Modified adenoviruses for cancer vaccine development

Abstract

To provide a new and potent customizable immunotherapy (e.g., cancer immunovirotherapy) platform.SOLUTION: A method of stimulating a peptide-specific immune response in a subject in need thereof comprises administering, to the subject, adenoviral vectors comprising polypeptides attached onto a viral capsid, where the polypeptides have not been genetically encoded by the adenoviral vectors.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an adenoviral vector, the viral capsid of which is coated with a polypeptide capable of stimulating a peptide-specific immune response in a subject, and its use.Furthermore, the present invention relates to a method for treating a disease, such as cancer, by an adenoviral vector coated with a polypeptide that induces a peptide-specific immune response.Furthermore, the present invention relates to a method for coating an adenoviral vector with a specific peptide, and a method for identifying a peptide suitable for coating the capsid of an adenoviral vector. [Background technology]

[0002] Cancer is a fatal disease that requires more effective treatment. Oncolytic viruses that cause tumor destruction have attracted much interest because they are potentially safer and more effective than other standard therapies. However, the overall therapeutic effect in cancer patients has been modest. There have been many studies on modifying adenovirus vectors to find the optimal tool for treatment. One aspect of modulating the function of adenovirus is to modify the surface of the virus. Both genetic and non-genetic modifications of the surface of adenovirus are well known.

[0003] For example, Stevenson M et al. (Cancer Gene Therapy (2007) 14, 335-345) look at enhancing the delivery of adenoviral vectors to target sites. Stevenson et al. describe a study in which adenoviral vectors are targeted to infect cells via integrins selectively expressed on metastatic tumor cells. To this end, a laminin-derived peptide (-SIKVAV-) was incorporated onto the surface of the polymer-coated virus.

[0004] WO 2013 / 116778 describes an immunologically enhanced adenovirus for cancer. The adenovirus was modified by inserting a tumor antigen transgene into its genome so that the tumor antigen is expressed during the viral replication cycle and presented directly to MHC-1. This method requires the generation of a new virus for every different tumor antigen (e.g., a new virus must be cloned for every peptide you want to express), making personalized treatment very time-consuming, labor-intensive, and expensive.

[0005] Indeed, there is a need for simple and improved adenoviral tools and methods for therapy, especially personalized therapy. The present invention provides an adenoviral application for inducing an immune response in a subject using a virus as a delivery system for peptides, but without genetic manipulation of the virus.

[0006] The present invention relates to the use of oncolytic adenoviruses as a platform to deliver patient- and disease-specific peptides, resulting in the conversion of anti-capsid immunity to a peptide-specific immune response (eg, anti-tumor immunity). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Stevenson M et al., Cancer Gene Therapy (2007) 14, 335-345 [Patent documents]

[0008] [Patent Document 1] International Publication No. 2013 / 116778 Summary of the Invention

[0009] The present invention provides a novel and powerful customizable immunotherapy (e.g., cancer immunoviral therapy) platform. It provides adenoviral vectors with modified viral surfaces, their uses, and methods for treating diseases by stimulating peptide-specific (i.e., anti-peptide) immune responses, aiming to solve problems such as inefficient, time-consuming, expensive and labor-intensive adenoviral therapy and the incompatibility of adenoviral therapy with personalized medicine. The object of the present invention is achieved by an apparatus and a method, which are characterized by what is stated in the independent claims. Preferred embodiments of the present invention are disclosed in the dependent claims.

[0010] The present invention overcomes the problems of the prior art, such as the lack of specificity and immunodominance of oncolytic adenoviruses.

[0011] The immune response generated by adenovirus infection is mainly directed against the virus and not against the tumor. Moreover, the majority of viral immunity is directed against the capsid proteins. The present invention overcomes these problems. In fact, the present invention is based on the idea of ​​diverting viral immunity to tumors by coating the viral capsid with peptides derived from tumor proteins (Figure 3). Major histocompatibility complex I (MHC-1) restricted peptides carried on the oncolytic adenovirus capsid diverte capsid immunity to antitumor immunity.

[0012] Simply, if the peptide and virus are administered as a single physically linked entity, both the danger signal (virus) and the tumor antigen (peptide) will enter the same antigen-presenting cells for maximum antitumor efficacy. Clinical experience has already shown that peptide vaccination alone leads to transient and suboptimal immune responses that fail to control tumor growth. 1Also, although oncolytic viruses show promise as monotherapy, the immune response they induce is primarily targeted against the virus and not against the tumor. Even if peptide and virus are injected into the same anatomical location, they do not combine in a single therapeutic entity and therefore inefficiently enter the same cells. This is a critical aspect to achieve adequate and maximal immune activation. 2 The physical association of peptides and adenoviruses in a single therapeutic entity is a significant improvement over existing viral and peptide cancer vaccine technologies. In contrast to the prior art recombinant viruses engineered to express one tumor-associated antigen or peptide, the present invention makes it possible to achieve personalized medicines in a much faster and more cost-effective manner than before. Indeed, according to the present invention, the peptide attached on the viral capsid is not encoded by the adenoviral vector.

[0013] One aspect of the present invention is a technique that allows constant and rapid monitoring of tumor antigen presentation as small peptides (MHC-I restricted). The present invention utilizes disease (e.g., tumor)-specific and patient-specific peptides that are simultaneously presented to tumor cells (i.e., unmasked or edited after treatment) and dendritic cells (DCs) before and after adenoviral therapy. After identifying these specific peptides, they can be synthesized and loaded onto oncolytic adenoviral capsids to achieve high antitumor immunity. In this way, it is possible to ensure that tumors are effectively targeted by cytotoxic T cells (CTLs) even after virotherapy, and immune escape is not possible as the immune system targets the virus. Conversely, by comparing peptides that appear on DCs after virotherapy in the presence or absence of tumor, it is possible to exclude "virus-only" peptides and find peptides derived from tumor cells that induce CTL responses.

[0014] A personalized coated adenovirus can be obtained from a biopsy in just 2 weeks. This is possible because isolation and sequencing of the peptides from the MHC as well as automated synthesis is a rapid process and allows a large stockpile of virus (e.g., the same backbone virus for all peptides) for coating. The coating itself takes place in 1 hour, after which the coated adenovirus is ready for injection. This is a very unique feature of our system, since it avoids genetic manipulation of the virus, which would slow down the process making a "personalized vaccine approach" impossible.

[0015] The present invention also makes it possible to discover novel immunogenic tumor-specific peptides.

[0016] In addition to cancer therapy, the enveloped adenoviruses of the present invention can be used to treat other diseases in situations where a higher peptide-specific immune response is required.

[0017] The present invention relates to a method of stimulating a peptide-specific immune response in a subject in need thereof, comprising administering to the subject an adenoviral vector comprising a polypeptide attached on the viral capsid.The present invention also relates to a method of stimulating a peptide-specific immune response in a subject in need thereof, comprising administering to the subject an adenoviral vector comprising a polypeptide attached on the viral capsid, wherein said polypeptide is not genetically encoded by said adenoviral vector.

[0018] The invention further relates to an adenoviral vector comprising a polypeptide attached on the viral capsid for use in stimulating a peptide-specific immune response in a subject.The invention also relates to an adenoviral vector comprising a polypeptide attached on the viral capsid for use in stimulating a peptide-specific immune response in a subject, wherein the polypeptide is not genetically encoded by the adenoviral vector.

[0019] The present invention further relates to a method for treating cancer in a subject in need thereof, comprising administering to the subject an adenoviral vector capable of stimulating a peptide-specific immune response in the subject, the polypeptide being attached on the viral capsid.The present invention also relates to a method for treating cancer in a subject in need thereof, comprising administering to the subject an adenoviral vector capable of stimulating a peptide-specific immune response in the subject, the polypeptide being attached on the viral capsid, the polypeptide being not genetically encoded by said adenoviral vector.

[0020] The present invention also relates to an adenoviral vector comprising a polypeptide attached on the viral capsid, capable of stimulating a peptide-specific immune response in a subject, for use in treating cancer in a subject.The present invention also relates to an adenoviral vector comprising a polypeptide attached on the viral capsid, capable of stimulating a peptide-specific immune response in a subject, for use in treating cancer in a subject, wherein the polypeptide is not genetically encoded by said adenoviral vector.

[0021] Furthermore, the present invention relates to an adenoviral vector, the viral capsid of which is attached to a polypeptide, wherein the adenoviral vector attached to the polypeptide is capable of stimulating a peptide-specific immune response in a subject.

[0022] Furthermore, the present invention relates to a method for coating an adenovirus capsid, comprising covalently or non-covalently linking to the adenovirus capsid a polypeptide capable of stimulating a peptide-specific immune response in a subject.The present invention also relates to a method for modifying an adenovirus capsid, comprising covalently or non-covalently linking to the adenovirus capsid a polylysine-modified polypeptide, wherein the modified adenovirus vector is capable of stimulating a peptide-specific immune response in a subject.

[0023] Furthermore, the present invention relates to the use of polypeptides (e.g., polylysine-modified polypeptides) capable of stimulating a peptide-specific immune response in a subject to coat the capsid of an adenovirus by covalently or non-covalently attaching or linking the polypeptide to the capsid.

[0024] The adenoviral vectors and methods of the invention are used to convert anti-viral immunity to anti-peptide immunity. The modified viral vectors of the invention induce an anti-peptide response in a subject.

[0025] Furthermore, the present invention relates to a pharmaceutical composition comprising the adenoviral vector of the present invention.

[0026] The present invention further provides a method for identifying tumor-specific and MHC-I-specific polypeptides from a subject, comprising the steps of: i) infecting tumor cells of a subject with an adenoviral vector; ii) infecting dendritic cells of the subject with an adenoviral vector; iii) isolating MHC-I molecules from the tumor cells of step i) and from the dendritic cells of step ii) and identifying MHC-I associated polypeptides from both groups; iv) isolating MHC-I molecules from uninfected tumor cells and identifying MHC-I associated polypeptides; v) identifying the polypeptides presented by the infected and non-infected tumors of steps iii) and iv) and the dendritic cells of step iii); The present invention relates to a method comprising the steps of:

[0027] In the following, the invention will be explained in more detail by means of preferred embodiments with reference to the accompanying drawings. [Brief description of the drawings]

