Oncolytic adenoviruses encoding bispecific antibodies and methods and uses related thereto

Abstract

The invention relates to the fields of life science and medicine. In particular, the present invention relates to cancer therapy in humans. More specifically, the present invention relates to oncolytic adenoviral vectors encoding bispecific monoclonal antibodies. In addition, the present invention relates to methods and uses utilizing oncolytic adenoviral vectors and along with adoptive cell therapy.

Description

[0001] This application is a divisional application of patent application No. 201680016329.4, filed on March 17, 2016, with a priority date of March 17, 2015, entitled "Oncolytic Adenovirus Encoding Bispecific Antibodies and Related Methods and Uses". Invention Field

[0002] This invention relates to the fields of life sciences and medicine. Specifically, it relates to the treatment of cancer in humans. More specifically, it relates to oncolytic adenoviral vectors encoding bispecific monoclonal antibodies. Furthermore, this invention relates to methods and uses utilizing oncolytic adenoviral vectors in conjunction with adoptive cell therapy. Background of the Invention

[0003] New therapies for cancer treatment are constantly being developed. Adoptive cell therapy (ACT) is an effective approach for treating cancer, as well as other diseases such as infections and graft-versus-host disease. Adoptive cell transfer involves the passive transplantation of ex vivo cells (most commonly immune-derived cells) into a host to transfer the immunological function and characteristics of the graft. Adoptive cell transfer can be autologous, as is common in adoptive T-cell therapy, or allogeneic, as is typical for treating infections or graft-versus-host disease. Clinically, common implementations of this approach include transferring immune-boosting or tolerance-inducing cells, such as lymphocytes, to patients to increase immunity against viruses and cancer, or to promote tolerance in the context of autoimmune diseases such as type 1 diabetes or rheumatoid arthritis.

[0004] Adoptive transfer of autologous tumor-infiltrating lymphocytes (TILs) or genetically reoriented peripheral blood mononuclear cells has been successfully used to treat patients with melanoma and CD19-expressing hematologic malignancies. In ACTs, the most commonly used cell type is T cells, sometimes sorted for CD8+, but other variations include CD4+ cells, NK cells, delta-gamma T cells, regulatory T cells, and peripheral blood mononuclear cells. Cells can be unmodified, as in TIL therapy, or genetically modified. In TIL therapy, unsorted polyclonal cells are used. There are two common approaches to achieve genetic targeting of T cells to tumor-specific targets. One is to transfer T cell receptors with known specificity (TCR therapy) and a matching human leukocyte antigen (HLA, known as the major histocompatibility complex in rodents) type. The other is to modify cells with artificial molecules such as chimeric antigen receptors (CARs). This approach is HLA-independent and more flexible in targeting cell surface molecules. For example, single-chain antibodies can be used, and CARs can also incorporate co-stimulatory domains. However, CAR cell targets need to be on the target cell membrane, while TCR modification can utilize intracellular targets. In both TCR and CAR therapies, T cells are obtained from the patient's peripheral blood.

[0005] Despite advancements in adoptive cell therapy, clinical outcomes for adoptive T-cell therapy in non-melanoma solid tumors, which account for over 90% of human cancers and 95% of cancer deaths, have been disappointing. The primary reason is the highly immunosuppressive tumor microenvironment, which inactivates and anergizes T-cell grafts, inhibits local graft proliferation, and prevents the transport of adopted T-cells to the tumor. Currently, there are no effective tools to address this problem.

[0006] T-cell engagers have been used in cancer treatment. The main categories are trifunctional antibodies, chemically linked Fab, and bispecific T-cell engagers (BiTEs), the latter being the most advanced in clinical use (Baeuerle PA, Reinhardt C. Cancer Res. 2009 Jun 15; 69(12):4941-4). Although several BiTEs have been studied preclinically, and two (blinatumomab, an anti-CD19 BiTE, and solidumab, an anti-EpCAM Bite) are in clinical trials, many problems have arisen. One major problem is on-target-off-tumor toxicity, which leads to a high rate of adverse events, including a 12% toxicity mortality rate in the blinatumomab clinical trial (Topp MS et al. 2011, J Clin Oncol. Jun 20; 29(18):2493-8). Another issue is the insufficient concentration of BiTE at the target (tumor), which is particularly problematic in the context of solid tumors, where the mass of the tumor forms a barrier to BiTE penetration and concentration. This may explain why no formal response (reduction in tumor size that meets RECIST criteria) was observed in trials of solidomab. The best response was transient disease stabilization, achieved in 38% of patients (Walter M et al. 2012, J Clin Oncol 30, (suppl; abstr 2504)). Another problem with BiTE is its short half-life in humans, requiring continuous infusion, which is not a practical solution for routine use.

[0007] The use of oncolytic viral vectors armed with T-cell adjuvants for cancer therapy has been proposed. WO 2014138314 A1 (PCT / US2014 / 020935) and Yu et al. (2014, Mol Ther 22(1):102-11) describe oncolytic vaccinia virus encoding anti-EphA2 BiTe. Regarding vectored delivery of BiTE, single-chain molecules, including double-stranded constructs such as BiTE, cannot be automatically secreted from mammalian cells. In fact, the poor secretion of single-chain molecules and constructs such as BiTE has been a barrier to their application in gene therapy. Antibodies are typically produced by plasma cells of the B-cell lineage, and unsurprisingly, their production and release from epithelial tumor cells are problematic.

[0008] There is still room for improvement in the efficacy of oncolytic virus vectors, whether used alone or in combination with other therapies. Generally, this aims to ensure increased specificity and adequate tumor-killing ability in the treatment.

[0009] This invention provides an effective tool and method for cancer therapeutics by utilizing specific viral vectors, for example, in conjunction with adoptive cell therapy. Invention Overview

[0010] The object of this invention is to provide simple methods and tools for overcoming the aforementioned problems of inefficient, unsafe, and unpredictable cancer therapies. In one embodiment, the invention provides novel methods and means for cell therapy. The object of the invention is achieved through specific viral vectors, methods, and arrangements, which are characterized by the statements in the independent claims. Specific embodiments of the invention are disclosed in the dependent claims.

[0011] This invention proposes using specific oncolytic adenoviruses to address the problem of highly immunosuppressive tumor microenvironments that inactivate and render incapable of T-cell grafts, inhibit local graft proliferation, and prevent the transport of adoptively transferred T cells to the tumor. This invention is based on the surprising insight that oncolytic adenoviruses encoding bispecific T-cell adaptors (BiTE) can solve this problem. Figure 1 In particular, data relating to this invention indicate that adenoviruses can induce tumors in mice and danger signals in humans, as exemplified by the production of interferon gamma. Figure 2 This leads to a decrease in the expression of TIM3 (TIM3 is a key indicator of tumor immunosuppression). Figure 3 Importantly, even if adenovirus alone can generate danger signals in tumors, this is insufficient to recruit T cells to the tumor. Figure 4 Therefore, for optimal enhancement of adoptive cell therapy, it is necessary to use BiTE-armored oncolytic adenovirus (BTE). Figure 1 )

[0012] Notably, we have human data demonstrating that TIM3 expression and the ability of oncolytic adenovirus to downregulate TIM3 are associated with patient survival. This is strong data indicating that adenovirus-induced danger signaling leads to downregulation of tumor immunosuppression, which is associated with clinical benefit in patients. Figure 10 Importantly, not all oncolytic viruses are created equal, and in fact, vaccinia virus cannot generate danger signals in tumors, and is therefore incompatible with adenoviruses used for tumor immunotherapy by locally generating BiTE. Figure 5-6 ).

[0013] This invention solves the problems of poor systemic toxicity, poor local efficacy, and short half-life of BiTE by generating BiTE locally in the tumor via an adenovirus vector. This feature is particularly advantageous in the context of solid tumors. Figure 9 ).

[0014] Furthermore, this invention solves the problem of poor secretion of single-chain BiTE molecules in a surprising way: when using oncolytic adenoviruses that replicate only in tumor cells, and where the final step of replication is cell lysis, BiTE is released into the tumor microenvironment. Figure 8 In other words, the present invention solves the problem of BiTE secretion in a surprising way by utilizing oncolysis as a release device. According to the invention, BiTE secretion is not required, and in fact, it is not preferred, as another method, to restrict BiTE expression to the tumor (only tumor cells are lysed by the virus).

