering a temozolomide to a subject Temozolomide may be either oral or intravenous temozolomide, Autophagy inducing agents may be combined with immunomodulatory agents In one embodiment, the oncolytic adenovirus encoding an anti-CTLA4 mAb is combined with both a temozolomid and a temozolomid.

In one embodiment of the invention, the method or 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 CD20 positive cells and which may be selected from the group consisting of rituximab and other anti-CD20 monoclonal antibodies. "Neutralizing Antibody Therapy" refers to agents that are capable of inhibiting the development of antiviral antibodies 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. a group consisting of, but not limited to, cyclophosphamide, cyclosporine, azathioprine, methyl-prenizolone, etoposide, CD40L, CTLA4Ig4, FK506 (tacrolimus black), IL-12, IFN-gamma, interleukin , anti-CD8, anti-CD4 antibodies, myeloablation, and oral adenovirus proteins, the approach described in this application may also be combined with molecules capable of overcoming neutralizing antibodies, such as liposomes, lipoplexes, and polyethylene glycol, which may be Alternatively, neutralize These antibodies can be removed by an immunophoresis column consisting of adenovirus capsid 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 method or use further comprises administering agents which are capable of reducing regulatory T cells in the subject. "Regulatory T Cell Reduction Agents" refers to agents that reduce the number of cells identified as suppressor T cells or regulatory T cells. These cells have been identified to contain one or more of the following immunophenotype characters: CD4 +, CD25 +, FoxP3 +, CD127 and GITR + Such suppressor T-cell or regulatory T-cell depleting agents may be selected from the group consisting of anti-CD25 antibodies or chemotherapy agents, and may be useful in regulating T-cell counts. aCTLA4 is mainly effective in suppressing activity.

In a preferred embodiment of the invention, the method or use further comprises administering cyclophosphamide to the subject. Cyclophosphamide is a common chemotherapy agent that has also been used in some autoimmune diseases. In the present invention, cyclophosphamide can be used to enhance viral replication and the effects of aCTLA4-induced stimulation of NK cells and cytotoxic T cells to elicit an improved immune response to the tumor. It can be used as intravenous bolus doses or orally in small doses metronomically or in combination.

In the present invention, a human IgG2 subtype was selected to ensure that the CTLA-4 inhibitory mAb does not kill activated cytotoxic T cells, which may be useful for therapy. IgG2 produces minimal complement activation and Ab-dependent cell-mediated cytotoxicity (Bruggemann M. et al. J Exp Med 1987; 166: 1351-1361) and also reduces the possibility of cytokine syndrome in human use (Ribas A. et al., Oncologist 2007; 12: 873-83).

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

In the present invention, the oncolytic adenovirus is armed with a fully human CTLA-4 specific monoclonal antibody. In this approach, tumor cells die due to viral replication and direct anti-tumor activation of anti-CTLA4 mAb and direct pro-apoptotic tumor cell killing. An additional benefit may arise from the release of tumor antigen due to viral replication, which can potentially improve anti-CTLA-4 mAb therapy by potentially allowing a more specific immune response against tumor targets (Hodi FS et al., N Engl J Med 2010; 363; 8: 711-23; Mokyr MB et al., Cancer Res 1998; 58: 5301-4; Volochok JD and Saenger Y., Oncologist 2008; 13 Suppl 4: 2-9). Also, the side effects of the treatments do not overlap, which could allow for increased efficacy without increasing toxicity. It is shown that oncolytic adenoviruses can efficiently express a functional anti-CTLA4 mAb as a transgene in cancer cell lines (Figure 2). It was also found possible to combine replication of the oncolytic adenovirus with anti-CTLA4 mAb and obtain increased cell killing (Figures 3-4). This is of concern because inhibition of CTLA-4 by the mAb results in increased production of IFN-γ and Tissue Compatibility Complex (MHC) class I molecules, which can potentially inhibit viral replication (McCart JA et al., Gene Ther 2000; 7: 1217-23; Nakamura H et al., Cancer Res 2001; 61: 5447-52). Viral cololysis, together with expression of anti-CTLA4 mAb, resulted in higher antitumor activity in vivo than either treatment alone (Figure 4). There have been previous reports on the treatment of cancer patients with oncolytic viruses expressing granulocyte-macrophage growth factor (GMCSF) in combination with low-dose metronomic cyclophosphamide to reduce T-Regs (Cerullo V et al., Cancer Res; 70: 4297-30; Koski A et al., Mol Ther. Jul 27, 2010 [Epub ahead of print], PMID: 20664527). In addition, cell-mediated administration of mouse anti-CTLA-4 from GM-CSF-secreting tumor cell immunotherapy activates potent antitumor responses and prolonged overall survival, with less evidence of systemic autoimmunity (Simmons AD et al., 12 Cancer Immunol. -70). Therefore, the effectiveness of the approach can be further enhanced by a multimodal approach using conventional cancer therapies, e.g., irradiation + chemotherapy, vaccines, e.g., GM-CSF or / and T-Reg reductions, e.g., cyclophosphamide. This is the first time that it has been shown that full length mAb can be produced from an oncolytic adenovirus. In addition, it is the first fully human anti-CTLA4 mAb expressed in a tumor-replicating model. No side effects were seen in the mouse study performed. The results of PBMCs in cancer patients justify the conversion of Ad5 / 3-A24aCTLA4 into a clinically oncolytic viral vector for the treatment of patients with advanced cancer. Because the p16-Rb pathway is deficient in many, if not all, solid tumors (Sherr CJ., Science 1996; 274: 1672-724), Ad5 / 3-A24aCTLA4 and other A24-deficient adenoviruses of the invention are suitable for most types of cancer-responsive treatment. Embodiments of the invention which include, in addition to Δ24: η, hTERT or E2F promoters extend the utility of the present invention to virtually all cancers. In addition, the addition of a promoter may allow higher therapeutic doses, fewer side effects, and enhanced systemic use. Importantly, the addition of CpG residues to the viral genome may increase the anti-tumor immune response.