[0028] [Figure 1] Schematic diagram of the present invention. Modified adenoviruses are capable of replicating and killing cancer cells while redirecting the anti-viral immune response against tumors. [Diagram 2] Immunodominance of anti-adenovirus (left bar) versus tumor (right bar) responses is shown. Mice) C57BL / 6 mice bearing B16-OVA tumors were treated with PBS (mock), Ad5D24 (unmodified oncolytic virus) and (Ad5D24-CpG, a highly immunogenic oncolytic virus). T-cells from tumors were harvested and IFN-gamma ELISPOT was performed to assess anti-tumor and anti-adenovirus responses. Cancer patients) IFN-gamma ELISPOT was performed on PBMCs from patients treated with GMCSF armed oncolytic adenovirus (Ad5D24-GMCSF)15. PBMCs were stimulated with Ad5-derived peptides (anti-viral) and survivin-derived peptides (anti-tumor) prior to ELISPOT. [Figure 3A]The coated adenovirus of the present invention has advantages over existing technologies. A) Oncolytic adenovirus has the ability to trigger APCs, presenting not only viral antigens (resulting in an antiviral response) (another antigen presented on the cells in figure A) but also tumor antigens (another antigen presented on the cells in figure A) that lead to antitumor immunity as a side effect. Antitumor T cells are represented as the two lowest cells of the T cell group. B) The coated adenovirus of the present invention, because its capsid is covered by MHC-I-available tumor-specific antigens (peptides), prefers tumor antigen presentation (any antigen presented on the cells in figure B). In this way, anticapsid immunity can be converted into antitumor immunity. Antitumor T cells are represented as the four lowest cells of the T cell group. As used herein, APC refers to antigen-presenting cells, TAA refers to tumor-associated antigens, and "PRR activation" refers to pattern recognition receptor activation. PRRs are proteins expressed by cells of the innate immune system to identify pathogen-associated molecular patterns associated with, for example, microbial pathogens. [Figure 3B] Same as above. [Figure 4] Figure 1 shows the upregulated bio-functional network of dendritic cells exposed to oncolytic adenovirus. Human primary dendritic cells were harvested and cultured with IL4 and GMCSF for 2 weeks. Cells were pulsed with oncolytic adenovirus (Ad5D24) at 10VP / cell. After 72 hours, total RNA was collected and analyzed on an Agilent SurePrint G3 human 8x60k (mRNA). Data was then analyzed using Ingenuity Pathway software. [Diagram 5]A schematic diagram depicting the discovery of novel immunogenic tumor-associated MHCI-restricted peptides is shown. The different conditions allow matching tumor-expressed peptides with the same tumor peptides presented by dendritic cells. This is a key feature in the system to facilitate the identification of immunogenic peptides. A) Dendritic cells were pulsed with tumor oncolysate to present tumor antigens. B) Unpulsed dendritic cells were matured and analyzed. This serves as a control for the later removal of self-peptides presented by DCs. C) An infected tumor cell line (same as condition A) was infected with an oncolytic adenovirus and analyzed before complete lysis (less than 48 hours). This condition helps to distinguish whether the adenovirus has a significant effect on the quality of the presented tumor antigen. D) This is an uninfected tumor presenting tumor antigens and self-peptides (of course these two can be the same) on MHCI. [Figure 6] A schematic diagram of OVA-specific coated virus is shown. A) In this case, since all processed peptides of chicken ovalbumin (OVA) are known, the virus was coated with an OVA-specific immunogenic peptide (SIINFEKL) (SEQ ID NO: 1). Then, other coated viruses such as SIINFDL (SEQ ID NO: 2) (antagonist) and FILKSINE (SEQ ID NO: 3) (scrambled) as well as non-coated viruses were generated to be used as controls. B) Once the proof of concept was demonstrated, we started to study II generation adenoviruses coated with different peptides. (PeptiCRAd refers to peptide-coated oncolytic adenovirus.) [Figure 7] FIG. 1 shows a schematic diagram depicting three different strategies for generating peptide-coated oncolytic adenoviruses. [Figure 8]Figure 8A shows the complex formation between oncolytic adenovirus and tumor-specific peptide and the interaction between modified epitopes and oncolytic adenovirus. Figure 8B shows the complex formation between Ad5D24 oncolytic adenovirus and tumor-specific peptide. "Z-potential" curve) 1x1010 viral particles were combined with different concentrations of positively charged tumor-specific peptide. After the reaction, the Z-potential of single particles was measured. "Size" curve) 1x1010 viral particles were combined with different concentrations of positively charged tumor-specific peptide. The size of single particles was then measured and reported as a function of peptide concentration. When the Z-potential is from -20mV to +20mV, there is a dramatic change in the size of the complex, showing high polydispersity (high probability of virus aggregation), but this state returns to normal at high concentrations of peptide, and the complex (PeptiCRAd) is completely coated and has no possibility of forming dipoles that promote the formation of aggregates (high polydispersity). FIG. 8B shows the interaction between the modified MHC-I epitope SIINFEKL and oncolytic adenovirus. The virus / peptide interaction was measured by SPR. APTES silica SiO2 sensor was coated with Ad5D24 and different concentrations (0.15, 0.3, 1.6, 2.2, 2.4 and 7.2 μM) of SIINFEKL (dotted line) or polyK-SIINFEKL (solid line) were injected into the flow system. The SPR signal response is shown in relation to the duration of the experiment. [Figure 9] This shows that the coated adenovirus Ad5D24 (PeptiCRAd) of the present invention exhibits enhanced cell killing activity compared to non-coated oncolytic viruses. A representative cell viability assay (MTS assay) was performed on a lung cancer adenocarcinoma cell line (A549). Cells were seeded on day 0 and infected at the indicated infection efficiency on day 1, and the test was stopped and analyzed on day 3. [Figure 10A]Figure 1 shows that OVA-specific adenovirus enhances OVA-specific immunity. Mice bearing subcutaneous B16-OVA tumors were injected intratumorally with PBS, oncolytic virus (Ad5D24), oncolytic virus + SIINFEKL peptide (unconjugated), or oncolytic virus + SIINFEKL (complexed as monoclonal, PeptiCRAd). A) Tumor growth was measured and recorded at the indicated time points. B) SIINFEKL-specific immunity was assessed by flow cytometry (pentamer analysis). [Figure 10B] Same as above. [Figure 11] The consistency of peptide coating technology is shown. The figure shows the net charge of two different oncolytic adenoviruses coated with modified peptide (6K-SIINFEKL). The two viruses used in this example are Ad5D24-CpG (an oncolytic adenovirus genetically modified to be rich in CpG islands) and Ad5D24-RFP, an oncolytic adenovirus that encodes red fluorescent protein to facilitate in vitro and in vivo imaging (RFP stands for red fluorescent protein). [Figure 12] The net charge of PeptiCRAd correlates with its size. In this example, we started with naked virus (net charge of approximately -25-30mV) and added increasing concentrations of peptide so that a complex formed, which we called PeptiCRAd. As more and more peptide was added, the net charge of the virus also changed from negative to positive values, and finally, when the complex PeptiCRAd formed, the net charge of the peptide-coated virus was approximately +30-35mV. [Figure 13]Cross-presentation of modified SIINFEKL analogs on MHC-I with or without adsorption onto viral capsids is shown. Spleens were collected from C57BL / 6 mice (H-2Kb) and single cell suspensions were prepared in RPMI-1640 growth medium containing 10% FBS. (A) A total of 2 × 106 splenocytes were incubated in 200 μl of medium containing unmodified SIINFEKL (positive control), aminocaproic acid-containing SIINFEKL-AHX-polyK (negative control), C-terminally extended SIINFEKL-polyK, or N-terminally extended polyK-SIINFEKL (0.19 μg / μl). After 2 h of incubation at 37 °C, cells were washed and stained with APC anti-H-2Kb conjugated to SIINFEKL or isotype control. (B) Fresh murine splenocytes were infected with OVA-PeptiCRAd (100 vp / cell + 37.5 μg peptide) and 37.5 μg SIINFEKL (positive control) or polyK-SIINFEKL as in (A). After 2 h of incubation, samples were washed and analyzed by flow cytometry. Data are shown as mean ± SEM (n = 2). Significance was assessed using one-way ANOVA with Bonferroni's multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001. [Figure 14]Figure 1 shows that PeptiCRAd retains intact oncolytic activity and displays increased infectivity in low CAR-expressing cell lines. (A) Cells were seeded at a density of 1x104 cells per well and infected with OVA-PeptiCRAd or naked Ad5D24 using different vp / cell ratios (0.1, 1, 10 and 100). PolyK-SIINFEKL peptide (dotted line, circle) was included as a control. Cell viability was then determined by MTS assay. Data are shown as mean ± SEM (n=3). (B) Viral infection studies by ICC. A total of 2x105 cells / well were seeded in 24-well plates and infected the next day with 100 μl of virus dilution (10 vp / cell) containing either OVA-PeptiCRAd or Ad5D24 (control). After 2 days of incubation, anti-hexon ICC was performed and 5 non-overlapping images were captured using a digital microscope. The average number of spots per field is shown. Data from a representative experiment are shown as mean ± SEM (n = 2–3). Significance was assessed using unpaired t-test with Welch's correction. *P<0.05, **P<0.01, ***P<0.001. [Figure 15-1]Antitumor effects of PeptiCRAd and immunological analysis of antigen-specific CD8+ T cells and DCs. (A) C57BL / 6 mice (n=6) were administered 3×105 B16-OVA cells in both flanks. Treatment began 9 days later and included saline solution (mock), peptide alone (SIINFEKL), virus alone (Ad5D24-CpG), a mixture of virus and peptide (Ad5D24-CpG+SIINFEKL) and virus-peptide complex (OVA-PeptiCRAd). Mice were treated three times (days 0, 2 and 7). Tumor size was then measured and expressed as mean ± SEM as a function of time. Statistical analysis was performed using two-way ANOVA with Bonferroni's multiple comparison test. *P<0.05, **P<0.01, ***P<0.001. Tumors, spleens and inguinal lymph nodes were harvested from mice (n=3–4) at two time points: day 7 (early) (B) and day 16 (late) (C). The percentage of SIINFEKL-specific CD8+ T cells was determined by gating out CD19+ cells. The percentage of CD8+OVA+ T cells is shown as mean ± SEM. (D) The mean tumor size at the end of the experiment (linear y-axis) was plotted against the mean percentage of double positive CD8+OVA+ T cells (log10X-axis). Pearson's r and r2 values ​​were also calculated and graphed for each set of samples. (E) The maturation profile and fold change in DCs exhibiting cross-presenting SIINFEKL on MHC-I molecules were determined. Mature DCs were defined as CD19-CD3-CD11c+CD86high cells. APC anti-mouse H-2Kb bound to SIINFEKL was used to follow the cross-presentation of SIINFEKL to MHC-I in selected pools of DC. [Figure 15-2] Same as above. [Figure 15-3] Same as above. [Figure 16-1]We show that targeting two tumor antigens with PeptiCRAd reduces the growth of both treated and distant untreated tumors. One primary tumor was implanted into the right flank of C57BL / 6 mice with 1x105 B16-F10 melanoma cells. Treatment began on day 10. On day 16, mice received 3x105 B16-F10 cells in the left flank. (A) Growth of primary (right) tumors was recorded and data are shown as mean ± SEM (n=5). Significance was determined using two-way ANOVA with Bonferroni's multiple comparison test. *P<0.05, **P<0.01, ***P<0.001. (B) Size of secondary (left) tumors at the end of the experiment is recorded on a log2 scale. Significance was assessed using the Mann-Whitney U test. *P<0.05, **P<0.01, ***P<0.001. (C) Spleens and inguinal lymph nodes were harvested and the levels of TRP-2- and hgp100-specific CD8+ T cells were measured in each organ by MHC-I pentamer staining. The percentage of epitope-specific CD8+ T cells found in each organ was normalized to mock and is expressed as the cumulative relative response for each experimental group. [Figure 16-2] Same as above. [Figure 17-1]Efficacy of PeptiCRAd in humanized mice bearing human melanoma. Triple knockout NGS mice were administered 2x106 human melanoma cells (SK-MEL-2) into each flank. When tumors reached a mean diameter of 4-5 mm, one group of mice (n=3) was administered human PBMCs from HLA-A-matched healthy donors and another group of mice (n=2) was not administered PBMCs. Mice were then treated with one of the following: i) saline (mock), ii) Ad5D24-GM-CSF, and iii) MAGE-A1 PeptiCRAd (days 0, 2, and 4). Tumor volumes in humanized mice (A) are shown as mean ± SEM. Significance was assessed using two-way ANOVA with Bonferroni's multiple comparison test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. (B) Area under the curve (AUC) versus tumor size for each group of humanized mice is shown. (C) Tumor volumes in non-humanized mice are recorded as mean ± SEM (****P<0.0001). [Figure 17-2] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Detailed Description of the Invention Tumor Immunology and Immunopeptidome Dendritic cells (DCs) are bone marrow-derived professional antigen-presenting cells. DCs are the antigen-presenting cells of choice for presenting tumor antigen epitopes to CD8+ and CD4+ T cells. 3 Exogenous antigens can be loaded onto MHC class I for "cross-presentation" to CD8+ T cells. 4 Cross-presentation is a phenomenon whose outcome is determined by the activation state of DCs. 5In cancer cells, the degree of DC maturation leading to tumor antigen cross-presentation is usually very low due to the unfavorable tumor microenvironment and tumor-derived immune suppression in local lymph nodes. These obstacles can be overcome by oncolytic virotherapy, as any tumor-destroying virus provides the necessary "danger signals" to drive DC activation and thwart tumor immune suppression to expose hidden immunogenic antigens. 6-8 .

