Coated oncolytic adenoviruses for cancer vaccines

Abstract

The present invention relates to adenoviral vectors and uses thereof wherein the viral capsid has been coated with a polypeptide capable of stimulating a peptide-specific immune response in an individual. Furthermore, the present invention relates to methods of treating diseases, such as cancer, by means of adenoviral vectors that have been coated with polypeptides capable of eliciting a peptide-specific immune response. The invention also relates to methods of coating adenoviral vectors by specific peptides and methods of identifying those peptides suitable for coating the capsid of an adenoviral vector.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202511457024.4 (22) Application Date 2015.05.18 (30) Priority Data 20145449 2014.05.19 FI (62) Divisional Application Data 201580026994.7 2015.05.18 (71) Applicant Valo Therapeutics Company Address Helsinki, Finland (72) Inventors Vincenzo Sruller Marcus Vajakos Kairamari Helvini Christian Capasso (74) Patent Agency Beijing Ying Sai Jia Hua Intellectual Property Agency Co., Ltd. 11204 Patent Attorney Wang Dazuo Hong Xin (51) Int.Cl. A61K 39 / 00 (2006.01) A61K 47 / 46 (2006.01) A61K 35 / 761 (2015.01) A61K 47 / 42 (2017.01) A61K 47 / 64 (2017.01) A61P 35 / 00 (2006.01) A61P 35 / 02 (2006.01) C12N 7 / 00 (2006.01) C12N 15 / 861 (2006.01) C12R 1 / 93 (2006.01) (54) Invention Title: Coated Oncolytic Adenovirus for Cancer Vaccine (57) Abstract: This invention relates to adenovirus vectors and their uses, wherein the viral capsid is coated with a polypeptide capable of stimulating a peptide-specific immune response in an individual. Furthermore, this invention relates to a method of treating diseases such as cancer using an adenovirus vector coated with a polypeptide capable of eliciting a peptide-specific immune response. This invention also relates to a method for coating an adenovirus vector with a specific peptide and a method for identifying peptides suitable for coating an adenovirus vector capsid. Claims 3 pages, Description 26 pages, Sequence Listing (electronic publication), Drawings 15 pages. CN 121313807 A 2026.01.13 CN 1 21 31 38 07 A 1. Use of an adenovirus vector comprising a polypeptide attached to a viral capsid in the preparation of a medicament for stimulating a peptide-specific immune response against tumors in an individual in need, wherein the polypeptide is not genetically encoded by the adenovirus vector but is covalently or non-covalently attached to the viral capsid, and the polypeptide is a class I major histocompatibility complex (MHC-I)-specific polypeptide or a class II major histocompatibility complex (MHC-II)-specific polypeptide, and is also tumor-specific and DC-specific polypeptides. 2. An adenovirus vector for use in stimulating a peptide-specific immune response against tumors in an individual, comprising a polypeptide attached to a viral capsid.3. A polypeptide on the viral capsid, wherein the polypeptide is not genetically encoded by the adenovirus vector, but is covalently or non-covalently attached to the viral capsid, and the polypeptide is a class I major histocompatibility complex (MHC-I)-specific polypeptide or a class II major histocompatibility complex (MHC-II)-specific polypeptide, and is tumor-specific and DC-specific polypeptide. 4. The adenovirus vector of claim 2, wherein two or more different N-terminal polylysine-modified polypeptides capable of stimulating a peptide-specific antitumor immune response in an individual are attached to the viral capsid. 5. The use as described in claim 1, wherein the cancer is selected from nasopharyngeal carcinoma, synovial carcinoma, renal carcinoma, connective tissue cancer, melanoma, lung cancer, colorectal cancer, brain cancer, laryngeal cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, retinal angiomatosis, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, oligodendroglioma, neuroblastoma, and brain (spinal) cancer. Meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, Ewing sarcoma, cancer of unknown primary site, carcinoid, gastrointestinal carcinoid, fibrosarcoma, breast cancer, Paget's disease, esophageal cancer, gallbladder cancer, head cancer, eye cancer, neck cancer, nephroblastoma, liver cancer, Kaposi's sarcoma, prostate 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, gastric cancer, thymic cancer, thyroid cancer, trophoblastic cancer, hydatidiform mole, uterine cancer, vaginal cancer, vulvar cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gingival cancer, cardiac cancer, lip cancer, meningeal cancer, oral cancer, neurogenic cancer, palatal cancer, parotid gland cancer, peritoneal cancer, pharyngeal cancer, pleural cancer, salivary gland cancer, tongue cancer, and tonsil cancer. 6. The use as described in claim 1, wherein the cancer is selected from colon cancer, rectal cancer, and small bowel cancer. 7. The use as described in claim 1, wherein the cancer is selected from cervical cancer and endometrial cancer. 8. The use as described in claim 1 or 3, wherein the individual is a human or animal. 9. The use as described in claim 1 or 3, wherein the adenovirus vector is administered via intratumoral injection, intra-arterial injection, intravenous injection, intrapleural injection, intracystic injection, intracavitary injection, or intraperitoneal injection, or oral administration. 10. The adenovirus vector as described in claim 2, wherein the cancer is selected from nasopharyngeal carcinoma, synovial carcinoma, renal cell carcinoma, connective tissue carcinoma, etc.Cancers of the tissues, including melanoma, lung cancer, colorectal cancer, brain cancer, laryngeal cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, retinal angiomatosis, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, oligodendroglioma, and neuroblastoma. Meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, Ewing sarcoma, cancer of unknown primary origin, carcinoid, gastrointestinal carcinoid, fibrosarcoma, breast cancer, Paget's disease, cervical cancer, esophageal cancer, gallbladder cancer, head cancer, eye cancer, neck cancer, nephroblastoma, liver cancer, Kaposi's sarcoma, prostate 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, gastric cancer, thymic cancer, thyroid cancer, trophoblastic carcinoma, hydatidiform mole, uterine cancer, endometrial cancer, vaginal cancer, vulvar cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, dental claims 1 / 3 page 2 CN 121313807 A Gingival cancer, cardiac cancer, lip cancer, meningeal cancer, oral cancer, neurogenic cancer, palatal cancer, parotid gland cancer, peritoneal cancer, pharyngeal cancer, pleural cancer, salivary gland cancer, tongue cancer, and tonsil cancer. 11. The adenovirus vector of claim 2, wherein the cancer is selected from colon cancer, rectal cancer, and small bowel cancer. 12. The adenovirus vector of claim 2, wherein the cancer is selected from cervical cancer and endometrial cancer. 13. The adenovirus vector of claim 2 or 4, wherein the individual is a human or animal. 14. The adenovirus vector of claim 2 or 4, wherein the adenovirus vector is administered via intratumoral injection, intra-arterial injection, intravenous injection, intrapleural injection, intracystic injection, intracavitary injection, or intraperitoneal injection, or oral administration. 15. A method of modifying an adenovirus capsid, wherein the method comprises covalently or non-covalently linking a polypeptide to the adenovirus capsid, wherein the polypeptide is a class I major histocompatibility complex (MHC-I)-specific polypeptide or a class II major histocompatibility complex (MHC-II)-specific polypeptide, and is also an antitumor-specific and DC-specific polypeptide, and wherein the modified adenovirus vector is capable of stimulating a peptide-specific antitumor immune response in an individual. 16. The use of claim 1 or 3, wherein the polypeptide has been attached to the capsid by electrostatic linking. 17. The use of claim 1 or 3, wherein the serotype of the adenovirus vector backbone is selected from serotype 3 or 5. 18. The use of claim 1 or 3, wherein the adenovirus vector comprises a 24 bp deletion or an E1 gene deletion, or the vector is a helper virus-dependent vector.19. The use of claim 1 or 3, wherein the adenovirus vector comprises one or more transgenes. 20. The use of claim 1 or 3, wherein the adenovirus vector comprises a capsid modification. 21. The use of claim 1 or 3, wherein the adenovirus vector is Ad5 / 3 or Ad5 / 35 comprising an Ad5 nucleic acid backbone and fibrous nodes, wherein the fibrous nodes are selected from Ad3 fibrous nodes, Ad35 fibrous nodes, Ad5 / 3 chimeric fibrous nodes, and Ad5 / 35 chimeric fibrous nodes. 22. The adenovirus vector of claim 2 or 4, wherein different N-terminal polylysine-modified polypeptides have been attached to the capsid via electrostatic linkage. 23. The adenovirus vector of claim 2 or 4, wherein the serotype of the adenovirus vector backbone is selected from serotype 3 or 5. 24. The adenovirus vector of claim 2 or 4, wherein the adenovirus vector comprises a 24 bp deletion or an E1 gene deletion, or the vector is a helper virus-dependent vector. 25. The adenovirus vector of claim 2 or 4, wherein the adenovirus vector comprises one or more transgenes. 26. The adenovirus vector of claim 2 or 4, wherein the adenovirus vector comprises a capsid modification. 27. The adenovirus vector of claim 2 or 4, wherein the adenovirus vector is Ad5 / 3 or Ad5 / 35 comprising an Ad5 nucleic acid backbone and fibrous nodes, wherein the fibrous nodes are selected from Ad3 fibrous nodes, Ad35 fibrous nodes, Ad5 / 3 chimeric fibrous nodes, and Ad5 / 35 chimeric fibrous nodes. 28. The method of claim 15, wherein two or more different polypeptides have been attached to the capsid via electrostatic linkage. 29. The method of claim 15, wherein the serotype of the adenovirus vector backbone is selected from serotype 3 or 5. 30. The method of claim 15, wherein the adenovirus vector comprises a 24 bp deletion or an E1 gene deletion, or the vector is a helper virus-dependent vector. 31. The method of claim 15, wherein the adenovirus vector comprises one or more transgenes. 32. The method of claim 15, wherein the adenovirus vector comprises a capsid modification. Claims 2 / 3 Page 3 CN 121313807 A 33. The method of claim 15, wherein the adenovirus vector is Ad5 / 3 or Ad5 / 35 comprising an Ad5 nucleic acid backbone and a fibrous node, wherein the fibrous node is selected from Ad3 fibrous nodes, Ad35 fibrous nodes, Ad5 / 3 chimeric fibrous nodes, and Ad5 / 35 chimeric fibrous nodes. 34. A pharmaceutical composition comprising the adenovirus vector of any one of claims 1, 2, or 15. Claims 3 / 3 Page 4 CN 121313807 A Field of Invention for Coated Oncolytic Adenovirus for Cancer Vaccines

[0002] This invention relates to adenovirus vectors and their uses, wherein the viral capsid is coated with a polypeptide capable of stimulating a peptide-specific immune response in an individual. Furthermore, this invention relates to methods for treating diseases such as cancer via adenovirus vectors, wherein the adenovirus vectors are coated with polypeptides capable of evoking a peptide-specific immune response. This invention also relates to methods for coating adenovirus vectors with specific peptides and methods for identifying those peptides suitable for coating the capsid of an adenovirus vector. Background of the Invention

[0004] Cancer is a deadly disease requiring more effective treatment. Oncolytic viruses are of interest because they have the potential to be safer and more effective than any other standard therapy. However, overall treatment outcomes are low in cancer patients. There has been considerable research on modifying adenovirus vectors to find optimal therapeutic tools. One aspect of modulating adenovirus function is modifying the surface of the virus. Genetic and non-genetic modifications of the adenovirus surface are well known.

[0005] For example, Stevenson M et al. (Cancer Gene Therapy (2007) 14, 335–345) focused on enhancing the delivery of adenovirus vectors to target sites. Stevenson et al. described a study in which an adenoviral vector targets infected cells via integrin selectively expressed on metastatic tumor cells. For this purpose, a laminin-derived peptide (-SIKVAV-) was incorporated into the surface of a polymer-coated virus.

[0006] WO2013 / 116778 describes an adenovirus for immune enhancement in cancer. The adenovirus is modified by inserting a tumor antigen transgene into the genome of the adenovirus in such a way that the tumor antigen is expressed during the viral replication cycle and presented directly to MHC-I. For personalized therapy, this method is very slow, laborious, and expensive because a new virus needs to be generated for each different tumor antigen (e.g., a new virus must be cloned for each peptide that is to be expressed).

[0007] In fact, there is a need for simple and improved adenoviral tools and methods for therapeutics, especially personalized therapy. The present invention provides an adenoviral application for guiding an immune response in an individual while utilizing adenovirus as a peptide delivery system without involving genetic manipulation of the virus.

[0008] This invention relates to the use of oncolytic adenoviruses as a platform for delivering patient-specific and disease-specific peptides, and thus for converting anti-capsid immunity into peptide-specific immune responses (e.g., anti-tumor immunity). Summary of the Invention

[0010] This invention provides a novel, efficient, and customizable immunoviral therapy platform (e.g., cancer immunoviral therapy). One object is to provide an adenoviral vector with a modified viral surface, its use, and a method of treating a disease by stimulating a peptide-specific (i.e., anti-peptide) immune responses to address, for example, the inefficient, slow, expensive, and laborious nature of adenoviral therapy.The invention addresses issues such as the unsuitability of adenovirus therapy for personalized medicine. The object of the invention is achieved through the arrangements and methods characterized by the contents set forth in the independent claims. Preferred embodiments of the invention are disclosed in the dependent claims.

[0011] Through the invention, the problems of the prior art, such as the lack of specificity and immunogenicity of oncolytic adenoviruses, can be solved.