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

eXAMPLES

Animals

All animal protocols were reviewed and approved by the Experimental Animal Committee of the University of Helsinki and the Provincial Office of Southern Finland. C57Black mice and NMRI nude mice were obtained from Taconic (Ejby, Denmark) at 4-5 weeks of age and were quarantined for at least one week prior to study. The mice were frequently monitored for their health, and as soon as any sign of pain or pain appeared, they were killed.

cell culture

Low-growth tumor cell culture of human head and neck squamous cell carcinoma (HNSCC) UT-SCC8 (27) was cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS (PromoCell GmbH, Heidberg). Germany), 1% non-essential amino acids (Gibco, Invitrogen, Carlsbad, California) with 2 mmol / l glutamine, 100 units / ml penicillin and 100 units / ml strepto-mycine (all from Sigma, St. Louis, MO). UT-SCC cells were used in low culture, typically 15-30 culture.

Transformed human embryonic kidney cell line 293, human lung cancer cell line A549, human ovarian cell line SKOV3-ip1 and human prostate cancer cell line PC-3MM2, and Jurkat (clone E6-1) human leukemic T-cell lymphoblastic cell strain -on, Manassas, VA, USA). All cells were maintained under the recommended conditions.

human samples

Peripheral blood mononuclear cells (PBMCs) in healthy subjects and patients with advanced metastatic tumors unresponsive to conventional treatments were obtained by written consent.

Statistical analysis

A two-tailed Student's t test was used and a p value <0.05 was considered significant.

Example 1. Formation of adenoviruses

Chimeric adenoviruses carrying a cDNA sequence encoding an IgG2-type anti-CTLA4 mAb were generated (Figure 1). The coding sequence for the anti-CTLA4 mAb was introduced into the 6.7K / gp19K deletion of adenovirus E3A by Ad5 / 3-A24aCTLA4 (SEQ ID. NO: 1), Ad5 / 3-hTERT-A24aCTLA4 (SEQ ID NO: 2). ), Ad5 / 3-hTERT-A24aCTLA4-CpG (SEQ ID NO: 3), Ad5 / 3-E2F-A24aCTLA4 (SEQ ID NO: 4), and Ad5 / 3-E2F-A24aCTLA4-CpG (SEQ ID NO: 3). : 5), or to generate a replication-defective adenovirus Ad5 / 3-aCTLA4 (SEQ ID NO: 6) into the deleted E1 driven by the CMV promoter.

Oncolytic adenoviruses were generated and amplified using standard adenovirus production methods (Kanerva A, et al., Mol Ther 2002; 5: 695-704; Bauerschmitz GJ, et al., Mol Ther 2006; 14: 164-74; Kanerva A and Hemminki A, Int J Cancer 2004; 110: 475-80; Volk AL et al., Cancer Biol Ther 2003; 2: 511-5). Briefly, either the E1 or E3 shuttle vector carrying the transgene and other groups (promoters, CpG, poly-A) were first generated and linked to the resque plasmid in bacterial cells containing human recombinases. Key Viral Properties Including Key Features of Control Viruses Ad5Luc1 (Kanerva A et al., Clin Cancer Res 2002; 8: 275-80) and Ad5 / 3-A24 (Kanerva A et al., Mol Ther 2003; 8: 449-58) is illustrated in Figure 1.

Plasmid pTHSN-aCTLA4 was constructed to generate Ad5 / 3-Ä24aCTLA4. pTHSN-aCTLA4 contains the heavy and light chains of the IgG2 type anti-CTLA4 mAb from the 6.7K / gp19K deleted genome in the E3 region. Equimolarity between heavy and light chains is achieved by an internal ribosome entry site (IRES) between the chains. pAdEasy-1.5 / 3-A24-aCTLA4 was generated by homologous recombination in Escherichia coli BJ5183 cells (Qbiogene Inc., Irvine, CA, USA) with FspU linearized pTHSN-aCTLA4 and pAdEasy-1.5 / 3-A24 linearized with S / fl: (Kanerva A et al., Clin Cancer Res 2002; 8: 275-80), a resque plasmid containing a serotype 3 protrusion and a 24 bp deletion in E1A. The Ad5 / 3-A24aCTLA4 genome was released by Pacl digestion and subsequent transfection into A549 cells. The virus was added with A549 cells and purified with cesium chloride gradients. Viral particle concentration was determined at 260 nm and standard TCID50 (Medium Tissue Culture Infective Dose) assay on 293 cells was performed to determine the titer of the infecting particles.