[0030] Oncolytic adenoviruses, also known as conditionally replicating adenoviruses (CRAds), are genetically engineered to replicate and kill only cancer cells. 9、10 It is known that virus-induced tumor apoptosis and / or necrosis releases abundant tumor-associated proteins that are normally inaccessible to antigen-presenting cells, facilitating efficient cross-presentation by tumor-associated DCs in tumor-releasing lymph nodes. 11-13 .

[0031] Although viral therapy of cancer is generally well tolerated, overall therapeutic efficacy remains modest. After examining the immunological effects of viral therapy, a clear advantage of the virus against tumors was observed in both mice and humans (Figure 2). By coating the adenoviral capsid with synthetic MHC-I-restricted tumor-specific peptides, we "trick" antigen-presenting cells (APCs) that present these tumor antigens as part of the virus. In other words, the present invention, which utilizes the adenoviral capsid as a scaffold to deliver MHC-I-restricted peptides, redirects the immune response away from the virus and toward the tumor.

[0032] As used herein, "major histocompatibility complex class I" molecules refer to one of the two major classes of major histocompatibility complex (MHC) molecules (the other being MHC class II), which are found on almost all nucleated cells of the body. Their function is to present fragments of proteins from within the cell to T cells. Healthy cells are ignored, while cells containing foreign proteins are attacked by the immune system. Class I MHC molecules bind peptides generated primarily from the degradation of cytoplasmic proteins by the proteasome. The MHC I:peptide complex then inserts into the plasma membrane of the cell. The peptides bind to the extracellular portion of the class I MHC molecule. Thus, the function of class I MHC is to present intracellular proteins to cytotoxic T cells (CTLs). However, class I MHC can also present peptides generated from exogenous proteins in a process called cross-presentation. As used herein, "MHC-I specific polypeptide" refers to peptides that bind to MHC-I, i.e., the extracellular portion of class I MHC molecules, and are presented to CTLs.

[0033] All MHC-I peptides (MIPs) are collectively referred to as the immunopeptidome 14 Recently, with the use of advanced technologies, it has become possible to begin to study the MHC-I immune peptidome. Compared to other strategies that attempt to broadly screen the entire immune peptidome, the key difference in the present invention is that it focuses on specific peptides that are simultaneously present on tumor cells both before and after treatment (i.e., peptides that are not masked or excluded after treatment) and on DCs after treatment (Figure 3).

[0034] A significant difference between the present invention and conventional peptide-based immunotherapy is that the present invention takes full advantage of the fact that viruses, especially adenoviruses, have a privileged means of interacting with DCs (and thus are not obligated to target DCs). Adenoviruses bind to several pattern recognition receptors (PRRs), Toll-like receptors (TLRs), and IL-1 receptors (IL-2 receptors). 16、17 , NOD-like receptor family 18and inflammasomes 19 stimulates DCs to favor stable antigen presentation and CTL activation 20 To this end, we show that human primary DCs pulsed with oncolytic adenoviruses activate pathways involved in cell adhesion, cell-cell interactions and signaling, maturation and antigen presentation, suggesting that adenoviruses can promote maturation and migration of immature primary dendritic cells (Figure 4).

[0035] As used herein, "stimulating a peptide-specific immune response" refers to triggering an immune response in which cells that express a particular peptide are attacked and destroyed. "Immune response" refers to a system that includes lymphocytes (i.e., white blood cells), T lymphocytes or B lymphocytes, or both. T lymphocytes attack antigens directly and help control the immune response. They also release chemicals known as cytokines to control the overall immune response. B lymphocytes are the cells that produce antibodies. Antibodies bind to specific antigens, making them easier for immune cells to destroy.

[0036] In one embodiment of the present invention, the one or more polypeptides attached on the viral capsid are selected from the group consisting of a fragment of tyrosinase-related protein 2 (TRP-2), a fragment of human melanoma antigen gp100 (hgp100), a fragment of melanoma-associated antigen A1 (MAGE-A1), SIINFEKL, polyK-SIINFEKL, SIINFEKL-polyK, SLFRAVITK (SEQ ID NO: 4), polyK-SLFRAVITK, SLFRAVITK-polyK, SVYDFFVWL (SEQ ID NO: 5), polyK-SVYDFFVWL, SVYDFFVWL-polyK, KVPRNQDWL (SEQ ID NO: 6), polyK-KVPRNQDWL and KVPRNQDWL-polyK. In one embodiment of the present invention, the one or more polypeptides attached on the viral capsid include SIINFEKL, SLFRAVITK, SVYDFFVWL or KVPRNQDWL. In further embodiments, polypeptide fragments of TRP-2 and hgp100 (e.g., SVYDFFVWL or KVPRNQDWL) are attached onto the adenovirus capsid. In one embodiment of the present invention, the polypeptide used in the present invention is polylysine (poly K) modified. Poly K as used herein may be selected from the group consisting of 3K-15K, 3K-10K, 3K-8K, 5K-8K, 5K-7K and 6K. As used herein, "polylysine modified polypeptide" refers to a polypeptide into which a polylysine sequence has been inserted. Addition of a polylysine sequence to a polypeptide changes the charge of the peptide, resulting in its absorption to the surface of the virus.

[0037] Adenovirus Vectors The peptide-coated adenovirus can be of any type and species of the adenoviridae family (e.g., not limited to human adenovirus). In one embodiment of the present invention, the adenovirus can replicate and kill cancer cells while diverting anti-viral immune responses to tumors (Figure 1). The cancer-destroying virus of the present invention coated with patient-derived tumor-specific immune-activating peptides enhances the conversion of anti-viral immunity to anti-tumor immunity.

[0038] The adenoviral vector used in the present invention can be any adenoviral vector suitable for treating humans or animals. Alternatively, various types of adenoviral vectors can be used according to the present invention. Also, the vector can be modified by any method known in the art, such as deleting, inserting, mutating or modifying viral regions. The vector can be made tumor-specific for replication. For example, the adenoviral vector can include E1, E3 and / or E4 modifications, such as inserting tumor-specific promoters, deleting regions and inserting transgenes.

[0039] In one embodiment of the present invention, the adenoviral vector is an oncolytic adenoviral vector. As used herein, "oncolytic adenoviral vector" refers to an adenoviral vector that can infect and kill cancer cells by selectively replicating in tumor versus normal cells. In one embodiment of the present invention, the vector can only replicate in cells that have defects in the Rb pathway, specifically the Rb-p16 pathway. These defective cells include all tumor cells of animals and humans. As used herein, "defects in the Rb-pathway" refers to mutations and / or epigenetic changes in any gene or protein of the pathway. Tumor-specific oncolytic adenoviruses may be engineered, for example, by deleting 24 base pairs (D24) of the constant region 2 (CR2) of E1. As used herein, "D24" or "24bp deletion" refers to the deletion of nucleotides corresponding to amino acids 122-129 of the vector according to Heise C. et al. (2000, Nature Med 6, 1134-1139). In one embodiment of the present invention, the adenoviral vector contains a 24bp deletion (oncolytic viruses) or an E1 gene deletion (second generation viruses) or the vector is a helper-dependent vector. The E1 gene deletion may be a partial or complete deletion of the E1 region. As used herein, "helper-dependent vector" refers to a vector that does not contain genes encoding enzymes and / or structural proteins necessary for replication and thus relies on the assistance of a helper virus to replicate.

[0040] The backbone of the adenoviral vector can be of any serotype. In one embodiment of the present invention, the serotype of the adenoviral vector backbone is selected from serotype 3 or 5. As used herein, "adenovirus serotype 5 (Ad5) nucleic acid backbone" refers to the genome of Ad5, and "adenovirus serotype 3 (Ad3) nucleic acid backbone" refers to the genome of Ad3.

[0041] Additionally, the vector may be a chimeric vector, such as an Ad5 / 3, Ad3 / 5 or Ad5 / 35 vector. As an example, an "Ad5 / 3 vector" refers to a chimeric vector that has portions of both the Ad5 and Ad3 vectors.

[0042] In one embodiment of the present invention, the adenoviral vector comprises a capsid modification (i.e., a modification of the nucleotide sequence that encodes the proteins that form the viral capsid). The adenoviral "capsid" refers to the protein shell of the virus. The capsid is composed of several oligomeric structural subunits of proteins called protomers.

[0043] Additionally, the fiber knob region of the vector may be modified. In one embodiment of the invention, the adenoviral vector is Ad5 / 3 or Ad5 / 35, comprising an Ad5 nucleic acid backbone and a fiber knob selected from the group consisting of an Ad3 fiber knob, an Ad35 fiber knob, an Ad5 / 3 chimeric fiber knob, and an Ad5 / 35 chimeric fiber knob.

[0044] In a particular embodiment of the invention, the oncolytic adenoviral vector is based on an adenovirus serotype 5 (Ad5) nucleic acid backbone and includes a D24 deletion, optionally a transgene and optionally a CpG site. In another embodiment, the oncolytic adenoviral vector is based on an adenovirus serotype 5 (Ad5) nucleic acid backbone and includes a modification of the capsid (e.g., Ad3 fiber knob), optionally a D24 deletion and optionally a transgene.

[0045] The insertion of exogenous elements can enhance the effect of vector in target cells.The use of exogenous tissue or tumor specific promoters is common in recombinant vectors, and they can also be utilized in the present invention.Suitable promoters are well known to those skilled in the art, and include, but are not limited to, hTERT, CMV, E2F.

[0046] The adenoviral vector may also cause the expression of any transgene (e.g., granulocyte macrophage colony stimulating factor (GM-CSF)). In one embodiment of the present invention, the adenoviral vector comprises one or more transgenes. One example of a suitable transgene is a cytokine, which controls the increased trafficking of immune cells at the site affected by the disease, e.g., at the tumor site. The cytokine used in the present invention may be selected from any cytokine known in the art. In one embodiment of the present invention, the transgene is selected from the group consisting of chemokines and cytokines and signal peptides for the recruitment or manipulation of immune stroma at the tumor site for T cells, dendritic cells, macrophages, natural killer cells. The viral vector of the present invention may encode one or several transgenes, e.g., cytokines (e.g., 2, 3, 4, 5 or more). The adenoviral vector may express, for example, a monoclonal antibody that specifically blocks an immunological checkpoint (e.g., CTLA4, PD1, PDL1).