[0012] The immune response generated by adenovirus infection primarily targets the virus rather than the tumor. Furthermore, most viral immunity is directed towards the capsid protein. The invention will overcome these problems. In fact, the invention is based on the idea that a viral capsid coated with a peptide derived from a tumor protein (see page 1 / 26 of CN 121313807 A) transfers viral immunity to the tumor (Figure 3). Major histocompatibility complex I (MHC-I) restricted peptides embedded in the oncolytic adenovirus capsid transfer capsid immunity to antitumor immunity.

[0013] In simple terms, when the peptide and the virus are administered as a single physically linked entity, both the danger signal (virus) and the tumor antigen (peptide) enter the same antigen-presenting cell to maximize the antitumor effect. Clinical experience has shown that single peptide vaccination only results in a temporary, suboptimal immune response that fails to control tumor growth.1 Correspondingly, although oncolytic viruses have shown potential as a monotherapy, the immune response they elicit primarily targets the virus rather than the tumor. Even when peptides and viruses are injected into the same anatomical location, they cannot enter the same cell because they are not linked in a single therapeutic entity—a crucial aspect for achieving adequate and maximal immune activation.2 The physical linking of peptides and adenovirus-like viruses in a single therapeutic entity represents a significant improvement over existing viral and peptide-based cancer vaccine technologies. Compared to prior art recombinant viruses engineered to express a tumor-associated antigen or peptide, the present invention enables personalized medicine to be achieved in a faster and more cost-effective manner. In fact, according to the present invention, the peptide linked to the viral capsid is not encoded by an adenovirus vector.

[0014] One aspect of the present invention is a technique that allows for constant and rapid monitoring of tumor antigen presentation as small peptides (MHC-I restricted). This invention utilizes disease-specific (e.g., tumor-specific) and patient-specific peptides simultaneously presented to tumor cells (i.e., unmasked or unedited after therapy) and dendritic cells (DCs) after adenovirus therapy. After identifying these specific peptides, they can be synthesized and embedded into the capsid of oncolytic adenoviruses to achieve high anti-tumor immunity. This may ensure that tumors are effectively targeted by cytotoxic T-cells (CTLs) after viral therapy, making immune escape impossible due to the immune system targeting the virus. Conversely, by comparing peptides presented on DCs after viral therapy in the presence or absence of tumors, it may be possible to eliminate the "virus-only" approach.Peptides, and identify those peptides derived from tumor cells that induce CTL responses.

[0015] Personalized coated adenoviruses can be obtained within as little as two weeks of biopsy, which is made possible because the isolation and sequencing of peptides from the MHC and the automated synthesis are rapid processes, and viruses (e.g., viruses with the same backbone for all peptides) can be accumulated in large quantities to await coating. Coating itself is completed within an hour, after which the coated adenovirus is ready for injection. This is a very unique feature of our system because it bypasses any genetic manipulation of the virus, which slows down the process and makes a “personalized vaccine approach” impossible.

[0016] The present invention also enables the discovery of novel immunogenic tumor-specific peptides.

[0017] In addition to cancer therapy, the coated adenoviruses of the present invention can be used to treat any other disease requiring a higher and peptide-specific immune response.

[0018] The present invention relates to a method for stimulating a peptide-specific immune response in an individual in need, wherein the method comprises administering to the individual an adenovirus vector comprising a polypeptide attached to a viral capsid. The present invention also relates to a method for stimulating a peptide-specific immune response in an individual in need, wherein the method comprises administering to the individual an adenoviral vector comprising a polypeptide attached to a viral capsid, wherein the polypeptide is not genetically encoded by the adenoviral vector.

[0019] The present invention also relates to an adenoviral vector comprising a polypeptide attached to a viral capsid for use in stimulating a peptide-specific immune response in an individual. The present invention also relates to an adenoviral vector comprising a polypeptide attached to a viral capsid for use in stimulating a peptide-specific immune response in an individual, wherein the polypeptide is not genetically encoded by the adenoviral vector.

[0020] The present invention also relates to a method for treating cancer in an individual in need, wherein the method comprises administering to the individual an adenoviral vector comprising a polypeptide capable of stimulating a peptide-specific immune response in the individual and having been attached to a viral capsid. The present invention also relates to a method of treating cancer in an individual in need, wherein the method comprises administering to the individual an adenoviral vector comprising a polypeptide capable of stimulating a peptide-specific immune response in the individual and having been attached to a viral capsid, wherein the polypeptide is not genetically encoded by the adenoviral vector.

[0021] Furthermore, the present invention relates to an adenoviral vector comprising a polypeptide for use in treating cancer in an individual, wherein the polypeptide is capable of stimulating a peptide-specific immune response in the individual and has been attached to a viral capsid. The present invention also relates to an adenoviral vector comprising a polypeptide for use in treating cancer in an individual, wherein the polypeptide is capable of stimulating a peptide-specific immune response in the individual and has been attached to a viral capsid, wherein the polypeptide is not genetically encoded by the adenoviral vector.

[0022] Furthermore, the present invention relates to adenovirus vectors wherein a viral capsid is attached with a polypeptide, and wherein the polypeptide-attached adenovirus vector is capable of stimulating a peptide-specific immune response in an individual.

[0023] Furthermore, the present invention relates to a method of coating an adenovirus capsid, wherein the method comprises covalently or non-covalently linking a polypeptide capable of stimulating a peptide-specific immune response in an individual to the adenovirus capsid. The present invention also relates to a method of modifying an adenovirus capsid, wherein the method comprises covalently or non-covalently linking a polylysine-modified polypeptide to the adenovirus capsid, wherein the modified adenovirus vector is capable of stimulating a peptide-specific immune response in an individual.

[0024] The present invention also relates to the use of polypeptides (e.g., polylysine-modified polypeptides) for coating adenovirus capsids, wherein the polypeptide is capable of stimulating a peptide-specific immune response in an individual by covalently or non-covalently attaching or linking the polypeptide to the capsid.

[0025] The adenovirus vectors and methods of the present invention are used to convert antiviral immunity into antipeptide immunity. The modified viral vectors of the present invention generate an antipeptide response in an individual.

[0026] The present invention also relates to pharmaceutical compositions comprising the adenovirus vectors of the present invention.

[0027] Meanwhile, the present invention relates to a method for identifying tumor-specific and MHC-I-specific polypeptides from an individual, the method comprising

[0028] i) infecting tumor cells of the individual with an adenovirus vector;

[0029] ii) infecting dendritic cells of the individual with an adenovirus vector;

[0030] iii) isolating MHC-I molecules from the tumor cells of step i) and the dendritic cells of step ii), and identifying MHC-I-related polypeptides from both groups;

[0031] iv) isolating MHC-I molecules from uninfected tumor cells, and identifying MHC-I-related polypeptides;

[0032] v) identifying those polypeptides presented by the infected and uninfected tumors of steps iii) and iv) and by the dendritic cells of step iii). Brief Description of the Drawings

[0033] The invention will now be described in more detail by way of preferred embodiments with reference to the accompanying drawings, in which

[0034] FIG1 shows a schematic diagram of the invention, wherein a modified adenovirus is capable of replicating and killing cancer cells while simultaneously transferring an antiviral immune response to the tumor.

[0035] Figure 2 shows the immunogenicity of the anti-adenovirus response (left column) versus the tumor response (right column). (Mice) C57BL / 6 mice bearing B16-OVA tumors were treated with PBS (simulant group), Ad5D24 (unmodified oncolytic virus), and (Ad5D24-CpG, a more immunogenic oncolytic virus). T cells from the tumor were harvested and subjected to IFNγ ELISPOT to assess antitumor and anti-adenovirus responses. (Cancer patients) IFNγ ELISPOT was performed on PBMCs from patients treated with GMCSF-equipped oncolytic adenovirus (Ad5D24-GMCSF).ELISPOT 15. Ad5-derived peptides (antiviral) and survivin-derived peptides (antitumor) are used to stimulate PBMCs prior to ELISPOT.

[0036] Figure 3 illustrates the advantages of the coated adenovirus of the present invention compared to the prior art. A) Oncolytic adenoviruses are capable of triggering APCs, thereby presenting not only viral antigens (leading to an antiviral response) (another antigen presented on the cells in Figure A), but also, as a side effect, tumor antigens (another antigen presented on the cells in Figure A), leading to antitumor immunity. Antitumor T cells are labeled as the two bottom cells of the T cell group. B) The coated adenovirus of the present invention will facilitate the presentation of tumor antigens (labeled as the two antigens presented on the cells in Figure B) because its capsid is covered with MHC-I ready-to-use tumor-specific antigens (peptides). In this way, anti-capsid immunity can be converted into antitumor immunity. Anti-tumor T cells were labeled as the bottom four cells of the T cell group. In this paper, APC refers to antigen-presenting cells, TAA refers to antigen-associated tumors, and “PRR activation” refers to pattern recognition receptor activation. PRR is a protein expressed by cells of the innate immune system that recognizes pathogen-associated molecular patterns, such as those associated with microbial pathogens.

[0037] Figure 4 shows the apical upregulated biological function network of dendritic cells exposed to oncolytic adenovirus. Human primary dendritic cells were harvested and cultured for two weeks with IL4 and GMCSF. Cells were pulsed with oncolytic adenovirus (Ad5D24) at 10 VP / cell. After 72 h, total RNA was collected and analyzed on Agilent SurePrint G3 human 8x60k (mRNA). The data were then analyzed using Ingenuity Pathway software.

[0038] Figure 5 shows a schematic diagram illustrating the discovery of novel immunogenic tumor-associated MHCI-restricted peptides. Different conditions allowed us to match the peptides expressed by the tumor with peptides from the same tumor presented by dendritic cells. This plays a key role in facilitating the identification of immunogenic peptides in the system. A) Dendritic cells pulsed with a tumor lysing agent to allow tumor antigen presentation. B) Unpulsed dendritic cells mature and are analyzed. This serves as a control for the subsequent elimination of self-peptides presented by DCs. C) Uninfected tumor cell lines (same as condition A) are infected with oncolytic adenovirus and analyzed before complete lysis (less than 48 h). This condition helps us distinguish whether the adenovirus has a significant impact on the quality of the presented tumor antigen. D) This is an uninfected tumor presenting tumor antigen and self-peptide (which can of course be the same) on MHCI.

[0039] Figure 6 shows a schematic diagram of OVA-specifically coated virus. A) In this case, since we understand chickenAll treated peptides of ovalbumin (OVA) were coated with the virus using an OVA-specific immunogenic peptide (SIINFEKL) (SEQ ID NO: 1). We then generated other coated viruses, such as SINFEKL (SEQ ID NO: 2) (antagonist) and FILKSINE (SEQ ID NO: 3) (randomized), as controls, along with the coated viruses. B) Once the proof of concept was proven, we began studies with second-generation adenoviruses coated with different peptides. (PeptiCRAd refers to peptide-coated oncolytic adenoviruses).

[0040] Figure 7 shows a schematic diagram illustrating three different strategies for generating peptide-coated oncolytic adenoviruses.

[0041] Figure 8 shows the complex formation between oncolytic adenoviruses and tumor-specific peptides, as well as the interaction between modified epitopes and oncolytic adenoviruses. Figure 8A shows the complex formation between Ad5D24 oncolytic adenovirus and tumor-specific peptides. (Z-potential line) 1 × 10¹⁰ viral particles were conjugated with different concentrations of positively charged tumor-specific peptides. The Z-potential of each individual particle was measured after the reaction. (Size line) 1 × 10¹⁰ viral particles were conjugated with different concentrations of positively charged tumor-specific peptides. The size of each individual particle was then measured and reported as a function of peptide concentration. A dramatic change in the size of the complex was observed when the Z-potential was between -20 mV and +20 mV, indicating high polydispersity (likely viral aggregation). However, this state returned to normal at higher peptide concentrations, suggesting that the complex (PeptiCRAd) was fully coated and unlikely to form dipoles that promote aggregation (high polydispersity). Figure 8B reveals the interaction between the modified MHC-I epitope SIINFEKL and the oncolytic adenovirus. Virus / peptide interactions were measured using SPR. The APTES silica SiO2 sensor was coated with Ad5D24 and injected into the flow system at increasing concentrations (0.15, 0.3, 0.6, 1.2, 2.4, and 7.2 µM) of SIINFEKL (dashed line) or poly-K-SIINFEKL (solid line). SPR signal responses were shown to be correlated with the duration of the experiment.

[0042] Figure 9 shows that the coated adenovirus Ad5D24 (PeptiCRAd) of the present invention exhibits enhanced cell-killing activity compared to uncoated oncolytic virus. Representative cell viability analysis (MTS analysis) was performed on the lung cancer adenocarcinoma cell line (A549). Cells were seeded on day 0, infected on day 1 at the specified multiplicity of infection, and the test was stopped and analyzed on day 3. Specification 4 / 26 pages 8 CN 121313807 A

[0043] Figure 10 shows that OVA-specific adenovirus enhances OVA-specific immunity. Subcutaneous B16-OVA-bearing tumorsMice were injected intratumorally with: PBS, oncolytic virus (Ad5D24), oncolytic virus + SIINFEKL peptide (uncomplexed), and oncolytic virus + SIINFEKL (complexed into a single entity, PeptiCRAd). A) Tumor growth was measured and reported at the time points shown. B) SIINFEKL-specific immunity was assessed by flow cytometry (pentamer analysis).

[0044] Figure 11 shows the consistency of the peptide coating technique. This figure shows the net charge of two different oncolytic adenoviruses coated with the modified peptide (6K-SIINFEKL). The two viruses used in this example are Ad5D24-CpG (a genetically modified oncolytic adenovirus rich in CpG islands in its genome) and Ad5D24-RFP, which is an oncolytic adenovirus encoding red fluorescent protein for facilitating in vitro and in vivo imaging; (RFP refers to red fluorescent protein).