To form Ad5 / 3-hTERT-A24aCTLA4, Ad5 / 3-hTERT-A24aCTLA4-CpG, Ad5 / 3-E2F-A24aCTLA4 and Ad5 / 3-E2F-A24aCTLA4-CpG, the anti-CTLA amplification product was extended. first to pTFISN or pTHSN-CpG and then recombined with pAd5 / 3-hTERT-E1A or pAd5 / 3-E2F-E1A (Bauerschmitz GJ, et al., Cancer Res 2008; 68: 5533- 9. Hakkarainen T, et al. Clin Cancer Res 2009; 15 (17): 5396-403.) The resulting plasmid was linearized with PacMI and transfected into A549 cells for amplification and capture. Viruses were added in A549 cells and purified with cesium chloride gradients. Viral particle concentration was determined at 260 nm and standard TCID50 (Medium Tissue Culture Infective Dose) assay on 293 cells was performed to determine the titer of the infecting particles.

All cloning steps were confirmed by PCR and several restriction di gestures. The pTHSN-αCTLA4 shuttle plasmid was sequenced. The absence of wild type E1 was confirmed by PCR. The E1 region, the transgene and the strand were checked for final virus by sequencing and PCR. All steps of virus production, including transfection, were performed on A549 cells to avoid the risk of wild-type recombination as previously described (Kanerva A. et al. 2003, Mol Ther 8, 449-58; Baeurschmitz GJ et al. 2006, Mol Ther 14 , 164-74). aCTLA4 is under the E3 promoter (especially under the control of endogenous viral E3A gene expression control elements), leading to replication-associated transgene expression starting about 8 h after infection. E3 is intact except for the 6.7K / gp19K \ n deletion.

To generate the non-replicating E1-deficient control virus Ad5 / 3-aCTLA4, both strands of the anti-CTLA4 mAb cDNA were ligated into pShuttle-CMV. Homologous recombination was performed between pAdEasy-1.5 / 3 plasmid (Krasnykh VN, et al., J Virol 1996; 70: 6839-46) carrying the entire adeno-virus genome and Pmel linearized pShuttle-CMV-aCTLA4. pAdEasy-1.5 / 3-aCTLA4. The Ad5 / 3-αCTLA4 genome was released with Pac1 and transfected with 29. The virus was added to A549 cells and purified with cesium chloride gradients. The viral particle concentration was determined at 260 nm and a plaque assay on 293 cells was performed to determine the infectious particles.

Example 2. Expression and Functionality of Adenoviruses Generated in vitro

Western blot analysis was used to confirm that the adenoviruses formed expressed anti-CTLA4 mAb. A549 or PC3-MM2 tumor cells were infected with Ad5 / 3-A24aCTLA4 or Ad5 / 3-aCTLA4 formed at a concentration of 10 viral particles (VP) per cell. After 48 hours, supernatants of virus-infected cells were filtered on 0.02 µm filters (Anotop, Whatman, England), 15 µl were run on 7.5% SDS-polyacrylamide gel electrophoresis (PAGE) under reducing or native conditions and was transferred to a nitrocellulose membrane. The membrane was incubated with goat anti-human IgG (heavy and light chains) (AbD Serotec, MorphoSys, Germany), washed and incubated with a secondary antibody coupled with horseradish peroxidase (Dako, Denmark). Signal detection was performed by confirmed chemiluminescence (GE Healthcare, Amersham, UK).

In Western blot analysis, Ad5 / 3-A24aCTLA4 and Ad5 / 3-aCTLA4 expressed the expected about 150 kDa anti-CTLA4 mAb under native conditions and about 50 kDa heavy chains and about 25 kDa light chains under denaturing conditions ( Figure 2A) in supernatants 48 hours after infection.

To compare the oncolytic virus Ad5 / 3-A24aCTLA4 or the anti-CTLA4 mAb expressed in replication-defective Ad5 / 3-aCTLA4, PC3-MM2 cells were plated at 20,000 cells per well and infected at 10 VP per cell, respectively. 24 h, 48 h and 72 h after infection, supernatants were harvested and analyzed by ELISAs for the amount of human IgG (Figure 0).

To ensure that Ad5 / 3-Ä24aCTLA4 and Ad5 / 3-aCTLA4 express functional anti-CTLA4 mAb, increased IL-2 production by stimulated Jurkat cells was examined as previously described (Lee KM et al., Science 1998 ; 282: 2263-68).

Jurkat cells (clone 6.1) were stimulated with 0.3 pg / ml ionomycin (Sigma-Aldrich Co.), 0.03 pg / ml phorbol myristyl acetate (PMA) (Sigma-Aldrich Co.) and 1 pg / ml. ml of recombinant human B7-Fc chimera (R&D systems) and treated with virus-infected PC3-MM2 cell supernatants filtered as a 0.02 µm filter (Anotop, Whatman, England). Forty-eight to eight hours after stimulation of Jurkat cells, the concentrations of interleukin-2 (IL-2) in the media were analyzed with a BD Cytometric Bead Array Human Cell Blood Protein Flex Set (Becton Dickinson) according to the manufacturer's instructions. Ten viral particles (VP) were used per cell and supernatants were collected 48 hours later. Mouse anti-human CTLA-4 (= CD152) mAb (BD Pharmingen ™, Europe) was used as a positive control. FCAP Array v.1.0.2 (Soft Flow) software was used for analysis. Figure 7 shows diagrams of functionality assays.