[0047] The transgene may be located at different positions in the adenoviral vector, for example, in a partially or completely deleted E3 region, under the E3 promoter or an exogenous promoter, in a partially or completely deleted E1 region, or under the E1 promoter or an exogenous promoter.

[0048] In one embodiment of the present invention, the adenoviral vector for coating is Ad5D24, Ad5D24CpG or Ad5D24-GMCSF. In Ad5D24-GMCSF, GM-CSF transgene is under the control of E3 promoter in place of deleted E3 region (i.e., deleted 6.7K / gp19K) (Cerullo V et al., 2010, Cancer Research 70:4297-4309). As used herein, CpG refers to the CpG moiety added to the adenoviral genome to make the virus more immunostimulatory. The insertion of CpG-rich region in the adenoviral backbone increases the ability of adenovirus to stimulate TLR9 in antigen-presenting cells, which leads to increased stimulation and maturation of T cells and NK activation (Nayak S, Herzog RW, Gene Ther. Mar Mar;17(3):295-304).

[0049] Viral vectors utilized in the present invention may also contain modifications other than those described above. Any additional components or modifications may be used in some cases, but are not required for the present invention.

[0050] Coating of adenovirus vectors According to the present invention, the capsid of adenovirus is coated with a synthetic polypeptide or peptide that can stimulate peptide-specific immune response in a subject. The polypeptide used to coat the adenovirus vector is not genetically encoded by said adenovirus vector. In this specification, the terms "polypeptide" and "peptide" are used interchangeably to refer to a polymer of amino acids of any length.

[0051] The polypeptide can be attached to the capsid by any known suitable chemical or biochemical method. In one embodiment of the present invention, the peptide is covalently or non-covalently attached onto the viral capsid. In another embodiment of the present invention, the polypeptide is attached to the capsid by electrostatic, disulfide or amide bond linkages or is co-delivered and attached to the capsid of a single nanoparticle. The nanoparticle may also be covalently or non-covalently attached to the capsid, for example, by electrostatic, disulfide or amide bond linkages. As used herein, "nanoparticle" refers to any particle with a size between 1 and 100 nanometers. The electrostatic attachment strategy takes advantage of the fact that the adenovirus capsid has a negative total charge, which means the synthesis of a positively charged peptide consisting of polylysine attached to a small linker that is attached to the peptide of interest. The first strategy has two potential advantages: 1) it is rapid (e.g., about 15-30 minutes at room temperature or about 20 minutes at room temperature), which may be an important feature of personalized drugs. 2) Significantly increase transduction of adenovirus complexed with cationic polymers. 26,29 .

[0052] The polypeptides attached on the viral capsid may all be the same peptide or may be different peptides selected from two or more different tumor antigens. In one embodiment of the present invention, the adenovirus is coated with two or more peptides. The peptides may be, for example, different MHC-I specific polypeptides of the same antigen, MHC-I polypeptides from different antigens, or a combination of MHC-I and MHC-II restricted peptides. In one embodiment of the present invention, the polypeptide attached on the viral capsid is selected from the group consisting of major histocompatibility complex of class I (MHC-I) specific polypeptides (polypeptides that bind to MHC-I), major histocompatibility complex of class II (MHC-II) specific polypeptides (polypeptides that bind to MHC-II), disease specific polypeptides (polypeptides associated with disease), tumor specific polypeptides (polypeptides associated with tumors or specific tumors) and DC specific polypeptides (polypeptides that bind to DCs). In a particular embodiment of the present invention, the polypeptide attached on the viral capsid is a tumor specific MHC-I restricted peptide. These peptides can be directly isolated from the patient's tumor by the process shown in FIG. 5. By utilizing the method of FIG. 5, the polypeptide bound on the viral capsid may be obtained from DCs that are simultaneously presented on the MHC-I of the tumor and fed oncolytics. As used herein, "tumor-specific polypeptide" refers to a polypeptide presented by tumor cells. As used herein, "DC-specific polypeptide" refers to a polypeptide presented by DCs. As used herein, "disease-specific polypeptide" refers to a polypeptide presented by cells with a disease phenotype or cells infected with a disease.

[0053] The polypeptide attached to the capsid of the adenoviral vector includes any polypeptide (e.g., tumor antigen or peptide derived therefrom) that is simultaneously presented by diseased or tumor cells and dendritic cells of a patient. An example of a suitable peptide includes, but is not limited to, gp100.

[0054] The concentration of the polypeptide on the capsid may vary, and in one embodiment of the invention the polypeptide is at a concentration of at least 500 nM.

[0055] According to the present invention, in the production of patient-specific polypeptide-coated adenoviruses, MHC-I-loaded peptides derived from diseased cells or tumors can be isolated, identified, synthesized and mixed into the capsid of DC-stimulating oncolytic adenoviruses. However, this method involves at least two steps. First, the most immunogenic polypeptides loaded on MHC-I are identified, and second, these polypeptides are loaded into the oncolytic adenovirus capsid.

[0056] Pharmaceutical Compositions The present invention provides therapeutic methods and uses for treating disorders, as well as pharmaceutical compositions for use in said methods and therapeutic applications. Such pharmaceutical compositions comprise the enveloped adenovirus alone or in combination with other agents, such as therapeutically active agent(s) and / or pharma- ceutical acceptable vehicle(s).

[0057] The pharma- ceutically acceptable vehicle may be selected from the group consisting of, for example, pharma-ceutically acceptable solvents, diluents, adjuvants, excipients, buffers, carriers, preservatives, fillers, stabilizers and thickeners. Optionally, other components that are usually found in the corresponding product may be included. In one embodiment of the present invention, the pharmaceutical composition comprises a polypeptide-coated adenovirus and a pharma-ceutically acceptable vehicle.

[0058] The pharmaceutical composition may be in any form, such as a solid, semi-solid or liquid form suitable for administration. The formulation may be selected from the group consisting of, but is not limited to, a solution, an emulsion or a suspension. The means and methods for formulating the pharmaceutical formulation are known to those skilled in the art and may be prepared in a manner known per se.

[0059] treatment Any disease or disorder that can be treated, slowed down, or ameliorated by stimulating a peptide-specific immune response against disease-causing abnormal cells is included within the scope of the present invention. In one embodiment of the present invention, the peptide-specific immune response is selected from the group consisting of anti-tumor (against primary and / or secondary tumors), anti-cancer (against primary and / or secondary malignant neoplasms), anti-infection and anti-viral immune response. In these cases, the immune response is against tumors (including both malignant and benign tumors and primary and secondary tumors), cancers (i.e., primary or secondary malignant neoplasms), infectious diseases (e.g., malaria), viruses (e.g., in the case of viral infections such as influenza, SARS-CoV, or HIV), and the like. For example, any cancer may be the target of the coated adenovirus of the present invention. In one embodiment of the invention, the cancer is selected from the group consisting of nasopharyngeal carcinoma, synovial carcinoma, hepatocellular carcinoma, renal carcinoma, cancer of connective tissue, melanoma, lung cancer, intestinal cancer, colon cancer, rectal cancer, colorectal cancer, brain tumor, throat cancer, oral cancer, liver cancer, osteosarcoma, pancreatic cancer, choriomas, gastrinomas, pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, adrenal gland cancer, anal cancer, bile duct cancer, bladder cancer, ureter cancer, brain tumor, oligodendroglioma, neuroblastoma, meningioma, spinal tumor, osteosarcoma, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, carcinoid of the gastrointestinal tract, fibrosarcoma, breast cancer, Paget's disease, cervical cancer, colorectal cancer, rectal cancer, esophageal cancer, biliary bladder cancer, head cancer, eye cancer, neck cancer, kidney cancer, Wilms' tumor, liver cancer, Kaposi's sarcoma, prostate cancer, lung cancer, testicular cancer, Hodgkin's disease, non-Hodgkin's lymphoma, oral cancer, skin cancer, mesothelioma, multiple myeloma, ovarian cancer, endocrine pancreatic cancer, glucagonoma, pancreatic cancer, parathyroid cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, gastric cancer, thymus cancer, thyroid cancer, choriocarcinoma (trophoblastic cancer), hydatidiform mole, uterine cancer, endometrial cancer, vaginal cancer, vulvar cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatin tumor, gum cancer, heart cancer, lip cancer, meningeal cancer, oral cancer, nerve cancer, palate cancer, parotid gland cancer, peritoneal cancer, pharyngeal cancer, pleural cancer, salivary gland cancer, tongue cancer, and tonsil cancer.

[0060] The term "treatment" or "treating" as used herein refers to the administration to a subject of at least an enveloped adenoviral vector or a pharmaceutical composition comprising an enveloped adenoviral vector. The term "treating" as used herein and terms derived therefrom do not necessarily mean 100% or complete cure or increase. Rather, there are various degrees of what one skilled in the art would recognize as having a potential benefit or therapeutic effect. In this regard, the methods and uses of the present invention can provide any degree of treatment or prevention of disease. Thus, "treating" includes not only complete cure, but also prevention, improvement or alleviation of disorders or symptoms associated with the disease in question, such as cancer, tumor, infectious disease or viral infection. The therapeutic effect may be evaluated by any method known to one skilled in the art, for example, by monitoring the patient's symptoms or disease markers in the blood.

[0061] As used herein, the term "subject" refers to a subject selected from the group consisting of an animal, a mammal, or a human. In one embodiment of the invention, the subject is a human or an animal.

[0062] The polypeptide-coated adenovirus is administered to the subject in a therapeutically effective amount to generate peptide-specific immune response.As used herein, the term "therapeutically effective amount" refers to the amount of coated adenovirus that minimizes the harmful effects of disease or disorder (e.g., cancer).The harmful effects include detectable or noticeable effects in the subject, such as pain, dizziness or swelling.

[0063] Only one administration of the coated adenoviral vector or pharmaceutical composition of the present invention may have a therapeutic effect. On the other hand, a treatment may include several administrations. The adenoviral vector or pharmaceutical composition may be administered 1-10 times, for example, for 2, 3, 4 or 8 weeks or during a treatment period. The length of the treatment period may vary, for example, lasting from 2 months to 12 months or more. In some cases, it is also possible to use several treatment periods for one patient.

[0064] The effective dose of the vector will vary depending at least on the subject in need of treatment, the type of disease and the stage of the disease. 8 Virus particles (VP) ~ approx. 1 x 10 14 VP, specifically about 1×10 9 VP~approx. 1×10 13 VP, more specifically about 5×10 9 VP~approx. 1×10 12 Can change with VP.

[0065] The administration of the coated adenovirus can be carried out by any suitable method known to those skilled in the art.In one embodiment of the present invention, the administration of the adenoviral vector is carried out by intratumoral, intraarterial, intravenous, intrapleural, intracystic, intracavitary or intraperitoneal injection or by oral administration.It is also possible to combine different administration routes.

[0066] The enveloped adenovirus may also be used together (simultaneously or sequentially) with other therapeutic agents or methods or combinations of treatments. For example, the method or use of the present invention may further include radiation therapy, chemotherapy, administration of other drugs or any clinical surgery.

[0067] Before classifying a human or animal patient as suitable for the treatment of the present invention, the clinician may test the patient. Based on results that deviate from normal and indicate a disease, such as cancer, the clinician may suggest the method or treatment of the present invention to the patient.