[0015] BiTE generation at the tumor site can recruit adoptive T cell grafts to the tumor. Figure 1 Binding to cell surface molecular receptors (e.g., CD3 receptors) activates graft cells at the tumor site. Furthermore, adenovirus oncolysis induces a danger signal that counteracts tumor immunosuppression. Together, these components achieve an anti-immunosuppressive effect that cannot be achieved by using any one component alone. Notably, adenovirus is unique among oncolytic viruses in its ability to induce anti-immunosuppressive danger signals by binding to pathogen-associated pattern recognition receptors. Moreover, adenovirus has a significant effect on T cells, whereas many other oncolytic viruses, such as vaccinia virus, are rather stealthy in this respect. In other words, vaccinia virus cannot be used to enhance adoptive cell therapy. Finally, this specification presents data showing that vaccinia virus is not a good platform for enhancing adoptive cell therapy, while adenovirus is the optimal device for counteracting tumor immunosuppression.

[0016] Antiviral immunization has been considered limiting for viral therapies, including oncolytic adenovirus. One implementation of antiviral immunization involves antiviral T cells. However, this invention surprisingly reveals that when oncolytic adenoviruses generate BiTE at the tumor site, antiviral T cells can retarget the tumor. This effect is amplified during treatment because oncolytic viral replication leads to the formation of additional antiviral T cells, which then also target the tumor via the virus-generated BiTE. Figure 7 ).

[0017] In one embodiment, the present invention relates to enhancing T-cell therapy with oncolytic adenovirus encoding BiTE. Oncolytic adenovirus is an optimal platform for using BiTE to enhance T-cell therapy due to the unexpected synergistic effect between oncolysis at the tumor site and the anti-immunosuppressive effect of BiTE expression.

[0018] This specification describes the construction of recombinant adenovirus vectors, methods related to adenovirus vectors, and their various applications. Furthermore, the adenovirus vector encoding a T-cell connective of the present invention can be combined with adoptive cell therapeutic agents for cancer treatment.

[0019] The advantages of this invention are achieved through a method of treating malignant tumors, which involves administering an effective amount of the adenovirus vector of this invention (e.g., alone or with TIL) to a patient with cancer to result in the regression or stabilization of the cancer.

[0020] This application relates to an oncolytic adenovirus vector, which contains

[0021] The deletion of nucleic acid sequences in the E3 region, and

[0022] The nucleic acid sequence encoding the bispecific monoclonal antibody is replaced with the nucleic acid sequence missing in the E3 region.

[0023] This application also relates to oncolytic adenovirus vectors, which contain

[0024] The deletion of nucleic acid sequences in the E3 region, and

[0025] The nucleic acid sequence encoding the bispecific monoclonal antibody is used to replace the missing nucleic acid sequence in the E3 region.

[0026] The bispecific monoclonal antibody comprises a single-chain variable fragment (scFv) specific to cell surface molecules and a scFv specific to tumor antigens.

[0027] In addition, the present invention relates to a pharmaceutical composition comprising an oncolytic adenovirus vector, wherein the oncolytic adenovirus vector comprises a deletion of a nucleic acid sequence in the E3 region and a nucleic acid sequence encoding a bispecific monoclonal antibody, replacing the deleted nucleic acid sequence in the E3 region.

[0028] Furthermore, the present invention relates to a combination of an oncolytic adenovirus vector and an adoptive cell therapy composition, wherein the oncolytic adenovirus vector comprises a deletion of a nucleic acid sequence in the E3 region and a nucleic acid sequence encoding a bispecific monoclonal antibody, replacing the deleted nucleic acid sequence in the E3 region.

[0029] Furthermore, this invention relates to a combination of the oncolytic adenovirus vector and the adoptive cell therapy composition of the present invention for the treatment of cancer.

[0030] Furthermore, the present invention relates to the oncolytic adenovirus vector of the present invention in conjunction with adoptive cell therapy compositions for treating cancer.

[0031] Furthermore, the present invention relates to an oncolytic adenovirus vector for treating cancer, used in conjunction with adoptive cell therapy compositions.

[0032] The present invention also relates to a method of treating cancer in a subject, wherein the method comprises administering the oncolytic adenovirus vector of the present invention to the subject.

[0033] The present invention also relates to an oncolytic adenovirus vector for enhancing the efficacy of adoptive cell therapy in subjects, said oncolytic adenovirus vector comprising a nucleic acid sequence encoding a bispecific monoclonal antibody with and without deletions in the E3 region and substitutions for the E3 deletion region.

[0034] The present invention also relates to a method for enhancing the efficacy of adoptive cell therapy in subjects by administering an oncolytic adenovirus to a subject in need, the oncolytic adenovirus vector comprising a nucleic acid sequence encoding a bispecific monoclonal antibody with and without an E3 region, wherein the subject has been or is to be administered adoptive cell therapy.

[0035] Furthermore, the present invention relates to the use of the oncolytic adenovirus vector of the present invention in the preparation of a medicament for treating cancer in subjects.

[0036] Furthermore, the present invention relates to the use of the oncolytic adenovirus vector of the present invention in the preparation of a medicament for increasing the efficacy of adoptive cell therapy in subjects.

[0037] The advantages of the arrangement of this invention include, but are not limited to, enhanced therapeutic efficacy and reduced side effects. Serious adverse events, even death, are prevented because the enhanced efficacy and antisuppressive effect of our method can reduce the need for preconditioning chemotherapy and / or radiation used in prior art methods, thus “making room” for metastatic cells and reducing tumor immunosuppression.

[0038] The present invention also relates to the following embodiments:

[0039] Implementation Method 1. An oncolytic adenoviral vector containing...

[0040] The deletion of nucleic acid sequences in the E3 region, and

[0041] The nucleic acid sequence encoding a bispecific monoclonal antibody replaces the missing nucleic acid sequence in the E3 region, wherein the bispecific monoclonal antibody comprises a single-stranded variable fragment (scFv) specific to cell surface molecules and an scFv specific to tumor antigens.

[0042] Implementation Method 2. The oncolytic adenovirus vector according to Implementation Method 1, wherein the backbone of the adenovirus vector is an adenovirus serotype 5 (Ad5) or adenovirus serotype 3 (Ad3) nucleic acid backbone.

[0043] Implementation Method 3. An oncolytic adenovirus vector according to any one of the foregoing embodiments, wherein the vector further comprises an E2F1 promoter for tumor-specific expression of E1A.

[0044] Implementation 4. An oncolytic adenovirus vector according to any one of the preceding embodiments, wherein the vector further comprises a 24bp deletion (D24) in the Rb binding constant region 2 of adenovirus E1.

[0045] Implementation Method 5. The oncolytic adenovirus vector according to any one of the foregoing embodiments, wherein the deletion of the nucleic acid sequence in the E3 region is the deletion of the viral gp19k and 6.7k reading frames.

[0046] Embodiment 6. An oncolytic adenovirus vector according to any one of the foregoing embodiments, wherein the vector comprises:

[0047] 1) E2F1 promoter for tumor-specific expression of E1A

[0048] 2) A 24bp deletion (D24) in the Rb binding constant region 2 of adenovirus E1;

[0049] 3) Deletion of nucleic acid sequences in the viral gp19k and 6.7k reading frames; and

[0050] 4) The nucleic acid sequence encoding the bipartite molecule, replacing the missing nucleic acid sequence as defined in point 3), wherein the bipartite molecule comprises a single-stranded variable fragment (scFv) specific to cell surface molecules and a scFv specific to tumor antigens.

[0051] Implementation Method 7. An oncolytic adenovirus vector according to any one of the foregoing embodiments, wherein the backbone is an Ad5 nucleic acid backbone, and the vector further comprises an Ad3 fiber knob.

[0052] Implementation Method 8. An oncolytic adenovirus vector according to any one of the foregoing embodiments, wherein the cell surface molecules are on immune effector cells.

[0053] Implementation Method 9. The oncolytic adenovirus vector according to Implementation Method 8, wherein the effector cells are T lymphocytes.