The anti-CTLA4 mAb binds to cell surface CTLA-4, preventing immunosuppressive interaction with recombinant B7 (rB7). This analysis indicates that the anti-CTLA4 activity of the mAb was found in supernatants of cells infected with Ad5 / 3-A24aCTLA4 and Ad5 / 3-aCTLA4 compared to the corresponding isogenic controls, Ad5 / 3-A24 and Ad5 / 3Luc1-infected cells. (Figure 2B). Recombinant anti-CTLA4 mAb was used as a positive control and was more potent than the supernatant collected from Jurkat cells.

To further verify that the virus-expressed anti-CTLA4 mAb is capable of blocking signaling via CTLA-4, a loss-of-function assay was performed. Jurkat cells were activated as above but 0.1 µg / ml recombinant human CTLA-4 / Fc chimera (R&D Systems) (rCTLA-4) was added to ionomycin, PMA and recombinant B7. rCTLA-4 binds to anti-CTLA4 mAb in culture medium, releasing cell surface CTLA-4 to interact with rB7 and to counteract T cell activation, which is seen as a decrease in IL-2 production. Loss of anti-CTLA4 mAb function was observed in the supernatant from cells infected with Ad5 / 3-A24aCTLA4 and Ad5 / 3-aCTLA4 compared to their respective isogenic unarmed controls Ad5 / 3-A24 or Ad5 / 3Luc1 (Fig. 2C). . In addition, supernatant was found to have the highest loss of function in Ad5 / 3-Ä24aCTLA4-infected tumor cells, even higher than the positive control. Taken together, these results indicate that infection of cancer cells with Ad5 / 3-A24aCTLA4 results in high expression of a functional, fully anti-human CTLA-4 mAb, leading to a decrease in T cell activity as measured by IL-2.

Example 3. Expression of CTLA-4 tumor cell lines and low growth tumor implants

Since it has been reported that almost 90% of tumor cell lines express CTLA-4 and that anti-CTLA-4 mAb could have direct antitumor activity (13), it was investigated whether this would also be true in tumor cell lines including the low-growth HNSCC tumor implants used. .

Indirect immunofluorescence was performed to analyze CTLA-4 on low-growth UT-SCC8 tumor cell culture or tumor cell lines A549, SKOV3-ip1 and PC3-MM2. Briefly, the cell pellet was incubated for 30 min at 4 ° C with mouse anti-human CTLA-4 mAb (BD Pharmingen ™, Europe) as the primary antibody followed by a further 30 min incubation at 4 ° C: Alexa Fluor® 488 asase secondary anti-mouse IgG (Invitrogen). Fluorescence intensity was measured on an LSR flow cytometer (BD Pharmingen ™, Europe). At least 40,000 cells / sample were counted. Clontech Discovery Labvvare Immunocytometry Systems (BD Pharmingen ™, Europe) and FlovvJo 7.6.1 software were used for analysis. The A549, SKOV3-ip1 and PC3-MM2 tumor cell lines expressed 99.5%, 96.6% and 96.6% CTLA-4, respectively, whereas low-growth tumor UT-SCC8 explants expressed 90.3%. CTLA-4 (Figure 2D).

Example 4. In vitro and in vivo assay for oncolytic efficacy of formed adenoviruses

The oncolytic potency (i.e., cell killing) of Ad5 / 3-A24aCTLA4 on different tumor cell lines and low growth tumor cell culture of HNSCC was evaluated. Low growth tumor cell culture or PC3-MM2, SKOV3-ip1 or A549 tumor cell culture of HNSCC was plated at 1.5 * 10 4 or 1.0 * 10 4 cells / well in 96-well plates. The next day, the viruses were diluted in DMEM containing 2% FCS at various concentrations (1, 10, 100, 1000 VP / cell), the cells were infected for one hour at 37 ° C, washed and incubated in DMEM with was 5% FCS. Cell viability was determined according to the manufacturer's protocol (Cell Titer 96 Aqueous One Solution Cell Proliferation Assay, Prome). The results are shown in Figure 3.

The oncolytic potency of Ad5 / 3-A24aCTLA4 was similar to that of the positive control virus Ad5 / 3-A24 in all cell lines. Also, replication-deficient Ad5 / 3-aCTLA4 had antitumor activity because tumor cells express CTLA4.

The oncolytic effect of Ad5 / 3-A24aCTLA4 resulted in 97.6%, 78.2%, 69.1% and 57.3% cell death by PC3-MM2, SKOV3-ip1, A549 and UT-SCC8, respectively (Figure 3). There was no statistical difference in the cytotoxicity of Ad5 / 3-A24aCTLA4 and its counterpart without transgene in E3 (Ad5 / 3-A24) in any of the tumor cell lines analyzed.

Non-replicating Ad5 / 3-aCTLA4 showed no cytotoxicity at low virus doses (Figure 3). However, cytotoxicity was observed at the highest Ad5 / 3 αCTLA4 values with maximal cell death rates of 96.2%, 74.3%, 49.1% and 56.15% for PC3-MM2, SKOV3-ip1, A549 and respectively. UT-SCC8 (Figure 3). This result is consistent with previously demonstrated direct binding of anti-CTLA-4 to CTLA expressed on the surface of cancer cells, leading to cell death (Contardi E et al., Int J Cancer 2005; 117: 538-50).