[0068] Identification of specific peptides for coating The present invention reveals a method for identifying at least tumor-specific and MHC-I-specific polypeptides from a subject. The method utilizes qualitative and quantitative studies, specifically on the MHC-I immunopeptidome of DCs and tumors exposed to tumor lysates in vitro. The methodology, briefly summarized in Figure 5, involves the isolation of MHC-I molecules from both DCs (virus-infected tumor cells) and tumor cells pulsed with tumor lysates in vitro, and the sequencing of MHC-associated polypeptides by mass spectrometry-based techniques. Immunologically relevant peptides are presented by both tumor lysate-pulsed dendritic cells and tumors. For example, the use of OVA-expressing model mice can facilitate validation of the system, and indeed well-known immunogenic OVA-derived peptides (e.g. SIINFEKL) derived from mouse experiments can serve as positive controls.

[0069] The subject's tumor cells before and after in vitro adenoviral infection are used in this method to block the polypeptides presented by the cells due to the viral infection. DCs pulsed with oncolysates in vitro are also used in this method to allow the presentation of tumor antigens. The advantage of using tumors as well as DCs pulsed with oncolysates for the isolation of tumor-specific peptides is that immunologically active peptides can be better identified (if the peptides are presented by both tumors and DCs, there is an efficient immune response). Isolation of MHC-I molecules from tumor cells and dendritic cells may be performed by any suitable isolation method in the art. Sequencing of the polypeptides can then be performed by any suitable mass spectrometry-based technique (e.g., LC-MS / MS) for identifying MHC-associated peptides. Polypeptides presented by both tumors and dendritic cells can be identified by comparing the polypeptides presented by these cells. The polypeptides common to the two groups, i.e., the polypeptides presented by lysate-pulsed DCs minus the polypeptides presented by non-pulsed DCs (to exclude DC self-peptides) and the polypeptides presented by virus-infected and non-infected tumors (to exclude virus-specific peptides), are suitable for coating adenovirus. The comparison of the polypeptides can be performed manually or by any bioinformatics method known to those skilled in the art. Optionally, in vitro, ex vivo and / or in vivo validation can be performed for any specific polypeptide or combination thereof. In one embodiment of the present invention, in addition to isolating MHC-I molecules from infected and non-infected tumor cells and infected dendritic cells, the method includes isolating MHC-I molecules from non-infected dendritic cells, identifying MHC-I-associated polypeptides, and identifying the polypeptides presented by the infected and non-infected tumors of steps iii) and iv), and the polypeptides presented by the infected dendritic cells of step iii) but not by the non-infected dendritic cells.In a particular embodiment of the invention, the infection of tumor cells and DCs with the adenoviral vector occurs in vitro. The adenoviral vector used in the methods of the invention can be any adenoviral vector, such as any of those vectors described in the previous section.

[0070] In one embodiment of the present invention, the method for identifying tumor-specific and MHC-I-specific polypeptides from a subject is used to select one or more tumor-specific and MHC-I-specific polypeptides for coating an adenovirus capsid. Any of these tumor-specific and MHC-I-specific polypeptides or a combination thereof can be used for coating.

[0071] It is obvious to those skilled in the art that as technology advances, the concept of the present invention can be implemented in various ways. The present invention and its embodiments are not limited to the above examples and can be modified within the scope of the claims. EXAMPLES

[0072] The following examples demonstrate, at least, the analysis of the tumor MHC-I immunopeptidome to isolate and select tumor-specific polypeptides, the generation and physical characterization of tumor-specific polypeptide-coated oncolytic adenoviruses, and the characterization of the coated adenoviruses in animal models (e.g., i) therapeutic efficacy, ii) ability to convert anti-viral immunity to anti-tumor immunity, and iii) ability to recruit cells of the immune system and promote T cell responses).

[0073] Preparation of oncolytic adenoviruses All oncolytic adenoviruses (OAds) were generated and propagated using standard protocols previously described (8). Briefly, viruses were amplified by infecting 70–80% confluent A549 cells in ten T175 flasks at a multiplicity of infection (MOI) of 30. Three days after infection, cells were harvested and lysed by four freeze (-80 °C) and thaw (37 °C) cycles. Adenoviral particles were then separated from cell debris and impurities by two ultracentrifugations (22,000 and 27,000 rpm) in a CsCl gradient. The recovered band was purified by dialysis overnight at 4 °C against A195 buffer with continuous stirring. Specifically, dialysis cassettes with a molecular weight cutoff of 10,000 kDa (Pierce, Life Technologies) were used. The purified viruses were harvested from the cassettes, aliquoted, and stored at -80 °C.

[0074] The integrity of the adenoviral genome was assessed by PCR using primers specific for the D24 deletion in the E3 and E1A genes.

[0075] Viral particle titers were measured using spectrophotometry, and infectious titers were measured by immunocytochemical staining, as described elsewhere in this section. Protein concentrations of viral preparations were determined by Bradford assay using Bio-Rad protein assay staining reagent concentrate (Bio-Rad Laboratories, Hercules, CA, USA). All spectrophotometric measurements were read using a SPECTROstar Nano spectrophotometer (BMG Labtech, Ortenber, Germany).

[0076] All viruses used in this study have been previously described: Ad5D24 is an adenovirus characterized by a 24-base pair deletion (D24) in the E1A gene (9), Ad5D24-CpG is an OAd with a CpG-rich genome in the E3 gene (30), and Ad5D24-GM-CSF is an OAd that expresses GM-CSF under the control of the viral E3 promoter (8).

[0077] Analysis of tumor MHC-I immunopeptidome to isolate and select tumor-specific peptides Method 1a: Murine CD11c+ bone marrow dendritic cells were collected from C57BL / 6 mice and cultured for one week. 23 The cells were then exposed to the following solutions: A) PBS as a control; B) Oncolysates from B16-OVA cells (oncolysates are derived from B16-OVA cells infected with the oncolytic adenovirus Ad5D24 until complete lysis) C) B16-OVA cell lysates obtained by freezing and thawing the cells. At various time points, peptide-loaded MHC-I was isolated from live DCs using mild acid elution. 25 At the time of analysis, peptides were dissolved in aqueous solution and analyzed by nanoLC-MS / MS on a LTQ-Orbitrap Elite mass spectrometer (Thermo Fisher Scientific). Database searches were performed against the International Protein Index mouse database version 3.23 (http: / / www.ebi.ac.uk / IPI / IPIhelp.html), which contains 51536 sequences and 24497860 residues. Related peptides were grouped by peptides commonly present in both the lysate-pulsed DC group, excluding unpulsed DC (to exclude DC self-peptides), and the virus-infected B16-OVA group, excluding uninfected B16-OVA (to exclude virus-specific peptides).

[0078] Method 1b: We first reduced the complexity of the immunopeptidome in silico using method 1a: MHC-I class peptide prediction (http: / / www.syfpeithi.de / home.htm), protein functional annotation (http: / / david.abcc.ncifcrf.gov) and (http: / / www.ingenuity.com).

[0079] Oncomine analysis (https: / / www.oncomine.org) was used to show the expression levels of a given protein in different human cancers and cell lines. Most importantly, we validated the peptides using epitope tool prediction (17).

[0080] To experimentally select the most immunogenic peptides, we pulsed splenocytes, tumors and lymph nodes from C57BL / 6 mice with all the different peptides isolated from method 1a using mouse IFN-γ ELISPOT (Mabtech AB, Sweden).

[0081] Briefly, C57BL / 6 mice bearing B16-OVA tumors were treated with an oncolytic adenovirus (Ad5D24). One to two weeks after treatment, mice were euthanized, and organs and tumors were harvested and made into single cell suspensions for IFN-γ ELISPOT analysis (Mabtech, Palo Alto CA). After identifying the most immunogenic pool of peptides, custom tetramers or pentamers (Proimmune, UK) were generated for flow cytometer-based detection of specific CD8 T cells that recognize these peptides on MHC-I molecules.

[0082] Generation and physical characterization of tumor-specific peptide-coated oncolytic adenoviruses Since OVA-derived peptides are very well known, as a proof of concept, we first generated OVA-specific coated viruses (Figure 6). More specifically, we generated SIINFEKL coated adenovirus (SIINFEKL (SEQ ID NO: 1) is the most immunogenic OVA-derived peptide); SIINFEDL coated virus (SIINFEDL (SEQ ID NO: 7) is an antagonist of SIINFEKL peptide); FILKSINE coated virus (FILKSINE (SEQ ID NO: 3) is a scrambled peptide of SIINFEKL).

[0083] Method 2a: To generate peptide-coated oncolytic adenoviruses, different strategies were considered (Figure 7).

[0084] One uses electrostatic binding between the virus and the peptide, and the other two involve covalent binding between the virus and the peptide. I. Electrostatic interactions. Positively charged peptide complexed with a negatively charged viral capsid 26 . II. Covalent bonds. Disulfide bonds with cysteines of capsid proteins 27,28 . III. Covalent bond. Amide bond. Succinimidyl ester reaction with the amine groups of lysines in the capsid. 28 . Ligation methods are described in the corresponding references.

[0085] In one embodiment of the present invention, a peptide-enveloped oncolytic adenovirus was prepared as follows: PeptiCRAd complex formation All PeptiCRAd complexes described in this study were prepared by mixing oncolytic virus (described in the "Oncolytic Adenovirus Preparations" section) with polyK epitope in a 1:500 ratio according to the following protocol (see Figures 8A and 12): i) the corresponding number of micrograms of protein present per microliter of virus preparation used was calculated, ii) 500 μg of peptide was then added per microgram of viral protein, iii) after vortexing, the mixture was incubated at room temperature (RT) for 15 min, and iv) the solution was vortexed and used for assays or animal injections. New PeptiCRAd were prepared before each experiment using fresh reagents. All viruses and peptides required prior to incubation were diluted in sterile Milli-Q water adjusted to pH 7.4. The PeptiCRAd was then diluted in the buffer required for the assay.

[0086] Method 2b: The infectivity of this peptide-coated virus of method 2a was evaluated in vitro by luciferase assay and qPCR in different cell lines (human and mouse). 30 To evaluate infectivity, a panel of heterogeneous tumor cell lines with different levels of CAR expression were infected with different concentrations of luciferase-expressing coated virus (Ad5D24-Luc) (1, 10, 100, 1000VP / cell), with uncoated virus always used as a control. Luciferase expression was quantified at different time points. Simultaneously, total DNA was harvested and viral DNA replication was quantified by qPCR. In vitro oncolytic activity was tested by TCID50 and MTS assays. 31 .

[0087] In one embodiment of the present invention, infectivity was determined by ICC as follows. Infectivity assay by ICC Tumor cells were placed at 2.0 × 10 5Cells were seeded in 24-well plates in three or five replicates. The next day, cells were infected with 100 μl of virus dilution. Plates were then centrifuged at 1,000 rcf for 90 min at 37°C, followed by incubation for 48 h. After the incubation period, the medium was removed and cells were fixed by incubating with 250 μl of ice-cold methanol for 15 min. After removing the methanol, cells were washed three times with 300 μl of PBS supplemented with 1% bovine serum albumin (BSA). Cells were then stained with 250 μl of mouse monoclonal anti-hexon antibody (Novus Biologicals, Littleton, CO, USA) diluted 1:2,000 for 1 h at room temperature in the dark. Cells were then washed and stained with 250 μl of biotin-streptavidin-conjugated goat anti-mouse antibody diluted 1:500 in PBS / 1% BSA for 1 h at room temperature in the dark. Afterwards, cells were incubated with 250 μl of extravidin-peroxidase (Sigma-Aldrich, St. Louis, MO, USA) diluted 1:200 for 30 min at RT. Cells were washed extensively and DAB staining solution (Sigma-Aldrich, St. Louis, MO, USA) was prepared according to the manufacturer's instructions. A total of 250 μl of DAB staining solution was then applied to each well and cells were monitored under a microscope for the appearance of dark spots. Once an optimal signal-to-noise ratio was reached, the reaction was stopped by the addition of PBS / 1% BSA (500 μl per well). For each replicate (i.e., well), five images of non-overlapping fields were acquired using an AMG EVOS XL microscope (AMG Group, Life Technologies). The infection titer was determined using the following formula: Infectious titer = x * (well area) / (field area) * 1 / (dilution factor) * (1 ml) / (applied dilution volume) For comparison of infectivity, data were expressed as the average number of spots in each field.