[0045] Figure 12 shows the correlation between the net charge of PeptiCRAd and its size. In this example, we started with naked virus (net charge approximately -25 to -30 mV) and then added increasing concentrations of peptides to form a complex we called PeptiCRAd. It was shown that the more peptides we added, the more the net charge of the virus changed from negative to positive, and at the end, when the PeptiCRAd complex was formed, the net charge of the peptide-coated virus was approximately +30 to +35 mV.

[0046] Figure 13 shows the cross-presentation of modified SIINFEKL analogs adsorbed to or not adsorbed to MHC-I on the viral capsid. 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 spleen cells were incubated with 200 µl of the following media: unmodified SIINFEKL (positive control), SIINFEKL-AHX-polyK containing aminocaproic acid (negative control), C-terminal derived SIINFEKL-polyK, or N-terminal extended polyK-SIINFEKL (0.19 μg / µl). After incubation at 37°C for 2 h, the cells were washed and stained with SIINFEKL-bound APC anti-H-2Kb or an allotype control. (B) Similar to (A), fresh mouse spleen cells were infected with OVA-PeptiCRAd (100 vp / cell + 37.5 µg peptide) and 37.5 µg SIINFEKL (positive control) or polyK-SIINFEKL. After incubation for 2 h, the samples were washed and analyzed by flow cytometry. Data are presented as mean ± SEM (n=2). Significance was assessed using one-way ANOVA and Bonfrani multiple comparison test; P < 0.05, P < 0.01, P < 0.001.

[0047] Figure 14 shows that PeptiCRAd retained its full oncolytic activity and exhibited increased infectivity in cell lines with low CAR expression. (A) Cells were seeded at a density of 1 × 10⁴ cells / well and infected with OVA-PeptiCRAd or naked Ad5D24 at different vp / cell ratios (0.1, 1, 10, and 100). Peptidopeptidyl K-SIINFEKL (dashed line, circle) was included as a control. Cell viability was then determined by MTS analysis. Data are presented as mean ± SEM (n=3). (B) Viral infectivity was studied by ICC. A total of 2 × 10⁵ cells / well were seeded into 24-well plates and infected the next day with 100 µl of viral dilution (10 vp / cell) containing OVA-PeptiCRAd or Ad5D24 (control). After two days of incubation, anti-hexagon ICC was performed, and five non-overlapping images were acquired using digital microscopy. The mean number of spots in each field of view is presented. Data from representative experiments are presented as mean ± SEM (n=2–3). Significance was assessed using unpaired t-tests with Welch correction; P<0.05, P<0.01, P<0.001.

[0048] Figure 15 shows the antitumor efficacy of PeptiCRAd and immunoassays of antigen-specific CD8+ T cells and DCs. (A) C57BL / 6 mice (n=6) received 3 × 10⁵ B16-OVA cells in each flank. Treatment began after 9 days and included saline solution (simulated group), peptide alone (SIINFEKL), virus alone (Ad5D24-CpG), a mixture of virus and peptide (Ad5D24-CpG+SIINFEKL), and a virus-peptide complex (OVA-PeptiCRAd). Mice were treated three times (on day 0, day 2, and day 7). Tumor size was then measured and presented as mean ± SEM as a function of time. Statistical analysis was performed using two-way ANOVA and Bonfrani's multiple comparison test; P<0.05, P<0.01, P<0.001. Tumors, spleens, and inguinal lymph nodes were collected from mice (n=3–4) at two time points: day 7 (early) (B) and day 16 (late) (C). The proportion of SIINFEKL-specific CD8+ T cells was then confirmed by gating CD19+ cells. The percentage of CD8+ OVA+ T cells is presented as mean ± SEM. (D) The mean tumor size (linear y-axis) at the end of the experiment was plotted against the mean percentage of double-positive CD8+ OVA+ T cells (log10 x-axis). Pearson r-values ​​and r2-values ​​were also calculated and compared with the mean percentage of double-positive CD8+ OVA+ T cells.Plotting of each sample group. (E) Determining the fold change in DCs displaying a mature spectrum and cross-presenting SIINFEKL on their MHC-I molecules. Mature DCs were defined as CD19-CD3-CD11c+CD86 high-density cells. APC anti-mouse H-2Kb bound to SIINFEKL was used to track cross-presentation of SIINFEKL on MHC-I in the selected DC library.

[0049] Figure 16 shows the reduction in the growth of treated and distal, untreated tumors by targeting two tumor antigens with PeptiCRAd. A primary tumor was transplanted into the right flank of a C57BL / 6 mouse using 1×10⁵ B16-F10 melanoma cells. Treatment began on day 10. On day 16, the mouse received 3×10⁵ B16-F10 cells in its left flank. (A) Growth of the primary tumor (right) is reported, data presented as mean ± SEM (n=5). Significance was determined using two-way ANOVA and Bonfrony multiple comparison test; P<0.05, P<0.01, P<0.001. (B) The size of secondary tumors (left) at the end of the experiment was reported as log2 proportions. Significance was assessed using the Mann-Whitney U test; P<0.05, P<0.01, P<0.001. (C) Spleen and inguinal lymph nodes were harvested, and the levels of TRP-2-specific CD8+ T cells and hgp100-specific CD8+ T cells in each organ were determined by MHC-I pentamer staining. The percentage of epitope-specific CD8+ T cells present in each organ was normalized for the mimic group and presented as the cumulative relative response for each experimental group.

[0050] Figure 17 shows the efficacy of PeptiCRAd in humanized mice bearing human melanoma. Triple gene knockout NGS mice received 2 × 10⁶ human melanoma cells (SK-MEL-2) per flank. When the tumors reached a mean diameter of 4–5 mm, one group of mice (n=3) received human PBMCs from HLA-A-matched healthy donors, while another group of mice (n=2) did not receive PBMCs. The mice were then treated with one of the following (on days 0, 2, and 4): i) saline solution (simulated group), ii) Ad5D24-GM-CSF, and iii) MAGE-A1 PeptiCRAd. Tumor volumes in humanized mice (A) are presented as mean ± SEM. Significance was assessed using two-way ANOVA with Bonfrani's multiple comparison test; P < 0.0001. (B) For each group of humanized mice, the area under the curve (AUC) is presented relative to tumor size. (C) Tumor volumes in non-humanized mice are reported as mean ± SEM (P < 0.0001).

[0051] Detailed Description of the Invention

[0052] Tumor Immunology and Immunopeptidomics

[0053] Dendritic cells (DCs) are professional antigen-presenting cells derived from the bone marrow. DCs are the optimal antigen-presenting cells 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.5 In cancer cells, the degree of maturation of DCs leading to tumor antigen cross-presentation is usually very low due to the unfavorable tumor microenvironment and tumor-derived immunosuppression also located in local lymph nodes. These obstacles can be overcome by oncolytic virus therapy because the tumor-destroying virus both provides the necessary “danger signals” to drive DC maturation and interferes with tumor suppression to expose hidden immunogenic antigens.6-8

[0054] Oncolytic adenoviruses, also known as conditionally replicating adenoviruses (CRAds), are genetically modified to replicate and kill only cancer cells.9,10 It is known that virus-induced tumor apoptosis and / or necrosis lead to the release of large amounts of tumor-associated proteins that are normally difficult for antigen-presenting cells to access, which drives efficient cross-presentation of tumor-associated dendritic cells (DCs) in tumor-draining lymph nodes.

[0055] Viral therapies for cancer have been found to be generally well-tolerated; however, overall therapeutic efficacy remains low. A significant advantage of viruses over tumors has been observed in both mice and humans when monitoring the immunogenicity of viral therapies (Figure 2). Coating an adenovirus capsid with a synthetic MHC-I-restricted tumor-specific peptide "tricks" antigen-presenting cells (APCs) into presenting these tumor antigens as a part of the virus. In other words, the present invention utilizes an adenovirus capsid as a scaffold for delivering MHC-I-restricted peptides, diverting the immune response away from the virus and instead directing it towards the tumor. Instructions 6 / 26 pages 10 CN 121313807 A

[0056] The “Class I major histocompatibility complex” molecules used herein refer to one of the two major major histocompatibility complex (MHC) molecules (the other being Class II MHC) and are found on almost every nucleated cell in the body. Their function is to present fragments of intracellular proteins to T cells; healthy cells are ignored, while cells containing exogenous proteins are attacked by the immune system. Class I MHC molecules bind peptides primarily generated by the degradation of cytoplasmic proteins by the proteasome. The MHC I:peptide complex is then inserted into the cytoplasmic membrane. The peptide binds 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. The “MHC-I-specific peptide” used herein refers to those peptides that bind to MHC-I, the extracellular portion of Class I MHC molecules, and are presented to CTLs.

[0057] All MHC-I peptides (MIPs) are collectively referred to as the immune peptide group.14. Only recently, with the aid of advanced technology, has it become possible to begin studying the MHC-I immune peptidomome. A key difference in this invention compared to other strategies that attempt to broadly screen the entire immune peptidomome is that it focuses on delivering specific peptides simultaneously to tumor cells before and after therapy (i.e., those not masked or edited after therapy) and to dendritic cells (DCs) after therapy (Figure 3).

[0058] A significant difference between this invention and conventional peptide-based immunotherapies is that this invention fully utilizes the fact that viruses, especially adenoviruses, have specific means of interacting with DCs (therefore, it is not necessary to target DCs). Adenoviruses stimulate several pattern recognition receptors (PRRs), Toll-like receptors16,17, the NOD-like receptor family18, and inflammasomes19, causing DCs to favor strong antigen presentation and CTL activation20. To this end, we demonstrated that pulsed human primary DCs activated pathways involved in cell adhesion, cell-cell interactions and signal transduction, maturation, and antigen presentation, suggesting that adenoviruses can promote the maturation and migration of immature primary dendritic cells (Figure 4).

[0059] As used herein, “stimulated peptide-specific immune response” refers to an immune response in which cells presenting a specific peptide are attacked and destroyed. “Immune response” refers to a system involving lymphocytes (i.e., white blood cells), specifically either T lymphocytes or B lymphocytes, or both. T lymphocytes directly attack antigens and help control the immune response. They also release chemicals called cytokines, which control the entire immune response. B lymphocytes become antibody-producing cells. Antibodies attach to specific antigens and make it easier for immune cells to destroy them.

[0060] In one embodiment of the present invention, one or more polypeptides attached to the viral capsid are selected from: fragments of tyrosinase-associated protein 2 (TRP-2), fragments of human melanoma antigen gp100 (hgp100), fragments 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 invention, one or more polypeptides attached to the viral capsid include SIINFEKL, SLFRAVITK, SVYDFFVWL, or KVPRNQDWL. In another embodiment, polypeptide fragments of TRP-2 and hgp100 (e.g., SVYDFFVWL or...) are used.KVPRNQDWL) attaches to the adenovirus capsid. In one embodiment of the invention, the polypeptide used in the invention is polylysine (polyK) modified. As used herein, polyK can be selected from 3K-15K, 3K-10K, 3K-8K, 5K-8K, 5K-7K, and 6K. As used herein, "polylysine-modified polypeptide" refers to a polypeptide in which a polylysine sequence has been inserted. Adding a polylysine sequence to a polypeptide results in a change in peptide charge and the subsequent adsorption to the viral surface.

[0061] Adenovirus Vector

[0062] The adenovirus coated with the peptide can be any type and species of the Adenoviridae family (e.g., not limited to human adenovirus). In one embodiment of the invention, the adenovirus is capable of replicating and killing cancer cells while redirecting the antiviral immune response to the tumor (Figure 1). The cancer-destroying virus of the invention coated with a patient-derived tumor-specific immune-activating peptide enhances and transfers antiviral immunity to antitumor immunity. Specification 7 / 26 pages 11 CN 121313807 A

[0063] The adenovirus vector used in this invention can be any adenovirus vector suitable for treating humans or animals. Optionally, according to the invention, various types of adenovirus vectors can be used. Moreover, the vector can be modified in any manner known in the art, for example, by deletion, insertion, mutation, or modification of any viral region. In terms of replication, the vector can be made tumor-specific. For example, the adenovirus vector can contain modifications located in E1, E3, and / or E4, such as the insertion of a tumor-specific promoter, the deletion of a region, and the insertion of a transgene.

[0064] In one embodiment of the invention, the adenovirus vector is an oncolytic adenovirus vector. As used herein, "oncolytic adenovirus vector" refers to an adenovirus vector that is capable of infecting and killing cancer cells by selectively replicating in tumors (compared to normal cells). In one embodiment of the invention, the vector can replicate only in cells defective in the Rb- pathway, particularly the Rb-p16 pathway. These defective cells include all tumor cells in animals and humans. As used herein, “Rb-pathway defect” refers to mutations and / or epigenetic changes in any gene or protein within this pathway. Tumor-specific oncolytic adenoviruses can be engineered, for example, by deleting 24 base pairs (D24) of the E1 constant region 2 (CR2). As used herein, “D24” or “24 bp deletion” refers to, according to Heise C. et al. (2000, Nature Med 6, 1134–1139), the deletion of nucleotides corresponding to amino acids 122–129 of the vector. In one embodiment of the invention, the adenoviral vector comprises a 24 bp deletion (oncolytic virus) or an E1 gene deletion (second-generation virus), or the virus is a helper virus-dependent vector. The E1 gene deletion may be a partial deletion of the E1 region.Loss or complete absence. As used herein, “helper virus-dependent vector” refers to a vector that does not contain genes encoding enzymes and / or structural proteins required for replication, and therefore depends on the assistance of a helper virus for replication.