Example 5. Assay for antitumor activity of Ad5 / 3-A24aCTLA4

Adenovirus tumor growth suppression and apoptosis were determined in immune-deficient nude mice bearing human prostate cancer xenografts by treatment of mice with oncolytic adenoviruses expressing anti-CTLA-4 monoclonal antibody.

Human prostate implant xenografts were made by injecting 5x106 PC3-MM2 cells into the flanks of 5-6 weeks old female NMRI / nude mice (Taconic, Ejby, Denmark). After seven days, tumors (n = 8 / group, 5-8 mm in diameter) were injected using a volume of 50 µl three times every other day at a dose of 1x108 VP (days 0, 2 and 4) and control tumors were injected with DMEM alone. The formula (length x width2 x 0.5) was used to calculate tumor volume. On day 5, tumor cryosections were stained for anti-CTLA4 expression (human IgG) or apoptosis (active caspase-3) by immunohistochemistry. 4-5 µm cryosections of frozen tumors cast on Tissue Tek OCT (Sakura, Torrance, CA, USA) were prepared and fixed with acetone for 10 min at -20 ° C. Primary antibodies used were goat anti-human IgG (heavy and light chains) (AbD Serotec, MorphoSys) and rabbit monoclonal antibody against active caspase-3 at a dilution of 1: 200 for one hour at room temperature (BD Pharmingen Tm, AB559565). In addition, sections were incubated according to the manufacturer's instructions using the LSAB2 System-HRP kit (K0673, DakoCytomation, Carpinteria, CA, USA). Bound antibodies were visualized using 3,3'-diaminobenzidine (DAB, Sigma, St Louis, MO, USA). Finally, the sections were counterstained with hematoxylin and the water was removed in ethanol, clarified in xylene and sealed with Canadian balsam. Representative images were taken at 40 magnification using an Olympus DP50 color camera equipped with a Leica DM LB microscope. On day 7, human IgG levels in tumors and plasma of Ad5 / 3-A24aCTLA4 or Ad5 / 3-aCTLA4 treated mice were measured (Figure 4C). 81-fold more anti-CTLA4 mAb was detected in Ad5 / 3-A24aCTLA4-treated tumors compared to Ad5 / 3-aCTLA4-treated tumors (p <0.05). In addition, 43.3-fold more anti-CTLA4 mAb was detected in Ad5 / 3-A24aCTLA4-treated tumors than in the plasma of the same animals (p <0.05). The mean plasma concentration was 392.6 pg / g (SE 312.0), which is below the levels reported to be tolerated in humans treated with ipilimumab (561 pg / ml) and tremelimumab 450 pg / ml, respectively (Weber JS, et al. .J Clin Oncol 2008; 26: 5950-6; Ribas A, et al. J Clin Oncol 2005; 23: 8968-77; Tarhini AA, Iqbal F, Oncol Targets Ther 2010; 3: 15-25) (1 mL plasma weighs about 1 g). In Ad5 / 3-Ä24aCTLA4 tumors, the mAb concentration was 16,977 pg / g and thus much higher than in plasma.

Human anti-CTLA-4 mAb does not bind to murine CTLA-4 and xenograft assays require T-cell deficient nude mice. Thus, this model only measures oncolytic and proapoptotic effects. However, Ad5 / 3-Ä24aCTLA4 showed significant antitumor activity compared to mock control (p <0.01; Fig. 4A) in this aggressive subcutaneous xenograft model of prostate cancer. No other treated group showed a significant effect compared to mock control. No statistical difference (p = 0.43) was observed between Ad5 / 3-Ä24aCTLA4 and Ad5 / 3-Ä24, confirming in vitro results that expression of anti-CTLA4 mAb does not reduce viral antitumor efficacy .

Because CTLA-4 inhibiting antibodies are known to induce direct killing of tumor cells by initiating apoptosis, it was determined whether anti-tumor efficacy was associated with human anti-CTLA4 mAb production and subsequent increased apoptosis. Staining of human mAb with Ad5 / 3-A24aCTLA4 and Ad5 / 3-aCTLA4-treated tumors was observed but not with Ad5 / 3-A24 or Ad5 / 3-Luc1 (Fig. 4B). Expression of human mAb appears to correlate with increased apoptosis (Figure 4B). Thus, Ad5 / 3-Ä24aCTLA4-treated tumors express anti-CTLA-4 mAb leading to increased apoptosis.

Example 6. Immunomodulation of T Cells in Cancer Patients by Oncolytic Adenoviruses Expressing Anti-CTLA4 mAb

To extend preclinical findings to humans, PBMCs were studied in patients with advanced solid tumors that responded poorly to chemotherapy. PBMCs of cancer patients (patient I244 with chondroid melanoma, patient C26 with sputum cancer, patient M158 with mesothelioma, or patient X258 with cervical cancer) were stimulated with 0.3 pg / ml of ionomycin ( Co.), 0.03 pg / ml of phorbol myristyl acetate (PMA) (Sigma-Aldrich Co.) and 1 pg / ml of recombinant human B7-Fc chimeras (R&D Systems) to mimic tumor-induced immunosuppression. After stimulation, PBMCs were treated with 0.02 µM of PC3-MM2 cells infected with Ad5 / 3-A24aCTLA4, Ad5 / 3-A24, Ad5 / 3-aCTLA4 or Ad5 / 3-Luc1: n filter (Anotop, Whatman, England) with filtered supernatants.