[0088] Proof of method 2: Negatively charged adenovirus capsids were electrostatically coated with tumor-specific peptides. The complexes had a Z-potential variation proportional to the amount of peptide. This change in Z-potential indicated that positively charged peptides were bound to the viral capsid, which determined the charge reversal (dotted line in FIG. 8A). After all the negative charges of the capsid were saturated, the Z-potential seemed to plateau (circled line in FIG. 12). To proceed to in vitro and in vivo efficacy, homogeneous monodisperse complexes can be formed at polypeptide concentrations above 500 nM.

[0089] To further characterize the peptide-coated adenovirus complex, we performed several viability assays (MTS assays) to compare the cell killing effect of PeptiCRAd with that of non-coated oncolytic viruses (Figure 9). The results show that viral coating consistently results in unchanged or better cell killing activity compared to non-coated oncolytic viruses.

[0090] In one embodiment of the present invention, a viability assay was performed as follows. Viability assay Tumor cells were plated in growth medium containing 5% FBS at 1.0 × 10 4 Cells were seeded at 1000 cells / well. The next day, the medium was removed and cells were infected with 50 μl of virus diluted in growth medium containing 2% FBS for 2 h at 37°C. Afterwards, 100 μl of growth medium containing 5% FBS was added and cells were incubated again at 37°C. Growth medium was changed every other day. When the most infectious condition (100 vp / cell) showed extensive cytopathic effects (>90%), cell viability was measured by MTS assay according to the manufacturer's protocol (CellTiter 96 AQueous One Solution Cell Proliferation Assay; Promega, Nacka, Sweden). Spectrophotometric data were acquired using a Varioskan Flash Multimode reader (Thermo Scientific, Carlsbad, CA, USA).

[0091] Study design Sample size was determined using the following formula: n=1+2C(s / d)^2 where C is a constant based on α and β values, s is the estimated variable, and d is the observed effect (34). For all animal experiments, a power (1-β) of at least 80% and a significance (α) of 0.05 were considered. The criteria for stopping data collection were i) death of more than 60% of mice in one or more groups, and ii) total clearance of the tumor. All mice that died before the end of the experiment were excluded from the growth curves to maintain the statistical integrity of the analysis.

[0092] The aim of this study was to test whether OAd is an effective adjuvant for a peptide cancer vaccine approach using a melanoma model. In addition, two specific questions were raised: i) Can PeptiCRAd limit the growth of distant untreated tumors? ii) Can the efficacy of PeptiCRAd be enhanced by targeting multiple tumor antigens instead of a single tumor antigen? To answer these questions, we utilized immunocompetent or humanized mice bearing melanoma tumors. Mice were randomly assigned to each experimental group and no blinding was employed.

[0093] Cell lines, reagents and human samples The human lung cancer cell line A549, the human colorectal adenocarcinoma cell line CACO-2, the human malignant melanoma cell line SK-MEL-2, the human melanoma cell line HS294T, and the mouse melanoma cell line B16-F10 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). The cell line B16-OVA, a mouse melanoma cell line expressing chicken OVA (35), was kindly provided by Professor Richard Vile (Mayo Clinic, Rochester, MN, USA).

[0094] The A549, CACO-2 and B16-OVA cell lines were cultured in low glucose DMEM (Lonza, Basel, Switzerland), the HS294T cell line was cultured in high glucose DMEM (Gibco, Life Technologies, Carlsbad, CA, USA), the SK-MEL-2 cell line was cultured in EMEM (ATCC), and the B16-F10 cell line was cultured in RPMI-1640 (Gibco, Life Technologies). The medium was supplemented with 10% fetal bovine serum (FBS; Gibco, Life Technologies), 2 mM GlutaMAX (Gibco, Life Technologies) and 100 U / ml penicillin and 0.1 mg / ml streptomycin (Gibco, Life Technologies). The B16-OVA cell line was also cultured in the presence of 5 mg / ml geneticin (Gibco, Life Technologies) to ensure selection of OVA-expressing cells. During the culture period or when required for the assay, cells were washed with 1× phosphate-buffered saline (PBS) and detached by incubation with 1× TrypLE Express (Gibco, Life Technologies) for 3 min at 37°C.

[0095] SIINFEKL (OVA 257-264 ), polyK-SIINFEKL, SIINFEKL-polyK, polyK-AHX-SIINFEKL, polyK-SVYDFFVWL(TRP-2 180-188 ), polyK-KVPRNQDWL (hgp100 25-33 ) and polyK-SLFRAVITK(MAGE-A1 96-104 ) peptides were purchased from Zhejiang Ontores Biotechnologies Co. (Zhejiang, China). The purity of all peptides was estimated to be >80% and they were analyzed by mass spectrometry.

[0096] In the Examples section, polyK refers to 6K. The net charge of the peptides was calculated by the Peptide Property Calculator version 3.1 online tool (http: / / www.biosyn.com / PeptidePropertyCalculator / PeptidePropertyCalculator.aspx).

[0097] The genotype of the SK-MEL-2 cell line is HLA-A*03-*26, B*35-*38, C*04-*12. Buffy coat from a healthy donor was also obtained from the Finnish Red Cross Service and was genotyped as HLA-A*03-*03, B*07-*27, C*01-*07.

[0098] Characterization of enveloped adenoviruses in animal models Method 3a: We tested the efficacy, immunogenicity, toxicity and biodistribution of coated vs. non-coated common oncolytic viruses in vivo. Efficacy and immunogenicity were tested in C57BL / 6 mice bearing B16-OVA tumors. SIINFEKL coated virus showed a more robust anti-OVA response, which translated into more pronounced tumor control (efficacy), compared to other coated viruses (antagonist, scrambled and non-coated). At the same time, the trafficking of these cells into the tumor microenvironment was also evaluated via adaptive transfer of radiolabeled cells (DC and T cells). Finally, the toxicity and biodistribution of the modified adenoviral vectors were also studied.

[0099] To study the efficacy of coated viruses, different groups of C57BL / 6 mice (N=15 per group) bearing syngeneic B16-OVA tumors (2 tumors per mouse) were treated with: a) SIINFEKL coated virus, b) SIINFEDL coated virus, c) FILKSINE coated virus, and d) non-coated virus as a control. At different time points starting from day 3 after administration of the virus, 2 mice per group were euthanized and spleens, lymph nodes and tumors were harvested and made into single cell suspensions for ELISPOT, co-culture and flow cytometry analysis. At the same time, tumor growth was measured over time using standard calipers. Flow cytometry analysis directly revealed the amount of SIINFEKL-specific T cells in tumors, spleens and lymph nodes (both draining and not draining the tumor). For this analysis, we used SIINFEKL-specific pentamers (e.g., 31). A quantitative index of anti-OVA (anti-SIINFEKL) T cell activation was obtained by mouse IFN-γ ELISPOT. In co-culture experiments, the ability of T cells (taken from experimental mice) to kill B16 and B16-OVA was tested in vitro. Cells were co-cultured at different cell:target ratios and B16 and B16-OVA viability was assessed by MTS or MTT assays. In all these experiments, T cells taken from OT-I mice were used as control. Human adenoviruses are semi-permissive 33 The CMT64-OVA model, a murine tumor expressing OVA, was also used.

[0100] Method 3b: The antitumor activity and immunogenicity of viruses coated with i) the OVA-peptide (SIINFEKL (SEQ ID NO: 1)), ii) the B16 peptide TRP2 (SVYDFFVWL (SEQ ID NO: 5)), iii) the hgp100 peptide (KVPRNQDWL (SEQ ID NO: 6)) or iv) the novel peptides identified in method 1 were compared.

[0101] These viruses were tested for their ability and efficacy in inducing antitumor immune responses. Antiviral responses were compared with antitumor responses (ELISPOT and pentamer analysis). Their ability to induce immune responses against different epitopes (e.g., OVA virus induces TRP2 responses, epitope spreading) was also evaluated. The method used in this method has already been described in Method 3a.

[0102] Studies based on methods 2 and 3: As described in Figure 7 Strategy I, we generated OVA-specific PeptiCRAd (SIINFEKL-coated oncolytic adenovirus). Briefly, a synthetic SIINFEKL peptide was synthesized and attached to a polylysine linker (polyK-SIINFEKL) to give the peptide a net positive charge, and complexed with a naked virus that has a net negative charge 30 minutes before injection. This complex was then administered intratumorally to mice bearing subcutaneous B16-OVA tumors. Tumor growth was monitored, and at the end of the experiment, the mice were euthanized, tumors were collected, and OVA-specific T cells were quantified by flow cytometry (Figure 10).

[0103] This experiment demonstrates the superiority of the modified adenoviral vector of the present invention compared to virus alone and to virus and peptide administered separately, and also shows the importance of the correct formulation of the enveloped virus, since higher concentrations of peptide appeared to elicit fewer tumor-specific T cells (data not shown).

[0104] Second generation enveloped adenovirus Method 4: Second generation PeptiCRAds were generated by coating oncolytic viruses with more than one peptide to elicit stronger and more polyvalent immune responses. These new viruses were characterized as in Method 2, and efficacy was evaluated as described in Method 3. We then coated the cytokine-armed oncolytic adenoviruses with several types of polypeptides. The polypeptides could be either different MHC-I specific peptides of the same antigen, or MHC-I peptides from different antigens, or a combination of MHC-I and MHC-II restricted peptides.

[0105] Methods used to analyze enveloped oncolytic viruses Zeta potential and dynamic light scattering (DLS) analysis Coated oncolytic virus samples were prepared as described in the section titled "PeptiCRAd complex formation". Each sample was then vortexed and diluted to a final volume of 700 μl with sterile Milli-Q water adjusted to pH 7.4, after which the samples were transferred to polystyrene disposable cuvettes for complex size determination. Samples were then collected from the cuvettes and transferred to DTS1070 disposable capillary tubes (Malvern, Worcestershire, UK) for zeta potential measurements. All measurements were performed at 25°C using a Zetasizer Nano ZS (Malvern).

[0106] SPR The interaction of polyK-SIINFEKL or SIINFEKL with OAd was evaluated using SPR. The measurements were performed using a multiparametric SPR Navi™ 220A instrument (Bionavis Ltd, Tampere, Finland). The instrument contains a temperature-controlled dual fluidic channel with an integrated fluidic system and an autosampler for buffer and sample handling. Milli-Q water with pH adjusted to 7.4 was used as running buffer. Furthermore, a constant flow rate of 30 μl / min was used throughout the experiments and the temperature was set at +20°C. Laser light with a wavelength of 670 nm was used for surface plasmon excitation.