[0054] Implementation Method 10. An oncolytic adenovirus vector according to any one of the foregoing embodiments, wherein the tumor antigen is selected from Table 1 or the group consisting of mesothelin, EpCAM1 and MUC1.

[0055] Implementation Method 11. An oncolytic adenovirus vector according to any one of the foregoing embodiments, wherein the cell surface molecules are selected from CD3, CD8 and CD4.

[0056] Embodiment 12. An oncolytic adenovirus vector according to any one of the foregoing embodiments, wherein the tumor antigen is mesothelin and the cell surface molecule is CD3; the tumor antigen is EpCAM1 and the cell surface molecule is CD3; or the tumor antigen is MUC1 and the cell surface molecule is CD3.

[0057] Implementation Method 13. An oncolytic adenovirus vector according to any one of the foregoing embodiments, wherein the oncolytic adenovirus vector encodes two or more transgenes.

[0058] Implementation Method 14. The oncolytic adenovirus vector according to any one of the foregoing embodiments further comprises IL-2, TNFalpha or CD40L transgene.

[0059] Embodiment 15. A pharmaceutical composition comprising an oncolytic adenovirus vector according to any one of Embodiments 1-14.

[0060] Implementation Method 16. An oncolytic adenovirus vector according to any one of Implementation Methods 1-14, used for the treatment of cancer.

[0061] Implementation Method 17. The oncolytic adenovirus vector used in Implementation Method 16 is used in conjunction with an adoptive cell therapeutic composition.

[0062] Implementation Method 18. A method for treating cancer in a subject, wherein the method comprises administering an oncolytic adenovirus vector of any one of Implementation Methods 1-14 to the subject.

[0063] Implementation 19. The method according to Implementation 18, wherein the method further comprises administering the adoptive cell therapy composition to the subject.

[0064] Implementation 20. The oncolytic adenovirus vector used according to the method or method of Implementation 17 or 19, wherein the adoptive cell therapy composition comprises cell types selected from the group consisting of tumor-infiltrating lymphocytes (TILs), T-cell receptor-modified lymphocytes, and chimeric antigen receptor-modified lymphocytes.

[0065] Embodiment 21. The method or oncolytic adenovirus vector used according to any one of Embodiments 17 or 19-20, wherein the adoptive cell therapy composition comprises cell types selected from the group consisting of: T cells, CD8+ cells, CD4+ cells, NK cells, delta-gamma T cells, regulatory T cells, and peripheral blood mononuclear cells.

[0066] Embodiment 22. The method or oncolytic adenovirus vector used according to any one of Embodiments 17 or 19-21, wherein the adoptive cell therapy composition comprises T cells.

[0067] Implementation Method 23. The method or oncolytic adenovirus vector used according to any one of Implementation Methods 16-22, wherein the cancer is selected from the group consisting of: nasopharyngeal cancer, synovial cancer, hepatocellular carcinoma, renal cancer, connective tissue cancer, melanoma, lung cancer, bowel cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, throat cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, von Willebrand disease, etc. Hippel-Lindau disease, Zollinger-Ellison syndrome, renal cancer, anal cancer, bile duct cancer, bladder cancer, ureter cancer, brain cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, bone cancer, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, gastrointestinal carcinoid.tract, fibrosarcoma, breast cancer, Paget's disease, cervical cancer, colorectal cancer, rectal cancer, esophagus cancer, gallbladder cancer, head cancer, eye cancer, neck cancer, kidney cancer, Wilms' tumor, liver cancer, Kaposi's sarcoma, prostate cancer, lung cancer, testicular cancer, Hodgkin's disease, non-Hodgkin's lymphoma, oral cancer, skin cancer, mesothelioma, multiple myeloma, ovarian cancer, endocrine pancreatic cancer Cancer, glucagonoma, pancreatic cancer, parathyroid cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestinal cancer, stomach cancer, thymus cancer, thyroid cancer, trophoblastic cancer, hydatidiform mole, uterine cancer, endometrial cancer, vaginal cancer, vulva cancer, acoustic neuroma, mycosisFungoides, insulinoma, carcinoid syndrome, somatostatinoma, gum cancer, heart cancer, lip cancer, meninges cancer, mouth cancer, nerve cancer, palate cancer, parotid gland cancer, peritoneum cancer, pharynx cancer, pleural cancer, salivary gland cancer, tongue cancer, and tonsil cancer.

[0068] Implementation Method 24. The method or oncolytic adenovirus vector used according to any one of Implementation Methods 16-23, wherein the oncolytic virus vector and the adoptive cell therapy composition are administered to the subject simultaneously or sequentially in any order.

[0069] Implementation Method 25. The method or oncolytic adenovirus vector used according to any one of Implementation Methods 16-24 further includes administering simultaneous or sequential radiotherapy, monoclonal antibody, chemotherapy or other anticancer drugs or interventions to the subject.

[0070] Implementation Method 26. An oncolytic adenovirus vector according to any one of Implementation Methods 1-14, used to enhance the efficacy of adoptive cell therapy in subjects.

[0071] Implementation Method 27. A method for increasing the efficacy of adoptive cell therapy in a subject, which is carried out by administering an oncolytic adenovirus vector according to any one of Implementation Methods 1-14 to a subject in need, wherein adoptive cell therapy has been or will be administered to the subject. Brief description of the attached diagram

[0072] In the following, the invention will be described in more detail with reference to the accompanying drawings and specific embodiments, wherein...

[0073] Figure 1 This demonstrates the mechanism of action of T-cell therapy using an oncolytic adenovirus encoding the bispecific T-cell adaptor BiTE.

[0074] Figure 2This demonstrates the danger signals induced in tumors by adenovirus treatment. Treatment with a 5 / 3 chimeric adenovirus (an Ad5-based vector with a fiber knob from Ad3) induced danger signals in B16.OVA tumors, as demonstrated by interferon gamma expression. The binding of the adenovirus pathogen-associated molecular pattern (PAMP) to a toll-like receptor (TLR) on host cells can induce interferon-γ secretion, leading to rapid activation of both innate and adaptive immune responses. Therefore, adenoviruses can be used to generate immunogenic tumor phenotypes that are effectively recognized by the immune system.

[0075] Figure 3 This study demonstrates that adenoviruses possess anti-immunosuppressive effects in the tumor microenvironment. The 5 / 3 chimeric adenovirus exhibits anti-immunosuppressive activity in the B16.OVA tumor microenvironment. Tumors are highly resistant to immune attack, and even large numbers of adoptive metastatic tumor-specific OT-I T cells cannot overcome tumor immunosuppression. However, if mice are simultaneously treated with the 5 / 3 chimeric adenovirus, immunosuppressive molecules such as TIM-3 are downregulated in the tumor.

[0076] Figure 4 The enhancement of immunosuppression alone is insufficient to induce T cell transport to the tumor: BiTE is required. The enhancement of immunosuppression alone is insufficient to induce T cell transport to B16.OVA tumors. Intratumoral injection of 5 / 3 chimeric adenovirus induces CD8+ T cells in peripheral blood, but these cells do not effectively infiltrate the tumor. This poor T cell tumor transport highlights the limitations of oncolytic adenoviruses and adoptive T cell therapy as single agents, supporting the present invention's enhancement of adoptive T cell transport via oncolytic adenovirus expressing BiTe.

[0077] Figure 5 This study reveals that adenoviruses outperform vaccinia virus (VV) in inducing cellular antitumor immunity; this is a key feature for enhancing adoptive cell therapy. A comparison of the immunogenicity between adenovirus (Ad) and vaccinia virus (VV) is presented. Mice treated with 5 / 3 chimeric adenovirus showed higher levels of tumor-infiltrating CD8+ T cells in the spleen and B16.OVA compared to mice treated with double-deleted oncolytic Western reserve vaccinia virus (using this strain in Yu et al. Mol Ther 2014). Therefore, oncolytic adenoviruses appear to be an ideal expression platform for BiTe due to their inherent immunogenicity, particularly in the context of adoptive T-cell therapy.