In the Loss of function assay, 0.1 pg / ml recombinant human CTLA-4 / Fc chimeras (R&D Systems) was added to ionomycin, PMA and recombinant B7. Twenty-four hours after PBMC stimulation, concentrations of interleukin-2 (IL-2) or interferon-γ (IFN-γ) in media were analyzed with BD Cytometric Bead Array Human Soluble Protein Flex Set (Becton Dickinson) according to the manufacturer's instructions. Ten viral particles (VP) were used per cell and supernatants were harvested 48 hours later. A mouse monoclonal anti-human CTI_A-4 (= CD152) antibody (BD Pharmingen ™, Europe) was used as a positive control. FCAP Array v. 1.0.2 (Soft Flow) software was used for analysis.

In all four patient samples, the supernatant from oncolytic adenoviruses expressing anti-CTLA4 mAb was able to increase T cell activity as measured by IL-2 and interferon gamma (Figure 5). The principle of the assay is shown in Figure 7. Interestingly, although recombinant mAb was more potent in Jurkat cells, an immortalized T cell line, virus supernatants were more potent in patient samples. Similar results were obtained in the loss of function assay (Figure 8), the principle in Figures 7E-F.

Example 7. Effect of anti-CTLA4 mAb on PBMCs from healthy individuals.

The experiments described in Example 6 were also performed using PBMCs from healthy individuals. In contrast to cancer patients, supernatant from cells infected with Ad5 / 3-A24aCTLA4 did not increase the production of IL-2 or interferon-gamma. No significant changes were seen in the loss of function assay either. However, since the positive control, the recombinant mAb, was effective in both cases (increased and decreased IL-2 and gamma interferon in Figure 6; increased IL-2 p <0.05 and p = 0.206, increased INF- γ p <0.05 and p = 0.120, respectively, in donor 1 and donor 2 in Figure 6; decreased IL-2 p <0.001 and p <0.05, and decreased INF-γ p <0.001 and p <0.05, respectively. donor 1 and donor 2 in Figure 8; compared to rB7, rCTLA4. lonomycin and PMA only treated cells), this was assumed to be a dose effect. This is supported by the non-significant trend seen in Ad5 / 3-A24aCTLA4 in the loss-of-function assay (Fig. 8) and, in many cases, by a significant effect in the supernatant in Ad5 / 3-aCTLA4-infected cells (Figs. 6 and 8). However, the effect of anti-CTLA-4 mAb is significant in cancer patients, perhaps because they have a higher degree of ongoing immunosuppressive processes due to the presence of advanced tumor.

Example 8: Utility of a replicable template to increase expression of anti-CTLA4 mAb

The use of a replicable template to increase expression of anti-CTLA4 mAb compared to non replication-free virus was analyzed. PC3-MM2 cells were plated at a concentration of 20,000 cells per well and infected at 10 VP per cell with Ad5 / 3-A24aCTLA4 and Ad5 / 3-aCTLA4, respectively. At 24 h, 48 h and 72 h after infection, supernatants were harvested and analyzed by ELISA for the amount of human IgG. A threefold increase was observed with the oncolytic Ad5 / 3-A24aCTLA4 compared to the non-replicating Ad5 / 3-aCTLA4. Ad5 / 3-aCTLA4 (Figure 10).

One benefit of the oncolytic model is the higher level of transgene expression obtained. After expression of early genes, the viral genome is amplified and up to 10,000 copies of viral DNA are produced. This results in many more copies that can produce the transgene (Figure 10).

Example 9: Oncolytic adenoviral vectors with enhanced immune response

To further increase the immune response, oncolytic adeno virus containing TLR-9 (toll-like receptor 9) stimulating CpG molecules in the viral backbone (Figure 1) was studied using a mouse model of lung cancer xenograft. Hairless NMRI mice (5 mice per group, two tumors per mouse) were implanted with A549 cells and treated with 1x108 VP of CpG-rich oncolyte adenovirus Ad5-A24CpG, oncolytic adenovirus containing A24 deletion, oncolytic adenovirus containing recombinant oligos (ODN 2395, InvivoGen, USA). Tumor growth was measured every two days for 12 days as previously described. The CpG-rich virus Ad5-A24CpG was most effective in transmitting anti-tumor immunity (Figure 11).

Spleen cells harvested from the same mice were stimulated with UV-inactivated virus and co-cultured in a ratio of 1: 1 and 10: 1 with A549 cells. The MTS cell kill assay was performed at 72 hours. The percentage of A549 cells still alive at the given time is given. The result shown in Figure 12 shows that the CpG-modified virus was able to stimulate antigen-presenting cells and this resulted in an increased anti-tumor immune response. The response was so strong that it could be seen even in nude mice lacking T cells. Thus, even better results are expected in immunocompetent animals and humans. The use of TLR-9 stimulation (which induces anti-tumor immunity) is likely to be most significant with concomitant suppression of suppressive signals by aCTLA-4.