[0107] Prior to the SPR experiment, a sensor slide with a silicon dioxide surface was activated by plasma treatment for 3 min, and then coated with APTES ((3-aminopropyl)triethoxysilane) by incubating the sensor in 50 mM APTES in toluene solution for 1 h. The sensor was then placed in the SPR device, and OAd was immobilized in situ on the sensor surface of the test channel by injecting 50 μg / ml OAd in Milli-Q water (pH 7.4) for about 12 min, followed by washing with 20 mM CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) for 3 min. The second fluidic channel was used as a reference, and Milli-Q water (pH 7.4) was injected, followed by washing with CHAPS. The baseline was observed for at least 10 min before sample injection. PolyK-SIINFEKL or SIINFEKL was then injected simultaneously in increasing concentrations into both fluidic channels of the flow cell.

[0108] Cross-presentation experiments Fresh spleens were harvested from naive C57BL / 6 mice and passed through a 70 μm cell strainer (Fisher Scientific, Waltham, MA, USA). Red blood cells were lysed by incubating the samples with 5 ml of ACK lysis buffer (Life Technologies) for 5 min at room temperature. Splenocytes were then washed and prepared for the assay (2 × 10 cells in 800 μl of 10% RPMI-1640 medium for each condition tested). 6 cells). A total of 200 μl of SIINFEKL, polyK-SIINFEKL, SIINFEKL-polyK or SIINFEKL-AHX-polyK peptide dilutions (0.19 μg / μl) were added to splenocytes. To test the OVA-PeptiCRAd, infection conditions of 100 vp / cell were used (a total of 7.9 × 10 9vp). PeptiCRAd complexes were prepared as described in Method 2. Splenocytes were then incubated for 2 hours at 37°C. Afterwards, cells were washed extensively and incubated with APC anti-mouse H-2K conjugated to SIINFEKL. b or APC mouse IgG1 (κ isotype Ctrl) (BioLegend, San Diego, CA, USA). After incubation on ice for 30 min, samples were washed and analyzed by flow cytometry.

[0109] Flow cytometry analysis Tumors, spleens and lymph nodes of treated mice were collected, passed through a 70 μm cell strainer and cultured overnight in 10% RPMI-1640 medium. When necessary, samples were frozen in RPMI-1640 (with 10% FBS and 10% DMSO) and stored at -80 °C. Single cell suspensions were stained with fluorochrome-conjugated monoclonal antibodies and analyzed using a BD LSR II (BD Biosciences) or Gallios (Beckman Coulter) flow cytometer and FlowJo software (Tree Star, Ashland, OR, USA). Sterile PBS was used as staining buffer. Epitope-specific T cells were studied using MHC class I pentamer (ProImmune, Oxford, UK). Other antibodies used included: murine and human Fc block CD16 / 32 (BD Pharmingen), FITC anti-mouse CD8 and FITC anti-human CD8 (ProImmune), PE / Cy7 anti-mouse CD3ε, PE / Cy7 anti-mouse CD19, FITC anti-mouse CD11c, PerCp / Cy5.5 anti-mouse CD86, APC anti-mouse H-2Kb conjugated to SIINFEKL, and APC mouse IgG1 (κ isotype Ctrl) (BioLegend). All staining procedures were performed according to the manufacturers' recommendations.

[0110] statistical analysis Statistical significance was determined using GraphPad Prism 6 (GraphPad Software, Inc., La Jolla, CA, USA). A detailed description of the statistical methods used to analyze the data from each experiment is provided in the brief description of each figure.

[0111] Animal testing and ethical issues Animal experiments were performed under Finnish and European laws and legislation. The animal permit (ESAVI / 5924 / 04.10.03 / 2012) was revised and approved by the Finnish authorities (University of Helsinki Laboratory Animal Committee and Southern Finland Provincial Government). Fully immunocompetent C57BL / 6 mice were obtained from Scanbur (Karlslunde, Denmark) and were bred with the immunodeficient triple knockout NOD.Cg-Prkdc scid -IL2rg tm1Wjl A / SzJ mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA). All mice were purchased at 4–6 weeks of age and quarantined for 2 weeks prior to testing. Mice were kept in cages with isolated and regulated airflow and had food available ad libitum throughout the study period. Mouse health was monitored frequently and animals were sacrificed at the first sign of pain or distress. All procedures were performed in a biosafety level 2 cabinet under sterile conditions.

[0112] For efficacy experiments, tumor cells were harvested at 60–70% confluence (logarithmic growth phase) and injected subcutaneously (sc) into the flanks of mice. The number of tumor cells injected into each flank varied depending on the type of cell line, with 3 × 10 5 B16-OVA, 1 x 10 5 pcs B16-F10, and 2 x 10 6 The tumor growth was then tracked and tumor volume was measured using the formula:

[0113] In accordance with our license, humane endpoints were i) 25% weight loss, ii) tumor diameter >15 mm, and iii) overt signs of pain (tumor migration or reduction in ulceration). Euthanasia was performed by carbon dioxide inhalation followed by cervical dislocation.

[0114] result The negative charge of the adenovirus capsid can be used to conjugate positively charged immunogenic peptides to form PeptiCRAds. Adenovirus capsids have a very high net negative charge (36). Therefore, we hypothesized that positively charged MHC-I restricted peptides bind to the capsid by electrostatic interactions, coating the virus with immunologically relevant peptides (i.e., tumor-specific MHC-I restricted peptides). To test our hypothesis, we used the B16-OVA tumor model (37). This cell line expresses chicken ovalbumin (OVA) and presents the OVA-derived peptide SIINFEKL, used as a model epitope, on MHC-I.

[0115] To enable electrostatic interactions between the neutral hydrophobic SIINFEKL peptide and the negative viral surface, we added polylysine (polyK) chains to the peptide sequence. This chemical modification increased the net charge of the peptide under physiological conditions by 0 to + 6 mV. We then investigated the interaction between the viral capsid and the modified peptide by surface plasmon resonance (SPR). In particular, an APTES silica SiO2 sensor was coated with OAd and injected into a flow system with increasing concentrations of SIINFEKL or polyK-SIIN (Figure 8B). No increase in signal was observed for the unmodified peptide (Figure 8B, dashed line), whereas a concentration-dependent increase in signal was observed for the modified peptide (Figure 8B, solid line), indicating that the modification of the peptide significantly increased the interaction with the adenovirus capsid.

[0116] Next, the optimal concentration of peptide required to efficiently coat the viral surface was investigated. For this purpose, the net charge and hydrodynamic diameter of the virus-peptide complexes resulting from different OAd:peptide ratios (1:5, 1:50, 1:100 and 1:500) were evaluated. We observed a clear correlation between the amount of positive peptide in the reaction and the net charge of the complexes (Figure 8A). The lowest ratio (1:5) was able to increase the charge of the virus particles from -29.7 ± 0.5 to +6.3 ± 0.06 mV, but heavy aggregation was observed under these conditions. This aggregation is expressed in an increase in the size of the complexes (800 ± 13.5 nm). Above 1:5 the net charge increased and reached a plateau-like kinetics. In fact, we measured zeta potentials of +17.5±0.2, +18.4±0.1 and +18±0.8 mV for ratios of 1:50, 1:100 and 1:500, respectively. However, only the diameter of the complexes at the ratio of 1:500 was reduced below 120 nm, the normal diameter of adenovirus particles (Figure 8A). To facilitate reproducibility, the same experiment was repeated with concentrations of peptides rather than ratios (Figure 12).

[0117] Modified MHC-I epitopes adsorbed to PeptiCRAd are efficiently cross-presented To induce an effective cytotoxic T lymphocyte-mediated immune response, peptides must be cross-presented to naive CD8+ T lymphocytes via MHC-I on APCs. Therefore, we investigated whether the presence and position of poly-K chains affect the efficiency of cross-presentation. To this end, we pulsed ex vivo cultured splenocytes (from C57BL / 6 mice) with either native SIINFEKL or two different lysine-extended versions: poly-K-SIINFEKL (N-terminal extension) and SIINFEKL-polyK (C-terminal extension). As a negative control, we included extended SIINFEKL containing aminocaprone (AHX) residues, a well-known analogue of lysine that can inhibit the proteolytic activity of the proteosome. We then assessed the cross-presentation of SIINFEKL using an antibody that specifically recognizes SIINFEKL-loaded MHC-I (38).

[0118] As expected, 98.5% of SIINFEKL-pulsed splenocytes were positive for the presence of SIINFEKL on MHC-I molecules on the splenocyte membrane (Figure 13A). Interestingly, the position of the polyK chain in the sequence of the peptide significantly altered the percentage of stained cells. Indeed, 94.5% of splenocytes pulsed with the N-terminally extended peptide cross-presented SIINFEKL. In contrast, when splenocytes were pulsed with the C-terminally extended SIINFEKL-polyK, the stained population was reduced to 27.1%. When pulsed with the negative control SIINFEKL-AHX-polyK, only 1.36% of splenocytes cross-presented the SIINFEKL peptide. Based on these findings, we selected the N-terminally extended form (polyK-SIINFEKL) for further studies.

[0119] Next, we investigated whether the adsorption of modified SIINFEKL to the viral capsid could affect its cross-presentation. As in the previous experiment, we incubated mouse splenocytes with peptides SIINFEKL or polyK-SIINFEKL or OVA-PeptiCRAd. We found that the N-terminally extended polyK-SIINFEKL complexed with the OAd forming PeptiCRAd allowed efficient MHC-I-restricted presentation of the SIINFEKL peptide (Figure 13B).

[0120] PeptiCRAd exhibits unchanged infectivity and full oncolytic activity compared to unmodified virus OAd is able to selectively infect tumor cells and lyse them via the OAd replication cycle. Therefore, we investigated whether coating the viruses with modified peptides would affect their biological properties. We selected and tested a human colorectal adenocarcinoma cell line (CACO-2) expressing low levels of the coxsackie and adenovirus receptor (CAR) and two human melanoma cell lines (SK-MEL-2 and A2058) expressing high levels of CAR. We first performed an in vitro viability assay comparing OVA-PeptiCRAd with the unmodified virus Ad5D24 (Figure 14A), and the results showed no significant differences in terms of oncolytic activity. As expected, the most infectious condition (100 vp / cell) correlated with the lowest viability in all cell lines. Furthermore, we showed that the peptide polyK-SIINFEKL had no toxic effect on cells.

[0121] We next assessed the infectivity of PeptiCRAd by immunocytochemistry (ICC) assay using the same cell lines in vitro (Figure S14B). PeptiCRAd showed a significant increase in infectivity (P<0.01) compared to naked adenovirus in CACO-2 and A2058 cell lines, while no difference was observed in the SK-MEL-2 cell line. This increase is likely due to the different charges of PeptiCRAd and naked adenovirus (36).

[0122] Antitumor efficacy and immunological studies of the PeptiCRAd cancer vaccine in a murine model of melanoma To thoroughly study the antitumor effects of PeptiCRAd and the antitumor immunity it promotes, we first used a murine model of melanoma overexpressing chicken OVA (B16-OVA) (35). Specifically, B16-OVA was implanted into the flank of mice and then established tumors were treated. Experiments were performed using an OAd with a D24 deletion in E1A (Ad5D24) (37), and further immunity was boosted repeatedly with a CpG-rich adenovirus (Ad5D24-CpG) (39) (Figure 15). Test groups included mice treated with OVA-PeptiCRAd, unconjugated Ad5D24-CpG and SIINFEKL (Ad5D24-CpG+SIINFEKL), OAd (Ad5D24-CpG) or peptide (SIINFEKL) alone or saline (mock).