[0078] Figure 6Adenovirus was shown to be more effective than vaccinia virus in inducing antitumor immunity. Mice carrying syngeneic B16.OVA tumors were intratumorally injected with PBS, adenovirus, or vaccinia virus. Tumor cell samples were stained with pentamer-APC (which detects T-cell receptors specific to the SIINFEKL residues of ovalbumin) and evaluated by flow cytometry (n=3). Data indicated changes in antitumor T cells after adenovirus or vaccinia virus injection; adenovirus was more effective in inducing antitumor immunity, while vaccinia virus was actually immunosuppressive in the context of antitumor T cells.

[0079] Figure 7 This study revealed that BiTE delivered by oncolytic adenovirus targets all types of T cells against tumors, including antiviral T cells. In many patients, antiviral T cells far outnumber antitumor T cells (Kanerva A et al. ClinCancer Res. 2013 May 15;19(10):2734-44). They are generally considered counterproductive in the context of cancer therapy because a) they consume a major portion of the limited available immune response, and b) they can limit oncolytic virus replication. In contrast, our invention unexpectedly utilizes pre-existing and induced anti-adenoviral T cell immunity, as antiviral T cells target tumors (…). Figure 7 Since adenovirus-treated tumor TILs contain both anti-tumor and antiviral T cells, BiTe's CD3-scFV activates these T cells regardless of their endogenous specificity (independent of MHCI). Therefore, tumor-specific killing of these T cells is achieved via scFV specific to tumor cell surface antigens such as mesothelin, EpCAM1, and MUC1, and off-tumor / off-target reactivity is not expected. Thus, this approach redirects all CD8+ TILs (=anti-tumor and antiviral) to anti-tumor T cells through the binding of virally produced BiTe.

[0080] Figure 8The oncolytic adenovirus encoding functional antibodies, rather than the non-replicating adenovirus, resulted in effective antibody production and release from cancer cells. Cells were infected with the indicated adenovirus at 100 viral particles (VP) / cell, and antibody expression was analyzed by human IgG ELISA (A) or Western blot (B) several days later. At each specified time point post-infection, (A) oncolytic virus Ad5 / 3-OV-Ab (gray and black bars) showed significant production of functional antibodies from ovarian cancer SKOV-3 cells: antibody levels decreased in cell lysates (LYS) during progressive infection and cancer cell killing, and showed significant accumulation in the supernatant (SN). In contrast, the non-replicating virus Ad5 / 3-Ab failed to produce detectable antibodies in the supernatant, even though cell lysates showed evidence of antibodies on day 7 post-infection (white bars). Notably, cells treated with the non-replicating Ad5 / 3-Ab virus survived throughout the experiment, indicating a lack of active antibody secretion from cancer cells. (B) Western blot analysis of the supernatants of breast cancer BT-474 cells (left) and human embryonic 293 cells (right) 6 days after infection with the specified virus. Under reducing conditions, heavy chain (HC), light chain (LC), and full-length antibodies produced by the oncolytic virus Ad5 / 3-OV-Ab were observed in the supernatants of both cell lines. Non-replicating Ad5-Ab and Ad5 / 3-Ab viruses failed to show antibody release from BT-474 cells, which are not permitted to replicate. To confirm antibody expression of the non-replicating virus, we used human embryonic 293 cells (right), which also allow E1A-deficient adenovirus replication, followed by cell lysis and release of antibodies readily detectable by Western blot. The non-replicating control virus Ad5 / 3-Luc, encoding luciferase, was used as a negative control. HC and LC were detected using polyclonal goat anti-human IgG and donkey anti-goat IgG-HRP antibodies, respectively. Antibody affinity was lower for LC than for HC, resulting in a weaker signal. Bars represent mean ± SEM. **, P < 0.01; *, P < 0.05; all are Student's T-tests.

[0081] Figure 9 Oncolytic adenovirus encoding antibodies showed higher intratumoral antibody levels and lower systemic antibody levels compared to systemic antibody therapy. Intratumoral injection of oncolytic Ad5 / 3-OV-Ab virus (2 × 10⁻⁶) was performed on days 0, 4, 8, and 15. 8Nude mice / NMRI mice with subcutaneous N87 gastric cancer xenografts were treated with either VP / tumor or intraperitoneal injection of commercial antibodies (Ab; 0.3 μg / g) (n = 5 per group). Animal health was monitored, and tumor and blood samples were collected from mice sacrificed at days 32 and 40 (systemic Ab), day 46 (systemic Ab and Ad5 / 3-OV-Ab virus), and day 50 (Ad5 / 3-OV-Ab virus). A) End-point tumor and blood samples were measured by human IgG ELISA to assess antibody concentrations: Ad5 / 3-OV-Ab-treated mice sacrificed at days 46 and 50 after treatment showed significantly higher antibody concentrations still in tumors compared to mice sacrificed early at days 32, 40, and 46 (P < 0.001, left), while exhibiting much lower circulating levels (P < 0.001, right). B) Antibody levels in tumor and blood samples were compared for each individual animal to assess antibody distribution. In mice treated with Ad5 / 3-OV-Ab virus, the mean ratio of tumor to blood antibodies was greater than 1.0, while systemic Ab treatment resulted in a very low ratio below 0.01. Therefore, treatment with an antibody-expressing oncolytic virus can achieve improved intratumoral antibody concentrations while significantly reducing systemic exposure in animals. Notably, most virus-treated mice survived longer (up to 50 days), thus showing evidence of sustained local antibody production. Error bars represent mean ± SEM. **, P < 0.01, Student's T test.

[0082] Figure 10The expression of T-cell depletion markers and the immunosuppressive receptor TIM3 was reduced after oncolytic adenovirus treatment and associated with improved survival. Fifteen patients with advanced solid tumors were treated with oncolytic adenovirus in the context of the Advanced Therapy Access Program. Baseline and post-treatment tumor biopsies were analyzed by Illumina (HumanHT-12 v4 Expression BeadChips array) to identify differentially expressed genes by comparing gene expression levels. T-cell immunoglobulin mucin-3 (TIM3), a depletion marker and negative regulator of both innate and adaptive immune responses in tumors, was one of the top differentially expressed genes: TIM3 showed major downregulation in 5 patients (a change of 1.0, Δ[log2]) and minor downregulation in 4 patients (mean change of 0.38, Δ[log2]). Meanwhile, TIM3 downregulation was not shown in 6 patients, with 2 of them showing upregulation post-treatment. When comparing overall survival between these groups, patients with downregulated TIM3 (n = 9) showed significantly improved survival compared to patients with no change / upregulation in TIM3 (n = 6) (P = 0.004, time test). The median survival in the TIM3 downregulation and upregulation groups was 204 days and 64 days, respectively. Therefore, it appears that two-thirds of oncolytic adenovirus treatments lead to a reduction in the immunosuppressive receptor and depletion marker TIM3, which is strongly associated with prolonged overall survival.

[0083] Figure 11 This study demonstrates improved in vitro cell killing with the combination of TIL and oncolytic adenovirus. HapT1 cells were infected with oncolytic adenovirus (100 VP / cell) for 3 days prior to the addition of HapT1 TIL. Target cell viability was measured 24 hours after TIL addition. Error bars, SE. **** P<0.0001. Best killing was observed when T cells were stimulated with oncolytic adenovirus.

[0084] Figure 12 The study showed that, in the absence of BiTe molecules, TILs extracted from HapT1 tumors did not exhibit an additive effect in killing target cells when combined with oncolytic adenovirus. HapT1 cells were seeded in 96-well plates and incubated for 5 days with oncolytic adenovirus Ad5 / 3-E2F-d24, either alone or armored with human IL-2. TILs extracted from established HapT1 tumors were added to the cells at a 10:1 ratio, and cell viability was measured using the MTS assay after 24 hours. No synergistic effect was observed between the virus and TILs.