[0065] The backbone of the adenovirus vector can be any serotype. In one embodiment of the invention, the serotype of the adenovirus vector backbone is selected from serotype 3 or 5. As used herein, “adenovirus serotype 5 (Ad5) backbone” refers to the genome of Ad5, and “adenovirus serotype 3 (Ad3) backbone” refers to the genome of Ad3.

[0066] Furthermore, the vector can be a chimeric vector, such as an Ad5 / 3, Ad3 / 5, or Ad5 / 35 vector. For example, “Ad5 / 3 vector” refers to a chimeric vector having portions of both Ad5 and Ad3 vectors.

[0067] In one embodiment of the invention, the adenovirus vector includes capsid modifications (i.e., modifications in the nucleotide sequence encoding proteins that form the viral capsid). The “capsid” of an adenovirus refers to the protein coat of the virus. The capsid consists of several oligomeric structural subunits of proteins called protomers.

[0068] Furthermore, the fiber knob region of the vector can be modified. In one embodiment of the invention, the adenovirus vector is Ad5 / 3 or Ad5 / 35, comprising an Ad5 nucleic acid backbone and a fiber knob selected from Ad3 fiber knobs, Ad35 fiber knobs, Ad5 / 3 chimeric fiber knobs, and Ad5 / 35 chimeric fiber knobs.

[0069] In a specific embodiment of the invention, the oncolytic adenovirus vector is based on an adenovirus serotype 5 (Ad5) nucleic acid backbone and comprises D24 deletion, optionally transgenesis, and optionally CpG sites. In another embodiment, the oncolytic adenovirus vector is based on an adenovirus serotype 5 (Ad5) nucleic acid backbone and comprises capsid modifications (e.g., Ad3 fiber knobs), optionally D24 deletion, and optionally transgenesis.

[0070] The insertion of exogenous elements can enhance the effect of the vector in target cells. The use of exogenous tissue or tumor-specific promoters is common in recombinant viruses and can also be utilized in this invention. Suitable promoters are well known to those skilled in the art and include, but are not limited to, hTERT, CMV, and E2F.

[0071] The adenoviral vector can also induce the expression of any transgene (e.g., granulocyte-macrophage colony-stimulating factor (GM-CSF)). In one embodiment of the invention, the adenoviral vector comprises one or more transgenes. An example of a suitable transgene is a cytokine that manipulates increased delivery of immune cells to disease-affected sites, such as tumor sites. The cytokine used in the invention can be selected from any known cytokines in the art. In one embodiment of the invention, the transgene is selected from chemokines and cytokines and those involved in tumor sites, particularly T cells, dendritic cells, macrophages, etc. (See specification 8 / 26 pages 12 CN 121313807)A signal peptide that recruits or manipulates the immune matrix at the site of phages or natural killer cells. The viral vector of the present invention may encode one or more transgenes, such as cytokines (e.g., two, three, four, five or more). The adenovirus vector may, for example, express monoclonal antibodies that specifically block immune checkpoints (e.g., CTLA4, PD1, PDL1).

[0072] The transgene may be located at different locations on the adenovirus vector. The transgene may be located, for example, in a partially or completely deleted E3 region under an E3 promoter or a foreign promoter, or in a partially or completely deleted E1 region under an E1 promoter or a foreign promoter.

[0073] In one embodiment of the present invention, the adenovirus vector used for coating is Ad5D24, Ad5D24CpG or Ad5D24-GMCSF. In Ad5D24-GMCSF, the GM-CSF transgene is located at the site of the deleted E3 region (i.e., the deleted 6.7K / gp19K) under the control of the E3 promoter (Cerullo V et al. 2010, Cancer Research 70: 4297-4309). As used herein, CpG refers to the CpG portion added to the adenovirus genome to make the virus more immunostimulatory. Insertion of CpG-rich regions into the adenovirus backbone increases the adenovirus's ability to stimulate TLR9 in antigen-presenting cells, thereby increasing the ability of T cells to stimulate and mature and to activate NK cells (Nayak S, Herzog RW. Gene Ther. 2010 Mar;17(3):295-304.).

[0074] The viral vector used in this invention may also contain other modifications besides those described above. Any other components or modifications may be used optionally, but are not mandatory for the purposes of this invention.

[0075] Coated Adenovirus Vector

[0076] According to the invention, the adenovirus capsid is coated with a synthetic polypeptide or peptide capable of stimulating a peptide-specific immune response in an individual. The polypeptide used to coat the adenovirus vector is not genetically encoded by the adenovirus vector. Herein, the terms "polypeptide" and "peptide" are used interchangeably to refer to an amino acid polymer of any length.

[0077] The polypeptide can be attached to the capsid by any known suitable chemical or biochemical method. In one embodiment of the invention, the peptide has been covalently or non-covalently attached to the viral capsid. In another embodiment of the invention, the polypeptide has been attached to the capsid by electrostatic linkage, disulfide linkage, or amide bond linkage, or co-delivered and attached to the capsid in a single nanoparticle. Nanoparticles can also be covalently or non-covalently attached to the capsid, for example, by electrostatic linkage, disulfide linkage, or amide bond linkage. As used herein, "nanoparticle" refers to any particle with a size of 1 to 100 nanometers. The electrostatic linkage strategy utilizes the fact that the adenovirus capsid has a negative net total charge, which implies attachment to the small connector (which attaches to the target).The synthesis of positively charged peptides composed of polylysine. 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 play a key role in personalized medicine, and 2) the transduction of adenoviruses complexed with cationic polymers is significantly increased.26,29

[0078] The polypeptides attached to the viral capsid can all be the same peptide or different peptides selected from two or more different tumor antigens. In one embodiment of the invention, the adenovirus is coated with more than one peptide. The peptide can be, for example, a polypeptide specific to different MHC-I antigens, an MHC-I polypeptide from different antigens, or a combination of MHC-I and MHC-II restricted peptides. In one embodiment of the invention, the polypeptides attached to the viral capsid are selected from class I major histocompatibility complex (MHC-I)-specific polypeptides (polypeptides that bind to MHC-I), class II major histocompatibility complex (MHC-II)-specific polypeptides (polypeptides that bind to MHC-II), disease-specific polypeptides (polypeptides associated with a disease), tumor-specific polypeptides (polypeptides associated with a tumor or a specific tumor), and DC-specific polypeptides (polypeptides that bind to DCs). In a specific embodiment of the invention, the polypeptides attached to the viral capsid are tumor-specific MHC-I-restricted peptides. These peptides can be directly isolated from patient tumors, a process described in Figure 5. Using the method of Figure 5, the polypeptides to be attached to the viral capsid can be simultaneously presented to the MHC-I of the tumor and derived from DCs that have been supplied with tumor-lysing agents. 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. The term "Disease-Specific Specification 9 / 26 Page 13 CN 121313807 A Polypeptide" as used herein refers to a polypeptide presented by cells with a disease phenotype or cells infected with the disease.

[0079] The polypeptide to be attached to the adenovirus capsid vector includes any polypeptide (e.g., tumor antigens or peptides derived therefrom) presented simultaneously by diseased cells or tumor cells and dendritic cells of a patient. Examples of suitable peptides include, but are not limited to, gp100.

[0080] The concentration of the polypeptide on the capsid can vary, and in one embodiment of the invention, the concentration of the polypeptide is at least 500 nM.

[0081] According to the invention, in the production of a patient-suitable polypeptide-coated adenovirus, a diseased cell-derived or tumor-derived MHC-I loaded peptide can be isolated and identified, the peptide synthesized, and mixed into the capsid of a DC-stimulated oncolytic adenovirus. However, the method includes at least two steps. First, identifying the most immunogenic polypeptide loaded on MHC-I, and second, loading these polypeptides onto the oncolytic adenovirus capsid.

[0082] Pharmaceutical composition

[0083] This invention provides not only treatment methods and uses for treating conditions, but also pharmaceutical compositions for use in said methods and treatment uses. Such pharmaceutical compositions comprise a coated adenovirus, alone or in combination with other agents, such as one or more therapeutically effective amounts of the agent and / or one or more pharmaceutically acceptable mediators.

[0084] Pharmaceutically acceptable mediators may be selected, for example, from pharmaceutically acceptable solvents, diluents, adjuvants, excipients, buffers, carriers, preservatives, fillers, stabilizers, and thickeners. Optionally, any other components commonly found in corresponding products may be included. In one embodiment of the invention, the pharmaceutical composition comprises a polypeptide-coated adenovirus and a pharmaceutically acceptable mediator.

[0085] The pharmaceutical composition may be in any form suitable for administration, such as solid, semi-solid, or liquid form. Dosage forms may be selected from, but are not limited to, solutions, emulsions, or suspensions. Methods and approaches for formulating the pharmaceutical formulations of this invention are known to those skilled in the art and may be prepared in ways known per se.

[0086] Therapy

[0087] The scope of this invention includes any disease or condition that can be treated by a peptide-specific immune response to abnormal cell stimulation caused by the disease, the course of which can be slowed, or in which symptoms can be improved. In one embodiment of the invention, the peptide-specific immune response is selected from antitumor immune responses (for primary and / or secondary tumors), anticancer immune responses (for primary and / or secondary malignant tumors), anti-infective immune responses, and antiviral immune responses. In these cases, the immune response is correspondingly directed at tumors (including malignant and benign tumors and primary and secondary tumors), cancer (i.e., primary or secondary malignant tumors), infectious diseases (e.g., malaria), viruses (hypothetically viral infections, such as influenza, SARS-CoV, or HIV), etc. For example, any cancer can be a target of the coated adenovirus of the present invention. In one embodiment of the present invention, the cancer is selected from nasopharyngeal carcinoma, synovial carcinoma, hepatocellular carcinoma, renal carcinoma, connective tissue cancer, melanoma, lung cancer, intestinal cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, laryngeal cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, retinal angiomatosis, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, brain cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, bone cancer, osteochondroma, chondrosarcoma, Ewing sarcoma, cancer of unknown primary site, carcinoid, gastrointestinal carcinoid. Fibrosarcoma, breast cancer, Paget's disease, cervical cancer, colorectal cancer, rectal cancer, esophageal cancer, gallbladder cancer, head cancer, eye cancer, neck cancer, kidney cancer, nephroblastoma, liver cancer, Kaposi's sarcoma, prostate cancer, lung cancer, testicular cancer, Hodgkin's disease, non-Hodgkin's lymphomaTumors, 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 bowel cancer, gastric cancer, thymic cancer, thyroid cancer, trophoblastic cancer, hydatidiform mole, uterine cancer, endometrial cancer, vaginal cancer, vulvar cancer, acoustic neuroma, mycosis fungoides, pancreatic cancer, carcinoid syndrome, somatostatinoma, gingival cancer, cardiac cancer, lip cancer, meningeal cancer, oral cancer, neurogenic cancer, palatal cancer, parotid gland cancer, peritoneal cancer, pharyngeal cancer, pleural cancer, salivary gland cancer, tongue cancer, and tonsil cancer.

[0088] As used herein, the terms “treatment” or “treating” refer to at least the administration of a coated adenovirus vector or a pharmaceutical composition containing a coated adenovirus vector to an individual. As used herein, the term “treatment,” and terms derived therefrom, does not necessarily mean 100% or complete cure or enhancement. Rather, it refers to potential benefits or therapeutic effects of varying degrees, as recognized by those skilled in the art. In this respect, the methods and uses of the present invention can provide treatment or prevention of any degree of disease. Thus, “treatment” includes not only complete cure but also, for example, prevention, improvement, or relief of symptoms or symptoms associated with a disease of consideration (such as cancer, tumor, infectious disease, or viral infection). The therapeutic effect can be evaluated by any method known to those skilled in the art, such as by monitoring the patient’s symptoms or disease markers in the blood.

[0089] As used herein, the term “individual” refers to an individual selected from animals, mammals, or humans. In one embodiment of the invention, the individual is a human or an animal.

[0090] An adenovirus coated with a peptide is administered to the individual in a therapeutically effective amount that elicits a peptide-specific immune response. As used herein, the term “therapeutically effective amount” refers to an amount of coated adenovirus that minimizes the adverse effects of the disease or condition (e.g., cancer). Adverse effects include any detectable or noticeable effects in the individual, such as pain, dizziness, or swelling.

[0091] A single application of the coated adenovirus vector or pharmaceutical composition of the present invention can have a therapeutic effect. Alternatively, treatment may include multiple applications. For example, the adenovirus vector or pharmaceutical composition may be applied 1 to 10 times over 2, 3, 4, or 8 weeks, or during a treatment period. The length of the treatment period can vary, for example, it may last 2 to 12 months or longer. In some cases, several treatment periods may also be used for a single patient.

[0092] The effective dose of the vector depends at least on the individual requiring treatment, the type of disease, and the stage of the disease. The dose can range, for example, from about 1 × 10⁸ viral particles (VP) to about 1 × 10¹⁴ VP, specifically from about 1 × 10⁹ VP to about 1 × 10¹³ VP, and more specifically from about 5 × 10⁹ VP.VP changes from approximately 1 × 10¹² VP.

[0093] The coated adenovirus can be administered by any suitable method known to those skilled in the art. In one embodiment of the invention, the adenovirus vector is administered by intratumoral injection, intra-arterial injection, intravenous injection, intrapleural injection, intracystic injection, intracavitary injection, or intraperitoneal injection, or oral administration. Different routes of administration may also be combined.