Example 10. Safety and Efficacy of Oncolytic Adenovirus Vectors Containing Anti-CTLA-4 Monoclonal Antibodies in Human Cancer 1. Patients

Patients with advanced and difficult-to-treat solid tumors are invited to participate in the Advanced Therapy Access Program (ATAP) approved by the Finnish Center for Pharmaceutical Safety and Development (FIMEA).

Patients are selected from cancer patients with advanced solid tumors that did not respond well to standard treatments. Inclusion criteria were solid tumors that responded poorly to conventional treatments, a WHO performance score of 3 or less, and no major organ dysfunction. The criteria for rejection were transplantation, HIV, severe cardiovascular, metabolic or pulmonary disease, or other symptoms, findings, or diseases that prevented oncolytic viral therapy. Written informed consent was obtained and treatments were administered in accordance with good clinical practice and the Helsinki Declaration. The treatments are administered intravenously, intravenously or intraperitoneally, as appropriate. II. Treatments with an adenoviral vector encoding aCTLA-4 mAb

Oncolytic adenoviruses are produced according to clinical grade and patient treatment is initiated.

Treatment is given by intravenous injection. The total number of treatments is three to three weeks. The doses of the virus are selected based on the inventors' prior knowledge of other oncolytic viruses. However, other administration programs may be used as appropriate. For example, in the first round of serial therapy, patients receive a portion of the dose, e.g., four-fifths or two-fifths of the dose intravenously or intraperitoneally, and the remainder of the dose intravenously.

The virus is diluted in sterile saline at the time of administration under appropriate conditions. After the virus is administered, all patients are monitored overnight in hospital and then as outpatient patients. Physical examination and medical report were performed at each visit and clinically relevant laboratory values were monitored.

Treatment side effects are recorded and scored according to CTCAE (Common Terminology Criteria for Adverse Events) version 3.0. Because many cancer patients have symptoms due to the disease, the symptoms that occur earlier are not numbered unless they worsen. However, if the symptom became more severe, for example, if pre-treatment grade 1 changed to grade 2 after treatment, it will be numbered as grade 2.

Tumor size is determined by contrast-enhanced computed tomography (CT) scanning. Maximum tumor diameters are obtained. The Response Evaluation Criteria in Solid Tumors (RECIST1.1) applies to the entire disease, including injected and non-injected lesions. These criteria are: partial response PR (> 30% reduction in sum of tumor diameters), stable disease SD (no reduction / increase), progressive disease PD (> 20% increase). The number of non-PR-related tumor suppressors is assigned a number as low response (MR). Serum brand names were also determined as they rose from baseline, and the same percentages were used.

Patient serum samples are analyzed for immunological and virological parameters such as viral copy number over time in blood, induction of antiviral neutralizing antibodies, changes in antiviral and antitumor T cells, other immunological cell types, and changes in antitumor antibodies. In addition, adverse reactions are graded according to Common Terminology for Adverse Events v3.0 (CTCAE), neutralizing antibody titers are measured, and efficacy is evaluated using RECIST criteria for computed tomography (CT) (Therasse P et al. 2000, J Natl Cancer Inst 92 , 205-16PERCIST Criteria (Wahl et al. 2009 J Nucl Med 50 Suppl 1: 122S-50S) for positron emission computed tomography (PET-CT) All patients had progressive tumors prior to treatment and are in different states of the disease.

Claims (34)