[0085] Figure 13 (A and B) reveals the in vitro lytic activity of the combination of Ad5 / 3-E2F-d24-E3 virus with human CD3-specific EpCAM-targeting BiTE (anti-human EpCam, Cat#CABT-33295MH) and PBMC against the colon cancer cell line SW480. Figure 13A a) SW480 tumor cells were infected with Ad5 / 3-E2F-d24-E3 virus at increased VP (0, 01, 0, 1, 1, 10, 100, 1000 VP) and 10 ng BiTE. Effector cells (PBMCs) were added at an effector-to-target ratio of 5:1. Cell viability was measured at 48 hours post-infection using the MTS assay. Error bars represent SEM measurements in triplicate. Virus + Cells vs. Virus + PBMCs * P = 0.0184, Virus + Cells vs. Virus + PBMCs + BiTE *** P = 0.001. Figure 13B a) SW480 tumor cells were infected with 1000 VPs of Ad5 / 3-E2F-d24-E3 virus and 10 ng of BiTE. Effector cells (PBMCs) were added at an effector-to-target ratio of 5:1. Cell viability was measured at 48 hours post-infection using the MTS assay. Error bars represent SEM measurements in triplicate. Virus + Cells vs. Virus + PBMCs * P = 0.0184, Virus + Cells vs. Virus + PBMCs + BiTE *** P = 0.001.

[0086] Figure 14 Adenovirus or IL2-armored adenovirus was insufficient to accumulate T cells in tumors. Adenovirus treatment in combination with adoptive T cell transfer resulted in suboptimal T cell infiltration into B16.OVA melanoma tumors. Tumors collected 18 days after treatment initiation were analyzed by flow cytometry for ovalbumin-specific CD8+ T cells (OVA) and gp100-specific CD8+ T cells. OVA and gp100 are epitopes expressed on melanoma cells. Differences between treatment groups were not statistically significant and were not different from T-cell therapy alone (virus-free). Horizontal lines represent the mean.

[0087] Figure 15To reveal cytotoxic T cells in hamster pancreatic tumors. Oncolytic adenovirus failed to recruit cytotoxic CD8+ T cells to the tumor. Subcutaneous hamster pancreatic tumors (HapT1) were treated five times over 19 days with oncolytic adenovirus Ad5 / 3-E2F-d24, either alone or armored with human IL-2. On day 25, animals were sacrificed and tumor cells were labeled with a cross-reactive anti-rat CD8b PE antibody. (Sample number: simulated and unarmored n = 5, IL2 n = 1). Oncolytic adenovirus alone failed to recruit Cd8 cells to the tumor. IL2 appeared more promising, but the increase was not significant.

[0088] Figure 16 This study shows the results of re-challenge in immunocompetent hamsters. Hamsters previously cured with unarmored oncolytic adenovirus Ad5 / 3-E2F-d24 or with armored adenovirus treated with cytokines (TNFα, IL-2, or both) were resistant to the same tumor type (HapT1) rather than a different tumor type (DDT1-MF2). Naïve animals that had not previously encountered either cell line were used as controls. Armoring the virus with molecules capable of inducing antitumor immunity (e.g., BITE) is essential for inducing protective immunity (i.e., a sign of a memory response against tumor epitopes).

[0089] Figure 17 Demonstrates in vivo efficacy of armored or unarmored oncolytic adenovirus with or without T-cell therapy. On days 1 and 8, oncolytic adenovirus Ad5 / 3-E2F-d24 (1×10⁻⁶) was administered. 7 HapT1-infiltrated tumors were established through intratumoral injection of VP / tumor. On day 2, ex vivo-grown HapT1 tumor-infiltrating lymphocytes (1.5 × 10⁻⁶) were administered intratumorally. 6 (TIL / tumor). Error bars, SE. *p<0.05, **p<0.01. The best antitumor efficacy was observed when tumors were treated with oncolytic viruses, and TILs were also given.

[0090] Figure 18 This study presents hypothetical results for the in vivo antitumor efficacy of combined Ad-BiTE and OT1 T cell transfusion in immunocompetent mice carrying B16-OVA tumors. Percutaneously implanted B16-OVA tumors were treated with a single intraperitoneal injection of CD8-rich OT1 T cells, intratumoral injection of Ad-BiTE (1 × 10e9 VP / tumor), or both (0.25 × 10e6 cells / tumor). Viral injections were repeated every 7 days.

[0091] Figure 19The results showed that adenoviral delivery of the cytokines IL2 and TNFα enhanced the efficacy of adoptive cell therapy, providing a principle for including cytokines in oncolytic adenoviruses encoding BiTE. On day 1, C57 mice carrying B16-OVA tumors were treated intratumorally with 1 × 10e9 viral particles of armored adenovirus and intraperitoneally with 1.5 × 10e6 CD8-rich OT-1 T cells. Viral treatment continued every 7 days.

[0092] Figure 20 The construct design of the present invention is shown.

[0093] Figure 21 A diagram of the construction of the present invention is shown. Invention Details

[0094] Viral vector

[0095] The oncolytic adenovirus vector used in this invention can be any adenovirus vector suitable for treating humans or animals. As used herein, "oncolytic adenovirus vector" refers to an adenovirus vector capable of infecting and killing cancer cells through selective replication in tumors relative to normal cells.

[0096] In one embodiment of the invention, the adenovirus vector is a vector of a human virus. In one embodiment, the adenovirus vector is selected from Ad5, Ad3, and Ad5 / 3 vectors. As used herein, the expression "adenovirus serotype 5 (Ad5) nucleic acid backbone" refers to the genome of Ad5. Similarly, "adenovirus serotype 3 (Ad3) nucleic acid backbone" refers to the genome of Ad3. "Ad5 / 3 vector" refers to a chimeric vector that contains or has a portion of both Ad5 and Ad3 vectors. In one specific embodiment, the backbone of the adenovirus vector is an adenovirus serotype 5 (Ad5) or adenovirus serotype 3 (Ad3) nucleic acid backbone with a specific mutation. For example, the fibrillary regions of the vector may be modified. In one embodiment, the backbone is an Ad5 nucleic acid backbone that also contains Ad3 fibrillary knots. In other words, the construct has fibrillary knots from Ad3, while the remainder or a large portion of the remainder of the genome is from Ad5. (See example) Figure 20 ).

[0097] Adenoviral vectors 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, this makes the vector tumor-specific. For example, adenoviral vectors can contain modifications in E1, E3, and / or E4, such as the insertion of tumor-specific promoters (e.g., to drive E1), deletion of regions (e.g., E1 constant region 2, as used in “D24”, E3 / gp19k, E3 / 6.7k), and insertion of transgenes.

[0098] One method for generating tumor-specific oncolytic adenoviruses is to engineer a 24-base pair deletion (D24) affecting the constant region 2 (CR2) of E1. In wild-type adenoviruses, CR2 is responsible for binding to cellular Rb tumor suppressors / cell cycle regulators that induce the synthetic (S) phase, i.e., the DNA synthesis or replication phase. The interaction between pRb and E1A requires amino acids 121 to 127 of the conserved region of the E1A protein, which are deleted in this invention. The vector of this invention contains a deletion of nucleotides corresponding to amino acids 122-129 of the vector according to Heise C. et al. (2000, Nature Med 6, 1134-1139). Viruses with D24 are known to have a reduced ability to overcome the G1-S checkpoint and to replicate efficiently only in cells where such interaction is not necessary, for example, in tumor cells deficient in the Rb-p16 pathway, which includes most (if not all) human tumors. In one embodiment of the invention, the vector contains a 24bp deletion (D24) in the Rb-binding constant region 2 of adenovirus E1 (see [link]). Figure 20 ).

[0099] It is also possible to replace the E1A endogenous viral promoter with, for example, a tumor-specific promoter. In specific embodiments of the invention, the E1A endogenous viral promoter is replaced with, for example, an E2F1 promoter (e.g., in an Ad5-based vector) or an hTERT promoter (e.g., in an Ad3-based vector). In one embodiment, the vector contains an E2F1 promoter for tumor-specific expression of E1A.