[0094] The coated adenovirus can also be used (simultaneously or sequentially) with other therapeutic agents or treatments or combinations of treatments. For example, the methods or uses of the invention may also include radiotherapy, chemotherapy, administration of other drugs, or any clinical procedure.

[0095] Clinicians may examine patients before classifying human or animal patients for the treatments suitable for the invention. Based on deviations from normal and results indicating diseases such as cancer, clinicians may recommend the methods or treatments of the invention to patients.

[0096] Identification of Specific Peptides for Coating

[0097] The invention shows a method for identifying at least tumor-specific and MHC-I-specific peptides from an individual. This method employs qualitative and quantitative studies of the MHC-I immune peptide genome of tumor cells and dendritic cells (DCs) exposed to (especially in vivo) tumor lysates. The method is simply summarized in Figure 5, which involves isolating MHC I molecules from DCs of tumor cells and those from tumor-lysing in vitro pulses (virus-infected tumor cells) and sequencing MHC-related peptides using mass spectrometry-based techniques. The immune-related peptides are presented by both tumor cells and dendritic cells from tumor-lysing pulses. For example, the use of mouse models expressing OVA can facilitate system validation; in fact, known immunogenic OVA-derived peptides (e.g., SIINFEKL) are produced in mouse experiments and can serve as positive controls. Specification 11 / 26 pages 15 CN 121313807 A

[0098] Individual tumor cells before and after in vitro adenovirus infection are used in the method to prevent those peptides presented by the cells due to viral infection. DCs from tumor-lysing in vitro pulses are also used in the method to allow for the presentation of tumor antigens. To isolate tumor-specific peptides, the advantage of using not only tumor cells but also dendritic cells (DCs) pulsed with tumor lysing agents is to better identify immunologically active peptides (an effective immune response only occurs when the peptide is presented on both tumor cells and DCs). Isolation of MHC-I molecules from tumor cells and dendritic cells can be performed using any suitable isolation method in the art. Subsequently, peptide sequencing can be performed using any suitable mass spectrometry-based technique (e.g., LC-MS / MS) for the identification of MHC-related peptides. Peptides presented by both tumor cells and dendritic cells can be identified by comparing the peptides presented by these cells. Co-present peptides in both groups are identified by subtracting the peptides presented by unpulsed DCs from the peptides presented by the lysed DCs.Peptides (to eliminate the peptides of the dendritic cells themselves) and polypeptides presented by both virus-infected and uninfected tumors (to eliminate virus-specific peptides) are suitable for coating with adenovirus. Comparison of polypeptides can be performed manually or by any bioinformatics method known to those skilled in the art. Optionally, for any specific polypeptide or combination thereof, in vitro, ex vivo, and / or in vivo validation can be performed. In one embodiment of the invention, in addition to isolating MHC-I molecules from infected and uninfected tumor cells and infected dendritic cells, the method further includes isolating MHC-I molecules from uninfected dendritic cells and identifying MHC-I-related polypeptides; and identifying those polypeptides presented by the infected and uninfected tumors of steps iii) and iv) and the infected dendritic cells of step iii), but not by uninfected dendritic cells. In a specific embodiment of the invention, infection of tumor cells and dendritic cells with an adenovirus vector occurs in vitro. The adenovirus vector used in the method of the invention can be any adenovirus vector, such as any of the vectors described in the preceding sections.

[0099] In one embodiment of the invention, a method for identifying tumor-specific and MHC-I-specific polypeptides from an individual is used to select one or more tumor-specific and MHC-I-specific polypeptides coated with an adenovirus capsid. Any one or a combination of these tumor-specific and MHC-I-specific polypeptides can be used for coating.

[0100] It will be apparent to those skilled in the art that the concept of the invention can be implemented in various ways as a technical advancement. The invention and its embodiments are not limited to the examples described above, but can be varied within the scope of the claims.

[0101] Examples

[0102] The following examples at least demonstrate the analysis of tumor MHC-I immune peptides for isolating and selecting tumor-specific polypeptides, the generation and physical characterization of oncolytic adenoviruses coated with tumor-specific polypeptides, and the characterization of coated adenoviruses in animal models (e.g., i) therapeutic efficacy, ii) the ability to transfer antiviral immunity to antitumor immunity, and iii) the ability to recruit immune system cells and promote T cell responses.

[0103] Preparation of Oncolytic Adenovirus

[0104] As described previously (8), all oncolytic adenoviruses (OAd) were generated and propagated using a standard protocol. Briefly, the virus was amplified by infecting 10 T175 flasks with 70–80% confluence of A549 cells at a multiplicity of infection (MOI) of 30. Three days post-infection, cells were collected and lysed by four cycles of freezing (-80°C) and thawing (37°C). Adenovirus particles were then separated from cell debris and impurities by two ultracentrifugations (22,000 and 27,000 rpm) on a CsCl gradient.The recovered bands were purified by overnight dialyzing against A195 buffer at 4°C with continued stirring. Specifically, a dialysis cartridge (Pierce, Life Technologies) with a molecular weight cutoff of 10,000 kDa was used. The purified virus was recovered from the cartridge, aliquoted, and stored at -80°C.

[0105] The integrity of the adenovirus genome was assessed by PCR using primers specific to the D24 deletion in the E3 and E1A genes.

[0106] As described elsewhere in this section, the viral particle titer was determined spectrophotometrically, while the infectious titer was determined by chemical staining with immunocellular datasheets (page 12 / 26, CN 121313807 A). The protein concentration of the viral preparation was determined by Bradford assay using Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, Hercules, CA, USA). Spectrophotometer readings were performed using a SPECTROstar Nano spectrophotometer (BMG Labtech, Ortenber, Germany).

[0107] All viruses used in this study have been previously reported: Ad5D24 is an adenovirus characterized by a 24-base pair deletion (D24) in the E1A gene (9), Ad5D24-CpG is an OAd carrying a CpG-rich genome in the E3 gene (30), and Ad5D24-GM-CSF is an OAd expressing GM-CSF under the control of the viral E3 promoter (8).

[0108] Analysis of tumor MHC-I immunopeptide genome for isolation and selection of tumor-specific peptides

[0109] Method 1a:

[0110] Mice CD11c+ sorted bone marrow dendritic cells were harvested from C57BL / 6 mice and cultured for 1 week 23. Cells were then exposed to: A) PBS as a control, B) a tumor lysing agent from B16-OVA cells (the tumor lysing agent was derived from B16-OVA cells infected with oncolytic adenovirus Ad5D24 until complete lysis), and C) B16-OVA cell lysates obtained by freeze-thaw cycles.

[0111] At different time points, MHC-I25 cells loaded with peptides were isolated from live DCs using weak acid elution. For analysis, the peptides were dissolved in aqueous solution and analyzed by nano-LC-MS / MS on an LTQ-Orbitrap Elite mass spectrometer (Thermo Fisher Scientific). The results were compared to the International Protein Index, version 3.23, Mouse database (http: / / www.ebi.ac.uk / ).IPI / IPIhelp.html (containing 51,536 sequences and 24,497,860 residues) was used for database retrieval. The relevant peptides were in groups formed by peptides presented in two groups: DCs pulsed by lysate minus unpulsed DCs (to eliminate peptides of DCs themselves) and B16-OVA-infected subtracted from B16-OVA-uninfected (to eliminate virus-specific peptides).

[0112] Method 1b:

[0113] We first reduced the complexity of the immune peptidomome in a computer. MHC-I class peptide prediction (http: / / www.syfpeithi.de / home.htm). Functional annotation of proteins (http: / / david.abcc.ncifcrf.gov) and (http: / / www.ingenuity.com).

[0114] 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 our peptides using epitope prediction tools (

[17] ).

[0115] Experimentally, to select the most immunogenic peptides, we used mouse IFN-γ ELISPOT (Mabtech AB, Sweden) on splenocytes, tumors, and lymph nodes from C57BL / 6 mice collected and isolated using method 1a for all different peptide pulses.

[0116] Briefly, C57BL / 6 mice bearing B16-OVA tumors were treated with oncolytic adenovirus (Ad5D24). One to two weeks after treatment, the mice were euthanized, organs and tumors were collected, and degraded into single-cell suspensions for IFN-γ ELISPOT analysis (Mabtech, Palo Alto CA). Subsequently, once we identified some of the most immunogenic peptide libraries, we generated conventional tetramers or pentamers (Proimmune, UK) for flow cytometry-based detection of specific CD8 T cells that recognize these peptides on MHC-I molecules.

[0117] Generation and physical characterization of tumor-specific peptide-coated oncolytic adenoviruses

[0118] Since OVA-derived peptides are quite well known, as a proof of concept, we first generated OVA-specifically coated viruses (Figure 6). More specifically, we generated SIINFEKL-coated adenoviruses (SIINFEKL (SEQ ID NO: 1) is the most immunogenic OVA-derived peptide); SIINFEDL-coated viruses (SIINFEDL (SEQ ID NO: 7) is an antagonist of the SIINFEKL peptide, page 13 / 26 of the specification, 17 CN 121313807 A); and FILKSINE-coated viruses (FILKSINE (SEQ ID NO: 1) is an antagonist of the SIINFEKL peptide, page 13 / 26 of the specification, 17 CN 121313807 A); and FILKSINE-coated viruses (FILKSINE (SEQ ID NO: 1) is an antagonist of the SIINFEKL peptide, page 13 / 26 of the specification, 17 CN 121313807 A).ID NO: 3) is a disordered peptide of SIINFEKL.

[0119] Method 2a:

[0120] Different strategies were considered for generating peptide-coated oncolytic adenoviruses (Fig. 7).

[0121] One approach utilizes electrostatic binding between the virus and the peptide, while the other two involve covalent bonding between the virus and the peptide.

[0122] I. Electrostatic interaction. The positively charged peptide complexes with the negatively charged viral capsid 26.

[0123] II. Covalent bonding. Disulfide bond with cysteine ​​of the capsid protein 27, 28.

[0124] III. Covalent bonding. Amide bond. Succinimide ester reaction with amino group of lysine of the capsid 28.

[0125] The methods of connection are described in the corresponding references.

[0126] In one embodiment of the invention, peptide-coated oncolytic adenoviruses are prepared as follows: PeptiCRAd complex formation is performed according to the following protocol, all PeptiCRAd complexes described in this work are prepared by mixing oncolytic virus (as described under the title "Preparation of Oncolytic Adenoviruses") with poly-K-epitopes at a ratio of 1:500 (see Figures 8A and 12): i) calculating the corresponding micrograms of protein present for each microliter of viral preparation used; ii) then adding 500 µg of peptide for each microgram of viral protein; iii) after vortexing, incubating the mixture at room temperature (RT) for 15 min; and iv) vortexing the solution and using it for analysis or animal injection. Fresh PeptiCRAd is prepared using fresh reagents before each experiment. All dilutions of the virus and peptide required prior to incubation are performed in sterile Milli-Q water adjusted to pH 7.4. PeptiCRAd is then diluted with the buffer required for analysis.

[0127] Method 2b:

[0128] The infectivity of the peptide-coated virus from Method 2a was assessed in vitro by luciferase analysis and qPCR in different cell lines (human and mouse) 30. To assess infectivity, a group of different tumor cell lines with different CAR expression levels were infected with different concentrations of luciferase-expressing coated virus (Ad5D24-Luc) (1, 10, 100, 1000 VP / cell); uncoated virus was 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 analysis 31.

[0129] In one embodiment of the invention, infectivity was studied by ICC as follows: Tumor cells were seeded at 2.0 × 10⁵ cells / well in 24-well plates, in triplicate or in triplicate, for infectivity analysis by ICC. The next day, the cells were infected with 100 µl of viral dilution. Then, the plates were centrifuged at 1,000 rcf for 90 min at 37°C, and then incubated for 48 h.After incubation, the culture medium was removed, and the cells were fixed by incubation with 250 µl of ice-cold methanol for 15 min. Immediately after methanol removal, the cells were washed three times with 300 µl of PBS supplemented with 1% bovine serum albumin (BSA). The cells were then stained in the dark at RT for 1 h with 250 µl of mouse monoclonal anti-hexagon antibody (Novus Biologicals, Littleton, CO, USA) diluted 1:2,000. The cells were then washed and stained in the dark at RT for 1 h with 250 µl of biotin-streptavidin-conjugated goat anti-mouse antibody diluted 1:500 with PBS / 1% BSA. The cells were then incubated in RT for 30 min with 250 µl of extravidin-peroxidase (Sigma-Aldrich, St. Louis, MO, USA) diluted 1:200. Cells were thoroughly washed and DAB staining solution was prepared according to the manufacturer's instructions (Sigma-Aldrich, St. Louis, MO, USA). 250 µl of DAB staining solution was then applied to each well, and the appearance of black spots in the cells was monitored under a microscope. When the optimal signal-to-noise ratio was reached, the reaction was quenched by adding PBS / 1% BSA (500 µl / well). For each replicate (i.e., well), images of five non-overlapping fields of view were captured using an AMG EVOS XL microscope (AMG group, Life Technologies). The following formula, 14 / 26 pages, CN 121313807 A, was used to determine the infectious titer:

[0130] To compare infectiousness, the data are presented as the average number of spots in each field of view.