[0123] PeptiCRAd treatment significantly reduced tumor growth compared with sham treatment or a mixture of OAd and SIINFEKL (P < 0.01). At the end of the experiment, the mean volume of tumors in OVA-PeptiCRAd-treated mice was lower than those in all other groups (120.4 ± 31.6 mm). 3 Mock: 697.7±350mm 3 , SIINFEKL is 255±61.5mm 3 , and 713.7 ± 292.6 mm for Ad5D24-CpG. 3 , 489.7±73.2mm for Ad5D24-CpG+SIINFEKL 3;Figure 15A).

[0124] At two different time points (days 7 and 16 for early and late, respectively), mice were sacrificed and spleens, tumors and draining lymph nodes were collected for immune analysis. This analysis revealed the presence of a large population of SIINFEKL-specific CD8+ T cells (CD8+OVA+ T cells) in the inguinal draining lymph nodes in the group of mice treated with PeptiCRAd (7.4% at day 7 and 3.2% at day 16). The same analysis showed no dramatic differences in tumors at early time points, but a substantial increase was observed at later time points (0.23% for OVA-PeptiCRAd, 0.02% for mock, 0.03% for SIINFEKL, 0.01% for Ad5D24-CpG and 0.02% for Ad5D24-CpG+SIINFEKL at day 16; Figure 15B and C).

[0125] We next investigated the correlation between tumor size and the population of OVA-specific T cells (CD8+OVA+ T cells) in the spleen, lymph nodes and tumors. We calculated Pearson's r values ​​to assess the nature of the correlation (negative value, negative correlation, positive value, positive correlation) and observed a negative correlation between tumor volume and the extent of the anti-OVA response (Figure 15D). This suggests that the group of animals with smaller tumors matches the group of animals with a stronger population of CD8+OVA+ T cells. We then calculated the r for each set of samples to assess the strength of this correlation. 2 values ​​were calculated (spleen, r 2 =0.5719; lymph nodes, r 2 = 0.6385; tumor, r 2 = 0.7445). Interestingly, in the correlation analysis, the PeptiCRAd group (red dots in FIG. 15D) consistently showed the smallest tumor volume and the greatest immune response.

[0126] Finally, to gain a better understanding of the mechanism of PeptiCRAd, we investigated the mechanism of PeptiCRAd expression in mice by detecting mature DCs (CD19 - CD3 - CD11c +CD86 high At the later time points, the percentage of mature SIINFEKL-presenting DCs was significantly higher in mice treated with OVA-PeptiCRAd than in mice treated with unconjugated Ad5D24-CpG+SIINFEKL (P<0.05). Considering both time points, CD86 high OVA + Only PeptiCRAd treatment induced an increase in mature SIINFEKL-presenting DCs, as indicated by a 9.67-fold increase in the DC population (FIG. 15E).

[0127] These results suggest that maturation and expansion of epitope-specific DC pools may be the basis for the high antitumor efficacy of PeptiCRAd.

[0128] Multivalent PeptiCRAd exhibits enhanced antitumor activity against distant untreated melanoma One of the main advantages of using oncolytic vaccines is that it facilitates the elicited immune response to target not only the primary tumor but also disseminated metastases. For this reason, we investigated the antitumor efficacy of PeptiCRAd against untreated contralateral tumors in a murine model of melanoma. In a similar series of experiments, we also investigated whether targeting two tumor antigens (via a multivalent PeptiCRAd) rather than a single one would increase overall efficacy. Therefore, we used two tumor-specific MHC-I restricted epitopes: SVYDFFVWL (TRP-2 180-188 ;murine MHC-I molecule H-2K b (restricted to human gp100) and KVPRNQDWL (restricted to human gp100 25-33 or hgp100; murine MHC-I molecule H-2D b (40) was selected for these experiments. For these experiments, the highly aggressive melanoma B16-F10, which expresses both tumor antigens, was used. (41) As previously described for SIINFEKL, the peptide was modified at the N-terminus with a poly-K chain to promote attachment to the viral capsid.

[0129] First, 1×10 5 B16-F10 cells were implanted into the right flank of C57BL / 6 mice. Ten days later, treatments with i) saline (mock), ii) naked oncolytic virus (Ad5D24-CpG), and iii) double coated TRP-2-hgp100-PeptiCRAd were initiated. Treatments were administered intratumorally every 2 days, as shown in the schematic diagram in Figure 6A. Two days after the last round of injection, 3 × 10 5 Mice were injected with B16-F10 cells into the left flank and melanoma growth was followed. Mice treated with double-coated PeptiCRAd had significantly reduced tumor growth compared to controls (day 11; FIG. 16A) (P<0.001). Analysis of secondary and untreated tumors revealed the advantage of double-coated PeptiCRAd over all other groups. Notably, at the end of the experiment, secondary tumors in this group were significantly smaller compared to controls receiving saline solution or groups receiving only Ad5D24-CpG (P<0.01; FIG. 16B).

[0130] To better define the mechanism underlying these results, flow cytometry analysis was performed to examine the specific T cell responses to both epitopes. We observed that the cumulative population of epitope-specific CD8+ T cells was greater in mice treated with TRP-2-hgp100 PeptiCRAd than in all other groups (Figure 16C).

[0131] Taken together, these results demonstrate that the PeptiCRAd approach is effective against a poorly immunogenic and aggressive melanoma model. Furthermore, targeting multiple antigens has a strong effect on both treated and untreated tumors. Thus, it is possible to generate multivalent PeptiCRAds, offering the possibility to target different tumor antigens, thereby overcoming tumor immunological evasion.

[0132] PeptiCRAd exhibits enhanced efficacy and antitumor immunity in humanized mice bearing human tumors Finally, we sought to evaluate the efficacy of PeptiCRAd in a model that could provide information about the feasibility of translation to the clinical setting. Therefore, we chose a more capable humanized mouse model. To this end, we first developed a triple knockout mouse (NOD.Cg-Prkdc scid -IL2rg tm1Wjl Mice were implanted with the human melanoma cell line SK-MEL-2 in mice (n = 10, 100, 15 ... 96-104 We selected a peptide derived from the human GM-CSF gene (polyK-SLFRAVITK) and engineered it to allow interaction with the viral capsid (polyK-SLFRAVITK). Because we have extensively studied the human immune system in this experiment, we selected an OAd expressing human GM-CSF, which we have previously shown to have enhanced activity in immune response systems, including cancer patients (8).

[0133] The inventors found that MAGE-A1 PeptiCRAd showed increased efficacy compared to control treatment, as indicated by the rapid reduction in tumor volume (Figures 17A and B). Finally, the inventors investigated whether a stronger immune response could explain the increased antitumor effect of PeptiCRAd in this model. To this end, the inventors detected MAGE-A1 by pentamer staining. 96-104 specific CD8 + When we investigated the presence of T cells (Figure 17C), we found that human MAGE-specific T cells (CD8 + MAGE-A1 + ) group was the largest.

[0134] These data support our previous findings that PeptiCRAd exploits the natural immunogenicity of oncolytic viruses to stimulate tumor-specific immune responses, thereby improving the efficacy of cancer immunotherapy.

[0135] Analysis of MHC-I specific polypeptides for any disease and for the coating of adenovirus capsids and their use Any MHC-I specific polypeptides are identified by comparing the MHC-I restricted polypeptides represented by DCs and the infected diseased cells of the subject. One or more polypeptides presented by both cell populations are selected for coating the adenoviral vector.

[0136] Any adenoviral vector is chosen and coated according to any of the methods described in Method 2. The coated vector is used to treat a disease in a patient.

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Claims

1. An adenoviral vector comprising a polypeptide attached onto a viral capsid, the polypeptide being capable of stimulating a peptide-specific immune response in a subject, and further wherein the polypeptide is not genetically encoded by the adenoviral vector but is covalently or non-covalently attached to the capsid, and the polypeptide attached onto the viral capsid is a major histocompatibility complex class I (MHC-I)-specific and tumor-specific polypeptide, or a major histocompatibility complex class II (MHC-II)-specific and tumor-specific polypeptide.

2. The adenoviral vector of claim 1 , wherein the polypeptide is selected from the group consisting of anti-tumor and anti-cancer polypeptides.

3. The cancer is selected from the group consisting of nasopharyngeal cancer, synovial cancer, hepatocellular carcinoma, renal cancer, connective tissue cancer, melanoma, lung cancer, intestinal cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, throat cancer, oral cancer, liver cancer, osteosarcoma, pancreatic cancer, choriomas, gastrinomas, pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, adrenal gland cancer, anal cancer, bile duct cancer, bladder cancer, ureter cancer, oligodendroglioma, neuroblastoma, meningioma, spinal tumor, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, carcinoid of gastrointestinal tract, fibrosarcoma, breast cancer, Paget's disease, cervical cancer, esophageal cancer, gallbladder cancer, head cancer, eye cancer, neck cancer, 3. The adenoviral vector of claim 2, wherein the adenoviral agent is selected from the group consisting of 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, glucagonoma, parathyroid cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, gastric cancer, thymus cancer, thyroid cancer, choriocarcinoma, hydatidiform mole, uterine cancer, endometrial cancer, vaginal cancer, vulvar cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatin tumor, gingival cancer, cardiac cancer, lip cancer, meningeal cancer, oral cancer, nerve cancer, palate cancer, parotid gland cancer, peritoneal cancer, pharyngeal cancer, pleural cancer, salivary gland cancer, tongue cancer, and tonsil cancer.

4. The adenoviral vector according to any one of claims 1 to 3, wherein the subject is a human or an animal.

5. The adenoviral vector of any one of claims 1 to 4, wherein the adenoviral vector is formulated for intratumoral, intraarterial, intravenous, intrapleural, intravesical, intracavity or intraperitoneal injection, or oral administration.

6. 6. The adenoviral vector of claim 1, wherein the polypeptide is a polylysine-modified polypeptide or a positively charged polypeptide that is covalently or non-covalently linked to the adenoviral capsid.

7. The adenoviral vector of any one of claims 1 to 6, wherein the polypeptides are bound to the capsid by electrostatic, disulfide or amide bond linkages or are co-delivered and attached to the capsid in a single nanoparticle.

8. 8. The adenoviral vector of claim 1, wherein the polypeptides attached on the viral capsid are all the same polypeptide or are different polypeptides selected from two or more different polypeptides.

9. 9. The adenoviral vector according to claim 1, further comprising a disease-specific polypeptide or a dendritic cell-specific polypeptide as a polypeptide attached on the viral capsid.

10. 10. The adenoviral vector according to claim 1, wherein the serotype of the backbone of the adenoviral vector is selected from serotypes 3 or 5.

11. 11. The adenoviral vector of claim 1, wherein the adenoviral vector comprises a 24 bp deletion or an E1 gene deletion, or the adenoviral vector is a helper-dependent vector.

12. The adenoviral vector of claim 1 , wherein the adenoviral vector comprises one or more transgenes.

13. The adenoviral vector of claim 1 , wherein the adenoviral vector comprises a capsid modification.

14. 14. The adenoviral vector of any one of claims 1 to 13, wherein the adenoviral vector is Ad5 / 3 or Ad5 / 35, comprising an Ad5 nucleic acid backbone and a fiber knob selected from the group consisting of an Ad3 fiber knob, an Ad35 fiber knob, an Ad5 / 3 chimeric fiber knob and an Ad5 / 35 chimeric fiber knob.

15. A pharmaceutical composition comprising an adenoviral vector according to any one of claims 1 to 14.

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