[0100] The E3 region is not essential for viral replication in vitro, but the E3 protein plays an important role in regulating the host immune response, specifically suppressing both innate and specific immune responses. In one embodiment of the invention, the deletion of the nucleic acid sequence in the E3 region of the oncolytic adenovirus vector is the deletion of the viral gp19k and 6.7k reading frames. The gp19k / 6.7K deletion in E3 refers to the deletion of 965 base pairs from the adenovirus E3A region. In the resulting adenovirus construct, both the gp19k and 6.7K genes are deleted (Kanerva A et al. 2005, Gene Therapy 12, 87-94). The gp19k gene product is known to bind to and isolate the major histocompatibility complex I (MHC1, known as HLA1 in humans) molecule in the endoplasmic reticulum and prevent recognition of infected cells by cytotoxic T lymphocytes. Because many tumors lack HLA1 / MHC1, the absence of gp19k increases viral tumor selectivity (the virus is cleared from normal cells faster than wild-type virus, but there is no difference in tumor cells). 6.7K proteins are expressed on the cell surface, and they are involved in downregulating TNF-associated apoptosis-inducing ligand (TRAIL) receptor 2. (See also...) Figure 20 ).

[0101] Regarding specific embodiments of the invention, the deletion of both gp19k and 6.7K offers a surprising advantage. Since we are attempting to restore HLA / MHC expression for presenting tumor epitopes to adoptive T cells, gp19k expression is counterproductive, and in fact, HLA / MHC upregulation requires the deletion of gp19k. Regarding 6.7k, since one specific embodiment of the invention generates TNFalpha from a virus, and one of its antitumor activities is a direct antitumor pro-apoptotic effect (in both transduced and non-transduced bystander cells), the presence of 6.7k is counterproductive.

[0102] In one embodiment of the invention, one or more transgenes are placed in an E3 region with a gp19k / 6.7K deletion below the E3 promoter. This restricts transgene expression to tumor cells that allow viral replication and subsequent activation of the E3 promoter. In a specific embodiment, a nucleic acid sequence encoding a dimorphic molecule comprising a single-stranded variable fragment (scFv) specific to cell surface molecules and a scFv specific to tumor antigens is inserted at the location of the deleted nucleic acid sequence in the viral gp19k and 6.7k reading frames. In another embodiment of the invention, the E3 gp19k / 6.7K is retained in the vector, but one or more other E3 regions are deleted (e.g., E3 9-kDa, E3 10.2kDa, E3 15.2kDa, and / or E3 15.3kDa).

[0103] The E3 promoter can be any exogenous (e.g., CMV or E2F promoter) or endogenous promoter known in the art, particularly endogenous E3 promoters. While the E3 promoter is primarily activated through replication, some expression occurs when E1 is expressed. Because the selectivity of D24 viruses occurs after E1 expression (when E1 cannot bind Rb), these viruses do also express E1 in transduced normal cells. Therefore, regulating E1 expression to confine E3 promoter-mediated transgene expression to tumor cells is also crucial.

[0104] Specific embodiments of the present invention include an oncolytic adenovirus vector (e.g., Ad5 or Ad3 vector) whose replication is restricted to the p16 / Rb pathway by a dual-selectivity mechanism: an E2F (e.g., E2F1) tumor-specific promoter located preceding the adenovirus E1A gene, wherein the adenovirus E1A gene has been mutated in constant region 2, such that the resulting E1A protein cannot bind to Rb in the cell. Furthermore, the fibrils are modified with 5 / 3 chimerism to allow efficient entry into tumor cells. Additionally, a BiTE transgene, optionally placed along with other transgenes, is placed in the E3 region where the gp19k and 6.7k open reading frames have been deleted. This armored approach links transgene expression to viral replication without requiring a heterologous promoter. In specific embodiments, L (left)- and / or R (right)-ITR sequences may also be included in the vector. Inverted terminal repeat (ITR) sequences enable efficient viral genome replication and provide properties such as the ability to form hairpins.

[0105] In a specific embodiment of the present invention, the oncolytic adenovirus vector comprises:

[0106] 1) E2F1 promoter for tumor-specific expression of E1A

[0107] 2) A 24bp deletion (D24) in the Rb binding constant region 2 of adenovirus E1;

[0108] 3) Deletion of nucleic acid sequences in the viral gp19k and 6.7k reading frames; and

[0109] 4) A nucleic acid sequence encoding a bipartite molecule, replacing the missing nucleic acid sequence as defined in point 3), wherein the bipartite molecule comprises a single-stranded variable fragment (scFv) specific to cell surface molecules and an scFv specific to tumor antigens. (See also...) Figure 20 ).

[0110] Bispecific monoclonal antibodies (BsMAb, BsAb) are artificial proteins composed of fragments of two different monoclonal antibodies, and therefore capable of binding to two different types of antigens. In other words, bispecific antibodies combine two or more antigen recognition elements into a single construct capable of binding to two or more targets.

[0111] Examples of bispecific monoclonal antibodies include BsMAb, which is engineered to simultaneously bind to cytotoxic cells (using receptors such as CD3) and targets to be destroyed, such as tumor cells. First-generation BsMAb, known as trifunctional antibodies, have been developed. It consists of two heavy chains and two light chains, one from each of two different antibodies. Two Fab regions (arms) target two antigens. The Fc region (foot) is composed of the two heavy chains and forms a third binding site; hence the name. Other types of bispecific antibodies include chemically linked Fabs consisting only of Fab regions, and various types of bivalent and trivalent single-chain variable fragments (scFvs) (i.e., fusion proteins mimicking the variable domains of two antibodies). In a specific embodiment of the invention, the bispecific monoclonal antibody is selected from trifunctional antibodies and bivalent and trivalent single-chain variable fragments (scFvs). In one embodiment of the invention, the bispecific monoclonal antibody is a bivalent single-chain variable fragment. The bivalent single-chain variable fragment group comprises a bispecific T-cell adaptor (BiTE) and mAb2 (i.e., an antibody engineered to contain an Fcab antigen-binding fragment instead of the Fc constant region).

[0112] Bispecific T-cell adaptors (BiTEs) are a class of artificial bispecific monoclonal antibodies. They direct the host's immune system, more specifically, the cytotoxic activity of T cells targeting cancer cells. BiTEs are fusion proteins consisting of two single-chain variable fragments (scFvs) of different antibodies or amino acid sequences from four different genes on a single polypeptide chain of approximately 55 kilodaltons. One scFv binds to T cells via cell surface molecules (such as the CD3 receptor), while the other binds to tumor cells via tumor-specific molecules.

[0113] In a specific implementation, a bispecific monoclonal antibody is a dimorphic molecule comprising a single-chain variable fragment (scFv) specific to cell surface molecules and an scFv specific to tumor antigens. As used herein, "specific to cell surface molecules" refers to the ability to bind to a specific type of cell surface molecule. Also as used herein, "specific to tumor antigens" refers to the ability to bind to a specific type of tumor antigen.

[0114] In one embodiment of the invention, cell surface molecules are located on immune effector cells. As used herein, "immune effector cells" refers to cells selected from T cells, CD8+ cells, CD4+ cells, NK cells, delta-gamma T cells, regulatory T cells, and peripheral blood mononuclear cells. In one specific embodiment, the effector cell is a T cell, i.e., a T lymphocyte. In one embodiment, the cell surface molecules may be selected from CD3, CD8, and CD4.

[0115] In one implementation, the tumor antigen is selected from Table 1 or the group consisting of mesothelin, EpCAM1 and MUC1.

[0116] In one embodiment, the cell surface molecule is CD3, and the tumor antigen is selected from Table 1 or from mesothelin, EpCAM1, or MUC1. In another embodiment, the cell surface molecule is CD8, and the tumor antigen is selected from Table 1 or from mesothelin, EpCAM1, or MUC1. In yet another embodiment, the cell surface molecule is CD4, and the tumor antigen is selected from Table 1 or from mesothelin, EpCAM1, or MUC1. In a very specific embodiment, the tumor antigen is mesothelin, and the cell surface molecule is CD3; the tumor antigen is EpCAM1, and the cell surface molecule is CD3; or the tumor antigen is MUC1, and the cell surface molecule is CD3. In fact, specific examples of BiTe transgenes include anti-mesothelin-linker-anti-CD3, anti-EpCAM1-linker-anti-CD3, and anti-MUC1-linker-anti-CD3.

[0117] Table 1. Examples of tumor antigens suitable for use in this invention (http: / / cvc.dfci.harvard.edu / cvccgi / tadb / nomenclature.pl).