[0131] Supporting Method 2:

[0132] A negatively charged adenovirus capsid was coated with a tumor-specific peptide via electrostatic interaction. The Z-potential of the complex changes, and the Z-potential is proportional to the amount of peptide. This change in Z-potential indicates that the positively charged peptide binds to the viral capsid, determining the charge reversal (Fig. 8A, dotted line). Once all the negative charge on the capsid is saturated, the Z-potential appears to plateau (Fig. 12, circular line). The homogeneous monodisperse complexes used for in vitro and in vivo efficacy assays were formed from peptides at concentrations exceeding 500 nM.

[0133] To further characterize the peptide-coated adenovirus complex, we performed several activity analyses (MTS analyses) comparing the cytotoxic efficacy of PeptiCRAd to that of uncoated oncolytic viruses (Fig. 9). The results showed that viral coating always resulted in unchanged or better cytotoxic activity compared to uncoated oncolytic viruses.

[0134] In one embodiment of the invention, the activity analysis was performed as follows: Activity AnalysisTumor cells were seeded at 1.0 × 10⁴ cells / well in 96-well plates with growth medium containing 5% FBS. The next day, the medium was removed, and 50 µl of virus diluted in growth medium containing 2% FBS was used to infect the cells at 37°C for 2 h. Subsequently, 100 µl of growth medium containing 5% FBS was added, and the cells were incubated again at 37°C. The growth medium was changed every other day. Cell viability was determined by MTS analysis according to the manufacturer's instructions when the most infectious conditions (100 vp / cell) showed extensive cytopathic effects (>90%) (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).

[0135] Study Design

[0136] The sample size was determined using the following formula:

[0137] where C is a constant based on the values ​​of α and β, s is the estimated variable, and d is the effect to be observed (34). For all animal experiments, at least 80% power (1-β) and significance (α) of 0.05 were considered. The rules for stopping data collection were i) death of more than 60% of mice in one or more groups and ii) complete tumor clearance. All mice that died before the end of the experiment were excluded from the growth curve to maintain the statistical integrity of the analysis.

[0138] The goal of the study was to test whether OAD could represent an effective adjuvant for peptide cancer vaccine approaches using a melanoma model. In addition, two specific questions were raised: i) Can PeptiCRAd limit the growth of distal, untreated tumors? ii) Can the efficacy of PeptiCRAd be enhanced by targeting multiple tumor antigens rather than a single tumor antigen? To answer these questions, we used immune-active mice or humanized mice bearing melanoma tumors. Mice were randomly assigned to experimental groups and were not blinded.

[0139] Cell lines, reagents, and human samples

[0140] Human lung cancer cell line A549, human colorectal adenocarcinoma cell line CACO-2, human malignant melanoma cell line SK-MEL-2, human melanoma cell line HS294T, and mouse melanoma cell line B16-F10 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cell line B16-OVA (35) and mouse melanoma cell line expressing chicken OVA were kindly provided by Professor Richard Vile (Mayo) on page 15 / 26 of the instruction manual, CN 121313807 A.Clinic, Rochester, MN, USA).

[0141] A549, CACO-2, and B16-OVA cell lines were cultured in low-glucose DMEM (Lonza, Basel, Switzerland), HS294T cell line in high-glucose DMEM (Gibco, Life Technologies, Carlsbad, CA, USA), SK-MEL-2 cell line in EMEM (ATCC), and B16-F10 cell line in RPMI-1640 (Gibco, Life Technologies). All media were 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 genimycin (Gibco, Life Technologies) to ensure selection of OVA-expressing cells. During culture or when required for analysis, cells were washed with 1X phosphate-buffered saline (PBS) and separated by incubation at 37°C with 1X TrypLE Express (Gibco, Life Technologies) for 3 min.

[0142] SIINFEKL (OVA257-264), polyK-SIINFEKL, SIINFEKL-polyK, polyK-AHX-SIINFEKL, polyK-SVYDFFVWL (TRP-2180-188), polyK-KVPRNQDWL (hgp10025-33), and polyK-SLFRAVITK (MAGE-A196-104) peptides were purchased from Zhejiang Ontores Biotechnologies Co., Ltd. (Zhejiang, China). The purity of all peptides was estimated to be >80%, and they were analyzed by mass spectrometry.

[0143] In the Examples section, polyK refers to 6K.

[0144] The net charge of the peptide was calculated using the Peptide Property Calculator Ver. 3.1 online tool (http: / / www.biosyn.com / PeptidePropertyCalculator / PeptidePropertyCalculator.aspx).

[0145] The genotype of the SK-MEL-2 cell line is HLA-A 03-26; B35-38; C 04-12. Erythrocyte sedimentation rate (ESR) amber layers from healthy donors were also obtained from Finnish Red Cross services and the genotypes were determined to be HLA-A 03-03; B 07-27; C 01-07.

[0146] Characterization of coated adenovirus in animal models

[0147] Method 3a:

[0148] We tested the efficacy, immunogenicity, toxicity, and biodistribution of coated virus versus uncoated conventional oncolytic virus in vivo. Efficacy and immunogenicity were tested in C57BL / 6 mice bearing B16-OVA tumors. SIINFEKL-coated virus showed a stronger anti-OVA response compared to other uncoated viruses (antagonists, out-of-order, and uncoated), which was converted into more significant tumor control (efficacy). Simultaneously, the delivery of these cells to the tumor microenvironment was assessed through adoptive transfer of radiolabeled cells (DCs and T cells). Finally, the toxicity and biodistribution of the modified adenovirus vector were also investigated.

[0149] To investigate the efficacy of the coated virus, C57BL / 6 mice (N=15 per group) bearing different groups of syngeneic B16-OVA tumors (two tumors per mouse) were treated as follows: a) SIINFEKL-coated virus, b) SIINFEDL-coated virus, c) FILKSINE-coated virus, and d) uncoated virus as a control. At different time points from 3 days after virus administration, two mice in each group were euthanized, and the spleen, lymph nodes, and tumors were harvested into single-cell suspensions for ELISPOT, co-culture, and flow cytometry analysis. Tumor growth was measured over time using standard calipers. Flow cytometry analysis directly revealed the amount of SIINFEKL-specific T cells in tumors, spleen, and lymph nodes (tumor-draining and non-tumor-draining lymph nodes). For this analysis, we used SIINFEKL-specific pentamers (e.g., 31). Mouse IFN-γ ELISPOT also provided a quantitative indicator of anti-OVA (anti-SIINFEKL) T cell activation. In co-culture experiments, we tested the ability of T cells (harvested from experimental mice) to kill B16 and B16-OVA in vitro. Cells were co-cultured at different cell:target ratios, and the viability of B16 and B16-OVA was assessed by MTS or MTT analysis. In all these experiments, T cells harvested from OT-I mice were used as controls. The CMT64-OVA model, a mouse tumor expressing OVA (see manual page 16 / 26, 20 CN 121313807 A), was also used, in which human adenovirus was partially accepted.

[0150] Method 3b:

[0151] The antitumor activity and immunogenicity of viruses coated with the following were compared: i) OVA-peptide (SIINFEKL)(SEQ ID NO: 1)), ii) B16 peptide TRP2 (SVYDFFVWL (SEQ ID NO: 5)), iii) hgp100 peptide (KVPRNQDWL (SEQ ID NO: 6)), or iv) the novel peptides identified in Method 1.

[0152] The efficacy and ability of these viruses to induce antitumor immune responses were tested. Antiviral responses were compared with antitumor responses (ELISPOT and pentamer analysis). The ability to induce immune responses against different epitopes (e.g., OVA-virus triggering TRP2 response, epitope expansion) was also evaluated. The methods used in this study have been described in Method 3a.

[0153] Based on the studies of Methods 2 and 3:

[0154] We generated the OVA-specific PeptiCRAd (SIINFEKL-coated oncolytic adenovirus) described in Strategy I of Figure 7. In short, the synthesized SIINFEKL peptide was synthesized and attached to a polylysine linker (poly-K-SIINFEKL) to impart a positive net charge to the peptide, which was then complexed with a naked virus having a negative net charge 30 minutes before injection. The complex was then administered intratumorally to mice bearing subcutaneous B16-OVA tumors. Tumor growth was monitored and the mice were euthanized at the end of the experiment. The tumors were collected and OVA-specific T cells were quantified by flow cytometry (Figure 10).

[0155] This experiment demonstrates the superiority of the modified adenovirus vector of the present invention compared with the virus alone and separately administered virus and peptide. It also shows the importance of properly formulated coated virus, as using excessively high peptide concentrations appears to induce fewer tumor-specific T cells (data not shown).

[0156] Second-generation coated adenovirus

[0157] Method 4:

[0158] A second-generation PeptiCRAd was generated by coating an oncolytic virus with more than one single peptide (in order to elicit a stronger and multivalent immune response). These new viruses were characterized in the same manner as in Method 2, and their efficacy was evaluated in the same manner as in Method 3. Subsequently, we coated the cytokine-equipped oncolytic adenovirus with several peptides. The peptides could be 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 restrictive peptides.

[0159] Methods for analyzing the coated oncolytic virus

[0160] Z-potential and dynamic light scattering (DLS) analysis

[0161] Coated oncolytic virus samples were prepared as described under the heading “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 disposable polystyrene cups to determine the size of the complex. The samples were then recovered from the cups.The sample was transferred to a DTS1070 disposable capillary cell (Malvern, Worcestershire, UK) for Z-potential measurements. All measurements were performed at 25°C using a Zetasizer Nano ZS (Malvern).

[0162] SPR

[0163] The interaction between poly-K-SIINFEKL or SIINFEKL and OAD was evaluated using SPR. Measurements were performed using a multi-parameter SPR Navi™ 220A instrument (Bionavis Ltd, Tampere, Finland). This instrument includes a temperature-controlled dual-channel system with an integrated fluid system and an autosampler for buffer and sample handling. Milli-Q water, with its pH adjusted to 7.4, was used as the run buffer. Furthermore, a constant flow rate of 30 µl / min was used throughout the experiment, and the temperature was set to +20°C. A 670 nm laser was used for surface plasmon excitation.

[0164] Prior to the SPR experiment, the slider sensor with a silica surface was activated by: a 3-min plasma treatment followed by APTES coating by incubation in a toluene solution containing 50 mM APTES ((3-aminopropyl)triethoxysilane) for 1 h. The sensor was then placed in the SPR apparatus and immobilized in situ on the sensor surface of the test channel by injecting 50 µg / ml OAd into Milli-Q water (pH 7.4) for approximately 12 min, followed by washing with 20 mM CHAPS (3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate) for 3 min. The second channel was used as a reference and injected with Milli-Q water (pH 7.4), followed by washing with CHAPS. The baseline was observed for at least 10 min before sample injection. Then, poly-K-SIINFEKL or SIINFEKL was injected in parallel at increasing concentrations into both channels of the flow cell.

[0165] Cross-presentation experiment

[0166] Fresh spleens were collected from blank C57BL / 6 mice and induced to pass through a 70 µm cell filter (Fisher Scientific, Waltham, MA, USA). Red blood cells were lysed by incubating the sample with 5 ml ACK lysis buffer (Life Technologies) at RT for 5 min. Subsequently, spleen cells were washed and prepared for analysis (2 × 10⁶ cells in 800 µl 10% RPMI-1640 medium for each test condition). A total of 200 µl of the medium was added to the spleen cells.SIINFEKL, poly-K-SIINFEKL, SIINFEKL-poly-K, or SIINFEKL-AHX-poly-K peptide dilutions (0.19 μg / μl). For testing OVA-PeptiCRAd, infection conditions of 100 vp / cell were used (total 7.9 × 10⁹ vp mixed with 37.5 µg poly-K-SIINFEKL in 200 µl of 10% RPMI-1640). The PeptiCRAd complex was prepared as described in Method 2 below. Spleen cells were then incubated at 37°C for 2 h. Afterward, cells were thoroughly washed and stained with APC anti-mouse H-2Kb bound to SIINFEKL, or APC mouse IgG1,κ isotype controls (BioLegend, San Diego, CA, USA). After incubation on ice for 30 min, samples were washed and analyzed by flow cytometry.

[0167] Flow Cytometry Analysis

[0168] Tumors, spleens, and lymph nodes from treated mice were collected, passed through a 70 µm cell filter, and cultured overnight in 10% RPMI-1640 medium. If necessary, samples were frozen in RPMI-1640 (with 10% FBS and 10% DMSO) and stored at -80°C. Single-cell suspensions were stained with fluorescently 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 the staining buffer. Epitope-specific T cells were studied using class I MHC pentamers (ProImmune, Oxford, UK). Other antibodies used included the following: mouse and human Fc-blocked 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 and APC mouse IgG1,κ isotype control bound to SIINFEKL (BioLegend). All staining protocols were performed according to the manufacturer's recommendations.

[0169] Statistical Analysis

[0170] Statistical significance was determined using GraphPad Prism 6 (GraphPad Software, Inc., La Jolla, CA, USA). Detailed descriptions of the statistical methods used to analyze data from each experiment can be found in each brief description of the accompanying figures.