An oncolytic adenoviral vector, characterized in that it comprises an adenovirus serotype 5 (Ad5) nucleic acid backbone comprising a capsid modification, 2) a 24 base pair deletion (D24) in the Rb binding region of E1 and 3 nucleic acid sequences, CTLA-4-specific fully human monoclonal antibody (aCTLA-4 mAb), in place of the deleted gp19k / 6.7K in the E3 region. The oncolytic adenoviral vector of claim 1, further comprising one or more regions selected from the group consisting of E2, E4 and late regions. The oncolytic adenoviral vector according to claim 1 or 2, characterized in that it comprises the following regions: left ITR, partial E1, p1x, p1Va2, E2, VA1, VA2, L1, L2, L3, L4, partial E3, L5, E4 and right ITR. The oncolytic adenoviral vector according to claim 3, characterized in that the regions are in a sequential order in the 5'-3'-direction. The oncolytic adenoviral vector according to any one of the preceding claims, characterized in that a wild-type region is located upstream of the E1 region. The oncolytic adenoviral vector according to any one of the preceding claims, characterized in that the E1 region comprises a viral packaging signal. The oncolytic adenoviral vector according to any one of the preceding claims, characterized in that the nucleic acid sequence encoding the aCTLA-4 mAb is under the control of the viral E3 promoter. An oncolytic adenoviral vector according to any one of the preceding claims, characterized in that it further comprises a nucleic acid sequence encoding a tumor-specific human telomerase reverse transcriptase (hTERT) promoter or E2F promoter upstream of the E1 region. The oncolytic adenoviral vector according to any one of the preceding claims, characterized in that it further comprises a CpG site in the viral backbone. The oncolytic adenoviral vector according to claim 9, characterized in that the CpG site is in the E3 region. The oncolytic adenoviral vector according to any one of the preceding claims, characterized in that the E4 region is of the wild type. The oncolytic adenoviral vector according to any one of the preceding claims, characterized in that the capsid modification is Ad5 / 3-Chimerism, insertion of the integrin binding region (RGD) and / or modification of the heparin sulfate binding polylysine into the strand. The oncolytic adenoviral vector according to claim 12, characterized in that the capsid modification is Ad5 / 3 chimerism. The oncolytic adenoviral vector according to claim 12, characterized in that the capsid modification is an RGD-4C modification. The oncolytic adenoviral vector according to any one of the preceding claims, characterized in that it comprises at least one Expression cassette. The oncolytic adenoviral vector according to any one of the preceding claims, characterized in that it comprises only one expression cassette. The oncolytic adenoviral vector according to any one of the preceding claims, characterized in that the vector is capable of selectively replicating in cells defective in the Rb / p16 pathway. The oncolytic adenoviral vector according to any one of the preceding claims, characterized in that the vector is capable of selectively replicating in cells expressing telomerase. An in vitro cell, characterized in that it comprises an adenoviral vector according to any one of claims 1 to 18. Pharmaceutical composition, characterized in that it comprises an adenoviral vector according to any one of claims 1 to 18. The oncolytic adenoviral vector according to any one of claims 1 to 18 or the pharmaceutical composition of claim 20, characterized in that it functions as an in situ cancer vaccine. An adenoviral vector according to any one of claims 1 to 18 for use in treating cancer in a subject. A pharmaceutical composition according to claim 20 for use in treating cancer in a subject. The adenoviral vector of any one of claims 1 to 18 or the pharmaceutical composition of claim 20 for use in treating cancer in a subject, wherein the cancer is selected from the group consisting of nasopharyngeal cancer, synovial cancer, hepatocellular cancer, kidney cancer, connective tissue cancer, melanoma, lung cancer , colon cancer, rectal cancer, colorectal cancer, brain cancer, throat cancer, mouth cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, biliary cancer, bladder cancer, ureteral cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, osteochondromoma, chondrosarcoma, Evving's sarcoma, carcinoma of unknown location, carcinoma oid, fibro-sarcoma, breast cancer, Paget's disease, cervical cancer, esophageal cancer, gallbladder cancer, head cancer, eye cancer, neck cancer, kidney cancer, Wilms' tumor, Kaposi's sarcoma, prostate cancer, testicular cancer, Hodgkin's disease, Hodgkin's disease , mesothelioma, multiple myeloma, ovarian cancer, endocrine pancreas, glucagonoma, paratyroidinen cancer, penile cancer, aivolisäkesyöpä, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymic cancer, thyroid cancer, Trofo-blastisyöpä, hydatidiform mole, uterine cancer, endometrial cancer, vulval cancer, cancer, ulkosynnytinsyöpä, auditory nerve tumors , mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gingival cancer, heart cancer, lip cancer, membrane cancer, oral cancer, nerve cancer, cystic carcinoma, parathyroid cancer, cancer of the glands, glandular carcinoma, pleural cancer, pleural cancer. The adenoviral vector or pharmaceutical composition of claim 24 for use in treating cancer in a subject, characterized in that the subject is a human or animal. The adenoviral vector or pharmaceutical composition for use in the treatment of cancer according to claim 24 or 25, characterized in that the adenoviral vectors or pharmaceutical compositions are administered intravenously, intramuscularly, intravenously, intravenously, intraperitoneally or intracellularly, intracellularly or intracellularly. An adenoviral vector or pharmaceutical composition for use in the treatment of cancer according to any one of claims 24 to 26, characterized in that the oncolytic adenoviral vectors or pharmaceutical compositions are administered several times during a treatment period. The adenoviral vector or pharmaceutical composition of any one of claims 24 to 27 for use in treating cancer in a subject, characterized in that the subject is administered an oncolytic adenoviral vector having a different capsular filament protrusion than the vector used in previous treatment. The adenoviral vector or pharmaceutical composition for use in the treatment of cancer according to any one of claims 24 to 28, characterized in that the adenoviral vectors or pharmaceutical compositions are administered to the subject in parallel with radiation therapy or in parallel with chemotherapy or in parallel with other cancer therapies. The adenoviral vector or pharmaceutical composition for use in the treatment of cancer according to any one of claims 24 to 29, characterized in that the adenoviral vectors or pharmaceutical compositions are administered to the subject with an adjuvant selected from the group consisting of verapamil or another calcium channel blocker; an autophagy inducing agent; temozolomide; a substance capable of reducing regulatory T cells; cyclophosphamide and any combination thereof in the subject. The adenoviral vector or pharmaceutical composition for use in the treatment of cancer according to any one of claims 24 to 30, characterized in that the adenoviral vectors or pharmaceutical compositions are administered in combination with chemotherapy or anti-CD20 treatment or other neutralizing antibody blocking treatments. An in vitro method for producing a fully human monoclonal antibody to CTLA-4 in a cell, characterized in that the method comprises: a) delivering a vehicle comprising an oncolytic adenovirus vector according to any one of claims 1 to 18 and b) expressing the CTLA of said vector. -4-specific fully human monoclonal antibody in a cell. Use of an oncolytic adenoviral vector according to any one of claims 1 to 18 for the production of an anti-CTLA-4 mAb in a cell in vitro. The oncolytic adenovirus vector according to any one of claims 1 to 18 for use in enhancing a tumor-specific immune response and apoptosis in a subject.