[0118]

[0119]

[0120]

[0121]

[0122] In one embodiment, the vector of the present invention encodes a bispecific monoclonal antibody, but may also contain other transgenes. In a specific embodiment, the oncolytic adenovirus vector encodes two or more transgenes. A particular embodiment of the present invention includes an adenovirus vector encoding a bispecific T-cell adaptor and at least one cytokine. The cytokine used in the present invention may be selected from any cytokine known in the art. In a specific embodiment of the present invention, the cytokine is IL-2, TNFalpha, or CD40L. In practice, in addition to bispecific monoclonal antibodies, the oncolytic adenovirus vector may also include, for example, IL-2, TNFalpha, and / or CD40L transgenes.

[0123] Cytokines participate in immune responses through various mechanisms, including the recruitment of T cells to tumors. The nucleotide sequences encoding cytokine transgenes can originate from any animal such as humans, apes, rats, mice, hamsters, dogs, or cats, but specifically they are encoded by human sequences. The nucleotide sequences encoding transgenes can be modified to improve their function, or they can remain unmodified, i.e., wild-type nucleotide sequences.

[0124] Furthermore, the combination of an adenoviral vector encoding both BiTE and at least one cytokine with an adoptive cell therapy agent provides more effective results on a wider range of targets than previously assumed.

[0125] Other cytokines function by attracting and activating T cells and reducing tumor immunosuppression, while IL-2 induces the proliferation of T cell grafts. Therefore, IL-2 is produced locally at the tumor site where it is needed, rather than through systemic injection as is commonly done in T-cell therapy, which can cause side effects. This implementation method avoids a major problem with existing treatments (i.e., the toxicity of systemic IL-2). In fact, it prevents serious adverse events and even death because if IL-2 is produced while the virus replicates in the tumor, the separate addition of IL-2 used for proliferation and maintenance of transferred cells in existing methods after cell transfer to the patient is unnecessary. Local production at the tumor site also enhances the desirable effects of IL-2 (stimulation and proliferation of the graft) while reducing systemic exposure (the cause of adverse events). This invention provides a selective treatment with lower toxicity or damage to healthy tissues.

[0126] The transduction of danger signals provided by oncolytic virus replication and the activation of pathogen-associated molecular pattern recognition receptors by viral DNA, along with the effects of transgenics, can reduce tumor immunosuppression to a level where pretreatment can be omitted. Therefore, the major problems of existing technologies, namely the toxicity caused by pretreatment chemotherapy and radiation, can be avoided.

[0127] In one embodiment of the invention, the viral vector includes an internal ribosome entry site (IRES) or optionally a ribosome shunt site 2A between two transgenes. Thus, the IRES or ribosome shunt site 2A can be between any transgenes, such as between a bispecific monoclonal antibody and any cytokine. As used herein, “IRES” refers to a nucleotide sequence capable of initiating translation in the middle of a messenger RNA sequence during protein synthesis. The IRES can be derived from any virus, but in one embodiment of the invention, the IRES is derived from encephalomyocarditis virus (EMCV). As used herein, “ribosome shunt site 2A” refers to a translation initiation site where the ribosome physically bypasses a portion of the 5' untranslated region to reach the start codon. Both IRES and A2 enable the virus to produce two transgenes from a single promoter (E3 promoter). The IRES can be used, for example, in the following locations in adenovirus constructs ( Figure 20 The following are nucleotide sequences: aMesothelin-aCD3-IRES-IL2 (see SEQ ID NO: 1, 2, 3, 5, 6, 9); aMesothelin-aCD3-IRES-TNFα (see SEQ ID NO: 1, 2, 3, 5, 6, 7); aEpCAM-aCD3-IRES-IL2 (see SEQ ID NO: 1, 2, 3, 4, 5, 6); aEpCAM-aCD3-IRES-TNFα (see SEQ ID NO: 1, 2, 3, 4, 5, 7); aMUC1-aCD3-IRES-IL2 (see SEQ ID NO: 1, 2, 3, 5, 6, 8); aMUC1-aCD3-IRES-TNFα (see SEQ ID NO: 1, 2, 3, 5, 7, 8). The nucleotide sequences are derived from the adenovirus constructs of this invention and are presented in Table 2.

[0128] A schematic diagram illustrating a general layout of a viral genome that can be used, for example, in this invention is shown. Figure 20(Ad5 / 3-E2F-D24-transgenic). The nucleotide sequences of the viral vectors containing transgenic aMesothelin-aCD3 (e.g., aMesothelin-aCD3-IRES-IL2, see SEQ ID NO: 1, 2, 3, 5, 6, 9; aMesothelin-aCD3-IRES-TNFa, see SEQ ID NO: 1, 2, 3, 5, 6, 7), aEpCAM-aCD3 (e.g., aEpCAM-aCD3-IRES-IL2, see SEQ ID NO: 1, 2, 3, 4, 5, 6; aEpCAM-aCD3-IRES-TNFα, see SEQ ID NO: 1, 2, 3, 4, 5, 7), and aMUC1-aCD3 (e.g., aMUC1-aCD3-IRES-IL2, see SEQ ID NO: 1, 2, 3, 5, 6, 8; aMUC1-aCD3-IRES-TNFa, see SEQ ID NO: 1, 2, 3, 5, 7, 8) were constructed according to the sequences listed in Table 2. General methods for constructing adenovirus vectors are well known to those skilled in the art and are described, for example, in Koski et al. 2010 and Hemminki et al. 2015. These methods can also be used to construct the adenovirus vectors of the present invention.

[0129] In addition to the other advantages mentioned above, the present invention utilizing a viral vector containing at least one cytokine transgene has the following additional advantages: i) the cytokines and viruses themselves induce danger signals that recruit T cells and other immune cells to the tumor; ii) the cytokines induce T cell proliferation in the tumor and local lymphoid organs; iii) the cytokines and viruses themselves can induce T cell (both adopted T cell grafts and natural, innate anti-tumor T cells) proliferation in the tumor; iv) the cytokines and / or viruses induce upregulation of antigen-presenting molecules (HLA) on cancer cells, making them sensitive to T cell recognition and killing; and v) cytokine and viral replication advantageously alter the tumor microenvironment by reducing immunosuppression and cellular unresponsiveness.

[0130] The viral vector used in this invention may also include other modifications besides those described above. Any additional components or modifications may be used optionally, but are not mandatory for the purposes of this invention.

[0131] Inserting 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 vectors, and they can also be used in this invention.

[0132] Adoptive cell therapy

[0133] One method of the present invention is to develop a treatment for cancer patients using transfers of immune lymphocytes capable of reacting with and destroying cancer. Isolated tumor-infiltrating lymphocytes are cultured in large quantities in a culture and infused into the patient. In this invention, an adenoviral vector encoding at least a bispecific monoclonal antibody can be used to enhance the effect of the lymphocytes. As used herein, “enhancing the efficacy of adoptive cell therapy” refers to a situation where the adenoviral vector of the present invention, when used in conjunction with an adoptive cell therapy composition, produces a stronger therapeutic effect in a subject than the therapeutic effect of the adoptive cell therapy composition alone. Figure 1 This invention describes the mechanism by which T-cell therapy using an oncolytic adenovirus encoding a bispecific T-cell connective, BiTE, enhances efficacy. A specific embodiment of the invention is a method of treating a subject's cancer, wherein the method includes administering the oncolytic adenovirus vector of the invention to the subject, and further includes administering an adoptive cell therapy composition to the subject. The adoptive cell therapy composition and the vector of the invention are administered separately. Prior to the separate administration of the adoptive cell therapy composition and the adenovirus vector may be myeloablating or non-myeloablating pretreatment chemotherapy and / or radiation. Adoptive cell therapy aims to reduce or eliminate cancer in patients.