[0171] Animal Experimentation and Ethical Issues

[0172] Animal experiments were conducted in accordance with Finnish and European laws and legislation. The animal permit (ESAVI / 5924 / 04.10.03 / 2012) has been amended and approved by the Finnish authorities (the Laboratory Animal Committee of the University of Helsinki and the Provincial Government of Southern Finland). Fully immunocompetent C57BL / 6 mice were obtained from Scanbur (Karlslunde, Denmark), while immunodeficient triple knockout NOD.Cg-Prkdcscid-IL2rgtm1Wjl / SzJ mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA). All purchased mice were 4–6 weeks old and quarantined for 2 weeks prior to the study. Mice were kept in individual cages with controlled airflow and had unrestricted access to food throughout the study. The health of the mice was monitored frequently, and animals were euthanized at the first sign of pain or anxiety. All protocols were performed under sterile conditions in a biosafety level 2 room. Instructions for Use, Pages 18 / 26, 22 CN 121313807 A

[0173] For efficacy experiments, tumor cells were collected at 60-70% confluence (logarithmic growth phase) and injected subcutaneously (sc) into both flanks of mice. The number of tumor cells injected into each flank varied depending on the cell line type: 3×10⁵ B16-OVA, 1×10⁵ B16-F10, and 2×10⁶ SK-MEL-2. In all experiments, three treatment injections were administered. Tumor growth was then tracked, and tumor volume was determined using a formula.

[0174] In accordance with our license, the humane endpoints were as follows: i) 25% weight loss, ii) tumor diameter >15 mm, and iii) significant signs of pain (reduced mobility or tumor ulceration). Euthanasia was performed by carbon dioxide inhalation followed by cervical dislocation.

[0175] Results

[0176] The negative charge of the adenovirus capsid can be used to compound positively charged immunogenic peptides to form PeptiCRAd.

[0177] The adenovirus capsid carries a highly negative net charge (36), therefore, we hypothesize that a positively charged MHC-I-restricted peptide will bind to the capsid via electrostatic interactions, thereby covering the virus with an immune-associated peptide (i.e., a tumor-specific MHC-I-restricted peptide). 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, which we used as a pattern epitope on MHC-I.

[0178] To allow electrostatic interactions between the neutral, hydrophobic SIINFEKL peptide and the negatively charged viral surface, we added a poly-lysine (poly-K) chain to the peptide sequence. This chemical modification increased the net charge of the peptide from 0 to +6 under physiological conditions.mV. Next, we investigated the interaction between the viral capsid and the modified peptide by surface plasmon resonance (SPR). Specifically, we coated the APTES silica SiO2 sensor with OAD and injected incremental concentrations of SIINFEKL or poly-K-SIIN into the flow system (Fig. 8B). No increase in signal was observed with the unmodified peptide (Fig. 8B, dashed line), while a concentration-dependent increase in signal was observed with the modified peptide (Fig. 8B, solid line), demonstrating that peptide modification significantly increased the interaction with the adenovirus capsid.

[0179] Next, we investigated the optimal concentration of peptide required for effective coverage of the viral surface. To this end, we evaluated the net charge and hydrodynamic diameter of the virus-peptide complexes obtained from different OAD:peptide ratios (1:5, 1:50, 1:100, and 1:500). We observed a clear relationship between the amount of positively charged peptide and the net charge of the complex in the reaction (Fig. 8A). The lowest ratio (1:5) increased the viral particle charge from -29.7 ± 0.5 mV to +6.3 ± 0.06 mV, although severe aggregation was observed under these conditions, as indicated by the increase in complex size (800 ± 13.5 nm). Above 1:5, the net charge increased, reaching a plateau in the kinetics; indeed, we measured Z potentials of +17.5 ± 0.2 mV, +18.4 ± 0.1 mV, and +18 ± 0.8 mV for ratios of 1:50, 1:100, and 1:500, respectively. However, only at a ratio of 1:500 did the complex diameter decrease to below 120 nm, representing the normal diameter of adenovirus particles (Fig. 8A). The same experiment was repeated using peptide concentrations instead of ratios to improve reproducibility (Fig. 12).

[0180] Modified MHC-I epitopes adsorbed onto PeptiCRAd were efficiently cross-presented.

[0181] To induce an effective cytotoxic T-lymphocyte-mediated immune response, the peptide must be presented to naïve CD8+ T lymphocytes via MHC-I on the APC. Therefore, we investigated whether the presence and position of the poly-K chain could affect cross-presentation efficiency. For this purpose, we used native SIINFEKL or two different lysine-extended versions: poly-K-SIINFEKL (N-terminal extended) and SIINFEKL-poly-K (C-terminal extended) pulsed in vitro cultured spleen cells (from C57BL / 6 mice). As a negative control, we included extended SIINFEKL containing aminocaproic acid (AHX) residues, which are known lysine analogs capable of inhibiting proteasome proteolytic activity. Then, using an antibody that specifically recognizes MHC-I loaded with SIINFEKL, we evaluated the cross-presentation of SIINFEKL (38).

[0182] As expected, 98.5% of the splenocytes pulsed with SIINFEKL were positive for the presence of SIINFEKL on the MHC-I molecule of the splenocyte membrane (Fig. 13A). Interestingly, the position of the polyK chain in the peptide sequence significantly altered the proportion of stained cells. In fact, 94.5% of the splenocytes pulsed with the N-terminal extended peptide cross-presented SIINFEKL. In contrast, when splenocytes pulsed with the C-terminal extended SIINFEKL-polyK pulsed peptide, the stained population decreased to 27.1%. When the negative control SIINFEKL-AHX-polyK pulsed peptide was used, only 1.36% of the splenocytes cross-presented the SIINFEKL peptide. Based on these findings, we selected the N-terminal extended version (polyK-SIINFEKL) for further research.

[0183] Next, we investigated whether the adsorption of modified SIINFEKL onto the viral capsid affected its cross-presentation. As in previous experiments, we incubated mouse spleen cells with either the peptide SIINFEKL or poly-K-SIINFEKL or OVA-PeptiCRAd. We found that N-terminal extended poly-K-SIINFEKL complexed with OAD to form PeptiCRAd, allowing efficient MHC-I-restricted presentation of the SIINFEKL peptide (Fig. 13B).

[0184] PeptiCRAd showed unchanged infectivity and intact oncolytic activity compared to the unmodified virus.

[0185] OAd is able to selectively infect tumor cells and lyse them via the OAD replication cycle. Therefore, we investigated whether coating a virus with a modified peptide would affect its biological properties. We selected a human colorectal adenocarcinoma cell line (CACO-2) expressing low levels of Coxsackievirus and adenovirus receptor (CAR) and two human melanoma cell lines (SK-MEL-2 and A2058) expressing high levels of CAR to study. First, we performed an in vitro viability analysis comparing OVA-PeptiCRAd with unmodified viral Ad5D24 (Figure 14A), which showed no significant difference in oncolytic activity. As expected, the most infectious condition (100 vp / cell) was associated with the lowest viability across all cell lines. Furthermore, we demonstrated that pepti-K-SIINFEKL was not cytotoxic.

[0186] Next, we assessed the infectivity of PeptiCRAd using the same in vitro cell lines via immunocytochemistry (ICC) analysis (Figure 14B). However, we observed no differences in the SK-MEL-2, CACO-2, and A2058 cell lines, and PeptiCRAd showed a significant increase in infectivity compared to naked adenovirus (P<0.01).This increase may be due to the different charges of PeptiCRAd and naked adenovirus (36).

[0187] Antitumor efficacy and immunological study of PeptiCRAd cancer vaccine in mouse melanoma model

[0188] To thoroughly investigate the antitumor efficacy of PeptiCRAd and the antitumor immunity it promotes, we first used a mouse melanoma model overexpressing chicken OVA (B16-OVA) (35). Specifically, B16-OVA was implanted into the flanks of mice, and the established tumors were then treated. Experiments were conducted using OAD (Ad5D24) carrying D24 deletion in E1A (37), and then repeated with CpG-rich adenovirus (Ad5D24-CpG) (39) to further enhance immunity (Fig. 15). The study group included mice treated with OVA-PeptiCRAd, non-combined Ad5D24-CpG and SIINFEKL (Ad5D24-CpG+SIINFEKL), OAD alone (Ad5D24-CpG) or peptide (SIINFEKL), or saline solution (simulated group).

[0189] Compared with the simulated treatment or the mixture of OAD and SIINFEKL (P<0.01), PeptiCRAd treatment significantly reduced tumor growth. At the end of the experiment, the mean tumor volume in the OVA-PeptiCRAd-treated mice was lower than that in all other groups (120.4 ± 31.6 mm3 compared to 697.7 ± 350 mm3 in the simulated group, 255 ± 61.5 mm3 in SIINFEKL, and 713.7 ± 292.6 mm3 in Ad5D24-CpG and 489.7 ± 73.2 mm3 in Ad5D24-CpG+SIINFEKL; Figure 15A).

[0190] Mice were sacrificed at two different time points (day 7 and day 16 for the early and late time points, respectively), and spleens, tumors, and draining lymph nodes were collected for immunoassay. This analysis revealed a large population of SIINFEKL-specific CD8+ T cells (CD8+OVA+ T cells) in the inguinal draining lymph nodes in mice treated with PeptiCRAd (7.4% on day 7 and 3.2% on day 16). The same analysis showed no significant differences in tumors at early time points, but a substantial increase was observed at later time points (0.23% in OVA-PeptiCRAd on day 16 compared to 0.02% in the mock group, 0.03% in SIINFEKL, 0.01% in Ad5D24-CpG, and 0.02% in Ad5D24-CpG+SIINFEKL, Figures 15B and C).

[0191] We then investigated the correlation between tumor size and the populations of OVA-specific T cells (CD8+OVA+ T cells) in the spleen, lymph nodes, and tumors. We calculated Pearson r-values ​​to estimate the nature of the correlation (negative values, negative correlation; positive values, positive correlation) and observed a negative correlation between tumor volume and the degree of anti-OVA response (Fig. 15D), indicating that the animal group with smaller tumors corresponded to the animal group with a stronger CD8+OVA+ T cell population. Subsequently, r² values ​​were calculated for each sample group to assess the strength of this correlation (spleen, r² = 0.5719; lymph nodes, r² = 0.6385; tumor, r² = 0.7445). Interestingly, in the correlation analysis, the PeptiCRAd group (red dots in Fig. 15D) consistently showed the smallest tumor volume and the largest immune response.

[0192] Finally, to deepen our understanding of the mechanism of PeptiCRAd, we evaluated a population of mature DCs (CD19-CD3-CD11c+CD86 high-peptide) presenting SIINFEKL peptide on MHC-I in mouse spleen. At late time points, the proportion of mature SIINFEKL-presenting DCs in mice treated with OVA-PeptiCRAd was significantly higher (P<0.05) than in mice treated with non-compound Ad5D24-CpG+SIINFEKL. When considering both time points, PeptiCRAd was the only treatment that induced an increase in mature SIINFEKL-presenting DCs, as shown by a 9.67-fold increase in the CD86 high OVA+ DC population (Fig. 15E).

[0193] These results suggest that the expansion of the mature and epitope-specific DC repertoire may underlie the enhanced antitumor efficacy of PeptiCRAd.

[0194] Multivalent PeptiCRAd exhibited antitumor activity against distal, untreated melanoma.

[0195] A major advantage of using oncolytic vaccines is that the resulting immune response promotes targeting not only the primary tumor but also the spread of metastases. For this reason, we investigated the antitumor efficacy of PeptiCRAd against untreated contralateral tumors in a murine melanoma model. In the same experimental group, we also investigated whether targeting two tumor antigens (via multivalent PeptiCRAd) instead of a single one would increase the overall efficacy. Therefore, we selected two tumor-specific MHC-I-restricting epitopes to coat the oncolytic virus Ad5D24-CpG:SVYDFFVWL (TRP-2180-188; restricted to murine MHC-I molecule H-2Kb) and KVPRNQDWL (human gp10025-33, or hgp100; restricted to murine MHC-I molecule H-2Db (40)). For these experiments, weWe used highly aggressive melanoma B16-F10 cells, which express two tumor antigens (41). The peptides were modified with a polykine chain at their N-terminus to facilitate their adsorption to the viral capsid, as previously done for SIINFEKL.

[0196] We first implanted 1 × 10⁵ B16-F10 cells into the right flank of C57BL / 6 mice. After 10 days, we started treatment as follows: i) saline solution (simulated group), ii) naked oncolytic virus (Ad5D24-CpG), and iii) double-coated TRP-2-hgp100-PeptiCRAd. Intratumoral treatment was administered every two days, as illustrated in the schematic diagram in Figure 6A. Two days after the last round of injections, 3 × 10⁵ B16-F10 cells were injected into the left flank of the mice, and melanoma growth was tracked. Mice treated with double-coated PeptiCRAd showed significantly reduced tumor growth compared to controls (P<0.001) (day 11; Fig. 16A). Analysis of secondary and untreated tumors revealed the advantage of double-coated PeptiCRAd over all other groups. Specifically, at the end of the experiment, secondary tumors in this group were significantly smaller compared to those in controls that received saline or Ad5D24-CpG alone (P<0.01; Fig. 16B).

[0197] To better elucidate the mechanisms supporting these results, we performed flow cytometry analysis to investigate specific T-cell responses against the two epitopes. In mice treated with TRP-2-hgp100 PeptiCRAd, we observed an accumulation of more epitope-specific CD8+ T cells than in all other groups (Fig. 16C).

[0198] In summary, these results confirm that the PeptiCRAd approach is effective for both less immunogenic and more aggressive melanoma models. Furthermore, targeting multiple antigens produced strong effects on both treated and untreated tumors. Therefore, it is possible to generate multivalent PeptiCRAds, and they could provide us with the possibility of targeting different tumor antigens and thus overcoming certain immune evasions of tumors.

[0199] PeptiCRAds have shown enhanced efficacy and anti-tumor immunity in humanized mice bearing human tumors.

[0200] Finally, we wanted to evaluate the efficacy of PeptiCRAds in a model that could provide information on the feasibility of converting them to a clinical setting. Therefore, we chose a more refined humanized mouse model. For this purpose, triple gene knockout mice (NOD.Cg-Prkdcscid-IL2rgtm1Wjl / SzJ, or NSG) were first transfected with the human melanoma cell line SK-MEL-2. When the tumors reached a significant size, partially matched human peripheral blood mononuclear cells (PBMCs) from healthy donors were transfected into the tumor.In mice. One day later, the mice were treated with PeptiCRAd, uncoated virus, or saline solution. For this experiment, we selected a peptide derived from melanoma-associated antigen A1 (MAGE-A196-104; SLFRAVITK) and modified it to allow interaction with the viral capsid (poly-K-SLFRAVITK). In this experiment, since we were studying the full human immune system, we selected an OAd expressing human GM-CSF, which we have previously demonstrated to have enhanced activity in immune systems including cancer patients (8).

[0201] We found that MAGE-A1 PeptiCRAd showed increased efficacy compared to control treatment, as indicated by a rapid reduction in tumor volume (Figs. 17A and B). Finally, we investigated whether a stronger immune response could explain the increased antitumor efficacy of PeptiCRAd in this model. To this end, we investigated the presence of MAGE-A196-104-specific CD8+ T cells by pentamer staining (Fig. 17C), and we found the largest population of human MAGE-specific T cells (CD8+MAGE-A1+) in the spleen of mice treated with PeptiCRAd.

[0202] These data confirm our previous findings that PeptiCRAd utilizes the natural immunogenicity of oncolytic viruses to stimulate tumor-specific immune responses, thereby enhancing the efficacy of cancer immunoviral therapy.

[0203] Analysis of MHC-I-specific peptides for any disease and coating of adenovirus capsids and their uses

[0204] Any MHC-I-specific peptide was identified by comparing the MHC-I-restricted peptides represented by individual DCs and infected diseased cells. One or more peptides presented by the two cell groups were selected for coating adenovirus vectors.

[0205] Any adenovirus vector was selected and coated according to any of the methods described in Method 2.

[0206] The coated virus was used to treat the patient's disease.

[0207] References

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[0241] 34 . R . B . Dell , S . Holleran , R . Ramakrishnan , Sample size determination . ILAR journal / National Research Council , Institute of Laboratory Animal Resources43, 207 (2002) .

[0242] 35. M. W. Moore, F. R. Carbone, M. J. Bevan, Introduction of soluble protein into the class I pathway of antigen processing and presentation . Cell54, 777 (Sep 9, 1988) .

[0243] 36 . A. Fasbender et al ., Complexes of adenovirus with polycationic polymers and cationic lipids increase the efficiency of gene transfer in vitro and in vivo . The Journal of biological chemistry272 , 6479 (Mar 7 , 1997) .

[0244] 37 . C . Heise et al ., An adenovirus E1A mutant that demonstrates potent and selective systemic anti‑tumoral efficacy. Nature medicine6, 1134 (Oct, 2000) .

[0245] 38 . Y . Deng et al . , Assembly of MHC class I molecules with biosynthesized endoplasmic reticulum‑targeted peptides is inefficient in insect cellsand can be enhanced by protease inhibitors. J Immunol161, 1677 (Aug 15, 1998).

[0246] 39. V. Cerullo et al., An oncolytic adenovirus enhanced for toll‑like receptor 9 stimulation increases antitumor immune responses and tumor clearance. Molecular therapy: the journal of the American Society of Gene Therapy20, 2076 (Nov, 2012).

[0247] 40. WW Overwijk et al., gp100 / pmel 17 is a murine tumor rejection antigen: induction of "self"-reactive, tumoricidal T cells using high-affinity, altered peptide ligand. The Journal of experimental medicine188, 277 (Jul 20, 1998).

[0248] 41. W. W . Overwijk , N . P. Restifo, B16 as a mouse model for human melanoma. Current protocols in immunology / edited by John E. Coligan ... [et al., manual 25 / 26 pages 29 CN 121313807 A al.] Chapter 20, Unit 20 1 (May, 2001). Manual 26 / 26 pages 30 CN 121313807 A Figure 1 Figure 2 Figure 3A Manual Figure 1 / 15 pages 31 CN 121313807 A Figure 3B Figure 4 Manual Figure 2 / 15 pages 32 CN 121313807 A Figure 5 Manual Figure 3 / 15 pages 33 CN 121313807 A Figure 6A Figure 6B Figure 7 Manual Figure 4 / 15 pages34 CN 121313807 A Figure 8A Figure 8B Instruction Manual Drawings 5 / 15 Page 35 CN 121313807 A Figure 9 Figure 10A Instruction Manual Drawings 6 / 15 Page 36 CN 121313807 A Figure 10B Figure 11 Instruction Manual Drawings 7 / 15 Page 37 CN 121313807 A Figure 12 Figure 13A Instruction Manual Drawings 8 / 15 Page 38 CN 121313807 A Figure 13B Instruction Manual Drawings 9 / 15 Page 39 CN 121313807 A Figure 14A Figure 14B Instruction Manual Drawings 10 / 15 Page 40 CN 121313807 A Figure 15A Figure 15E Instruction Manual Drawings 11 / 15 Page 41 CN 121313807 A Figure 15B Figure 15C Instruction Manual Drawings 12 / 15 Page 42 CN 121313807 A Figure 15D Appendix to the Instruction Manual, Page 13 / 15, 43 CN 121313807 A Figure 16A Figure 16B Figure 16C Appendix to the Instruction Manual, Page 14 / 15, 44 CN 121313807 A Figure 17A Figure 17B Figure 17C Appendix to the Instruction Manual, Page 15 / 15, 45 CN 121313807 A Title: COATED ONCOLYTIC ADENOVIRUSES FOR CANCER VACCINES Abstract The present invention relates to adenoviral vectors, wherein the viral capsid has been coated with polypeptides, which are capable of stimulating a peptide-specific immune response in a subject and uses thereof. Furthermore, the present invention relates to methods of treating diseases, e.g., cancer, by adenoviral vectors which have been coated by polypeptides causing a peptide-specific immune response.Also the present invention relates to a method of coating adenoviral vectors by specific peptides as well as to a method of identifying those peptides suitable for coating the capsid of an adenoviral vector.

Claims

1. Use of an adenoviral vector containing a polypeptide attached to a viral capsid in the preparation of a medicament for stimulating an antitumor peptide-specific immune response in an individual in need, wherein the polypeptide is not genetically encoded by the adenoviral vector but is covalently or non-covalently attached to the viral capsid, and the polypeptide is a class I major histocompatibility complex (MHC-I)-specific polypeptide or a class II major histocompatibility complex (MHC-II)-specific polypeptide, and is also a tumor-specific and DC-specific polypeptide.

2. An adenovirus vector for use in stimulating an antitumor peptide-specific immune response in an individual, comprising a polypeptide attached to a viral capsid, wherein the polypeptide is not genetically encoded by the adenovirus vector but is covalently or non-covalently attached to the viral capsid, and the polypeptide is a class I major histocompatibility complex (MHC-I)-specific polypeptide or a class II major histocompatibility complex (MHC-II)-specific polypeptide, and is tumor-specific and DC-specific polypeptide.

3. The use as described in claim 1, wherein two or more different N-terminal polylysine-modified polypeptides capable of stimulating a peptide-specific antitumor immune response in an individual are attached to the viral capsid.

4. The adenovirus vector of claim 2, wherein two or more different N-terminal polylysine-modified polypeptides capable of stimulating a peptide-specific antitumor immune response in an individual are attached to the viral capsid.

5. The use as described in claim 1, wherein the cancer is selected from nasopharyngeal carcinoma, synovial carcinoma, renal carcinoma, connective tissue cancer, melanoma, lung cancer, colorectal cancer, brain cancer, laryngeal cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, retinal angiomatosis, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, Ewing sarcoma, cancer of unknown primary site, carcinoid, gastrointestinal carcinoid, fibrosarcoma, breast cancer, Paget's disease, esophageal cancer, gallbladder cancer, Head cancer, eye cancer, neck cancer, nephroblastoma, liver cancer, Kaposi's sarcoma, prostate 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, gastric cancer, thymic cancer, thyroid cancer, trophoblastic cancer, hydatidiform mole, uterine cancer, vaginal cancer, vulvar cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gingival 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.

6. The use as claimed in claim 1, wherein the cancer is selected from colon cancer, rectal cancer, and small bowel cancer.

7. The use as claimed in claim 1, wherein the cancer is selected from cervical cancer and endometrial cancer.

8. The use as described in claim 1 or 3, wherein the individual is a person or an animal.

9. The use as described in claim 1 or 3, wherein the adenovirus vector is administered by intratumoral injection, intra-arterial injection, intravenous injection, intrapleural injection, intracystic injection, intracavitary injection, or intraperitoneal injection, or oral administration.

10. The adenovirus vector of claim 2, wherein the cancer is selected from nasopharyngeal carcinoma, synovial carcinoma, renal carcinoma, connective tissue cancer, melanoma, lung cancer, colorectal cancer, brain cancer, laryngeal cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, retinal angiomatosis, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, Ewing sarcoma, cancer of unknown primary site, carcinoid, gastrointestinal carcinoid, fibrosarcoma, breast cancer, Paget's disease, cervical cancer, esophageal cancer, gallbladder cancer. Head cancer, eye cancer, neck cancer, nephroblastoma, liver cancer, Kaposi's sarcoma, prostate 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, gastric cancer, thymic cancer, thyroid cancer, trophoblastic cancer, hydatidiform mole, uterine cancer, endometrial cancer, vaginal cancer, vulvar cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gingival 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.

11. The adenovirus vector of claim 2, wherein the cancer is selected from colon cancer, rectal cancer, and small bowel cancer.

12. The adenovirus vector of claim 2, wherein the cancer is selected from cervical cancer and endometrial cancer.

13. The adenovirus vector of claim 2 or 4, wherein the individual is a human or animal.

14. The adenovirus vector of claim 2 or 4, wherein the adenovirus vector is administered by intratumoral injection, intra-arterial injection, intravenous injection, intrapleural injection, intracystic injection, intracavitary injection, or intraperitoneal injection, or orally.

15. A method for modifying an adenovirus capsid, wherein the method comprises covalently or non-covalently linking a polypeptide to the adenovirus capsid, wherein the polypeptide is a class I major histocompatibility complex (MHC-I)-specific polypeptide or a class II major histocompatibility complex (MHC-II)-specific polypeptide, and is also an antitumor-specific and DC-specific polypeptide, and wherein the modified adenovirus vector is capable of stimulating a peptide-specific antitumor immune response in an individual.

16. The use as described in claim 1 or 3, wherein the polypeptide has been attached to the capsid via electrostatic bonding.

17. The use as described in claim 1 or 3, wherein the serotype of the adenovirus vector backbone is selected from serotype 3 or 5.

18. The use as described in claim 1 or 3, wherein the adenovirus vector contains a 24bp deletion or an E1 gene deletion, or the vector is a helper virus-dependent vector.

19. The use as described in claim 1 or 3, wherein the adenovirus vector comprises one or more transgenes.

20. The use as described in claim 1 or 3, wherein the adenovirus vector comprises a capsid modification.

21. The use as described in claim 1 or 3, wherein the adenovirus vector is Ad5 / 3 or Ad5 / 35 comprising an Ad5 nucleic acid backbone and a fibrous node, wherein the fibrous node is selected from Ad3 fibrous nodes, Ad35 fibrous nodes, Ad5 / 3 chimeric fibrous nodes, and Ad5 / 35 chimeric fibrous nodes.

22. The adenovirus vector of claim 2 or 4, wherein different N-terminal polylysine-modified polypeptides have been attached to the capsid via electrostatic linkage.

23. The adenovirus vector of claim 2 or 4, wherein the serotype of the adenovirus vector backbone is selected from serotype 3 or 5.

24. The adenovirus vector of claim 2 or 4, wherein the adenovirus vector contains a 24bp deletion or an E1 gene deletion, or the vector is a helper virus-dependent vector.

25. The adenovirus vector of claim 2 or 4, wherein the adenovirus vector comprises one or more transgenes.

26. The adenovirus vector of claim 2 or 4, wherein the adenovirus vector comprises a capsid modification.

27. The adenovirus vector of claim 2 or 4, wherein the adenovirus vector is Ad5 / 3 or Ad5 / 35 comprising an Ad5 nucleic acid backbone and a fibrous knot, wherein the fibrous knot is selected from Ad3 fibrous knot, Ad35 fibrous knot, Ad5 / 3 chimeric fibrous knot and Ad5 / 35 chimeric fibrous knot.

28. The method of claim 15, wherein the two or more different polypeptides have been attached to the capsid via electrostatic bonding.

29. The method of claim 15, wherein the serotype of the adenovirus vector backbone is selected from serotype 3 or 5.

30. The method of claim 15, wherein the adenovirus vector contains a 24bp deletion or an E1 gene deletion, or the vector is a helper virus-dependent vector.

31. The method of claim 15, wherein the adenovirus vector comprises one or more transgenes.

32. The method of claim 15, wherein the adenovirus vector comprises a capsid modification.

33. The method of claim 15, wherein the adenovirus vector is Ad5 / 3 or Ad5 / 35 comprising an Ad5 nucleic acid backbone and a fibrous node, wherein the fibrous node is selected from Ad3 fibrous nodes, Ad35 fibrous nodes, Ad5 / 3 chimeric fibrous nodes, and Ad5 / 35 chimeric fibrous nodes.

34. A pharmaceutical composition comprising the adenovirus vector of any one of claims 1, 2 or 15.

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