[0134] Specific embodiments of the present invention relate to therapies using adenoviral vectors and adoptive cell therapy compositions (e.g., tumor-infiltrating lymphocytes, TCR-modified lymphocytes, or CAR-modified lymphocytes). T-cell therapy can be particularly employed in this invention, but any other adoptive therapy, such as NK-cell therapy or other cell therapies, can also be used. In fact, according to the present invention, the adoptive cell therapy composition can comprise unmodified cells, such as in TIL therapy or genetically modified cells. There are two common methods for achieving genetic targeting of tumor-specific targets by T cells. One is the transfer of T-cell receptors with known specificity (TCR therapy) and a matching human leukocyte antigen (HLA, known as the major histocompatibility complex in rodents) type. The other is the modification of cells with artificial molecules such as chimeric antigen receptors (CARs). This method is HLA-independent and more flexible in terms of targeting molecules. For example, single-chain antibodies can be used, and CARs can also incorporate co-stimulatory domains. However, the target of CAR cells needs to be on the membrane of the target cell, while TCR modification can utilize intracellular targets.

[0135] As used herein, "adoptive cell therapy composition" refers to any composition comprising cells suitable for adoptive cell transfer. In one embodiment of the invention, the adoptive cell therapy composition comprises a cell type selected from tumor-infiltrating lymphocytes (TILs), TCR (i.e., heterologous T cell receptor) modified lymphocytes, and CAR (i.e., chimeric antigen receptor) modified lymphocytes. In another embodiment of the invention, the adoptive cell therapy composition comprises a cell type selected from T cells, CD8+ cells, CD4+ cells, NK cells, Δ-γ T cells, regulatory T cells, and peripheral blood mononuclear cells. In yet another embodiment, TILs, T cells, CD8+ cells, CD4+ cells, NK cells, Δ-γ T cells, regulatory T cells, or peripheral blood mononuclear cells form the adoptive cell therapy composition. In a specific embodiment of the invention, the adoptive cell therapy composition comprises T cells. As used herein, "tumor-infiltrating lymphocytes" or TILs refer to leukocytes that have left the bloodstream and migrated into the tumor. Lymphocytes can be classified into three groups: B cells, T cells, and natural killer cells. In another specific embodiment of the invention, the adoptive cell therapy composition comprises T cells that have been modified with a target-specific chimeric antigen receptor or a specifically selected T cell receptor. As used herein, "T cell" refers to CD3+ cells, including CD4+ helper cells, CD8+ cytotoxic T cells, and γδ T cells.

[0136] In addition to suitable cells, the adoptive cell therapy compositions used in this invention may contain any other agents, such as pharmaceutically acceptable carriers, buffers, excipients, adjuvants, additives, preservatives, fillers, stabilizers and / or thickeners, and / or any components commonly found in the respective products. The selection of suitable ingredients and suitable methods of manufacture for formulating the compositions are common knowledge to those skilled in the art.

[0137] Adoptive cell therapy compositions can be in any suitable form for administration, such as solid, semi-solid, or liquid. Formulations can be selected from, but are not limited to, solutions, emulsions, suspensions, tablets, pills, and capsules. The composition is not limited to a specific formulation, but can be formulated into any known pharmaceutically acceptable formulation. Pharmaceutical compositions can be prepared by any conventional method known in the art.

[0138] The combination of the oncolytic adenovirus vector and adoptive cell therapy composition of the present invention refers to the combination of the oncolytic adenovirus vector and the adoptive cell therapy composition together, but used as separate compositions. It will be apparent to those skilled in the art that the oncolytic adenovirus vector and adoptive cell therapy composition of the present invention are not used as a single composition. In fact, the adenovirus vector is not used to modify adoptive cells, but rather to modify the target tumor, making the tumor more suitable for the desired effects of the cell graft. In particular, the present invention enhances the recruitment of the adoptive graft to the tumor and increases its activity therein. In a specific embodiment of the invention, the combined oncolytic adenovirus vector and adoptive cell therapy composition is administered to the subject simultaneously or in any sequential order.

[0139] cancer

[0140] The recombinant vector of the present invention is capable of replication in tumor cells. In one embodiment of the invention, the vector is capable of replication in cells and is defective in the Rb pathway, particularly the Rb-p16 pathway. These defective cells include all tumor cells in animals and humans. As used herein, “defective in the Rb pathway” refers to mutations and / or epigenetic changes in any gene or protein in this pathway. Due to these defects, tumor cells overexpress E2F, thus making Rb binding via E1A CR2 (which is typically required for efficient replication) unnecessary. Further selectivity of the adenoviral vector of the present invention is mediated by the E2F promoter, which is activated only in the presence of free E2F, as seen in Rb / p16 pathway defective cells. In the absence of free E2F, E1A transcription does not occur and the virus does not replicate. The inclusion of the E2F1 promoter is important for preventing E1A expression in normal tissues, which can cause toxicity directly and indirectly by allowing transgenic expression from the E3 promoter.

[0141] This invention relates to a method for treating cancer in a subject. In one embodiment of the invention, the subject is a human or animal, particularly an animal or human patient, more specifically a human or animal suffering from cancer.

[0142] The method of this invention can be used to treat any cancer or tumor, including both malignant and benign tumors, and both primary and metastatic tumors can be targets of this method. In one embodiment of the invention, the cancer is characterized by tumor-infiltrating lymphocytes. The tool of this invention is particularly suitable for treating metastatic solid tumors characterized by tumor-infiltrating lymphocytes. In another embodiment, the T-cell graft has been modified with a chimeric antigen receptor-specific T-cell receptor of the tumor or tissue.

[0143] As used herein, the terms “treatment” or “treatment” refer to the administration of at least an oncolytic adenovirus vector or at least an oncolytic adenovirus vector and adoptive cell therapy composition to a subject, preferably a mammalian or human subject, with the aim of not only achieving complete cure but also preventing, improving, or alleviating symptoms or signs associated with cancer or tumors. Treatment efficacy can be assessed by monitoring the patient’s symptoms, such as tumor markers in the blood, or, for example, tumor size or patient survival time.

[0144] In one embodiment of the present invention, the cancer is selected from nasopharyngeal cancer, synovial cancer, hepatocellular carcinoma, renal cancer, connective tissue cancer, melanoma, lung cancer, bowel cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, throat cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, and von Hippel-Lindau disease. Diseases, Zollinger-Ellison syndrome, renal cancer, anal cancer, bile duct cancer, bladder cancer, ureter cancer, brain cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, bone cancer, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, gastrointestinal carcinoidtract, fibrosarcoma, breast cancer, Paget's disease, cervical cancer, colorectal cancer, rectal cancer, esophagus cancer, gallbladder cancer, head cancer, eye cancer, neck cancer, kidney cancer, Wilms' tumor, liver cancer, Kaposi's sarcoma, prostate cancer, lung cancer, testicular cancer, Hodgkin's disease, non-Hodgkin's lymphoma, oral cancer, skin cancer, mesothelioma, multiple myeloma, ovarian cancer, endocrine pancreatic cancer Cancer, glucagonoma, pancreatic cancer, parathyroid cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestinal cancer, stomach cancer, thymus cancer, thyroid cancer, trophoblastic cancer, hydatidiform mole, uterine cancer, endometrial cancer, vaginal cancer, vulva cancer, acoustic neuroma, mycosisFungoides, insulinoma, carcinoid syndrome, somatostatinoma, gum cancer, heart cancer, lip cancer, meninges cancer, mouth cancer, nerve cancer, palate cancer, parotid gland cancer, peritoneum cancer, pharynx cancer, pleural cancer, salivary gland cancer, tongue cancer, and tonsil cancer.

[0145] Clinicians may examine patients before classifying them for treatment appropriate for this invention, whether human or animal. Based on deviations from normal conditions and findings revealing tumors or cancer, clinicians may recommend the treatment of this invention for the patient.

[0146] Pharmaceutical Composition

[0147] The pharmaceutical compositions of the present invention comprise at least one type of viral vector of the present invention. In one embodiment, the pharmaceutical composition of the present invention comprises an oncolytic adenovirus vector containing a nucleic acid sequence encoding a bispecific monoclonal antibody with a deletion of a nucleic acid sequence in the E3 region and a replacement nucleic acid sequence in the E3 region, wherein the bispecific monoclonal antibody comprises a single-stranded variable fragment (scFv) specific to cell surface molecules and an scFv specific to tumor antigens. Furthermore, the composition may comprise at least two, three, or four different vectors. In addition to vectors, the pharmaceutical composition may also comprise other therapeutically effective agents, any other agents such as pharmaceutically acceptable carriers, buffers, excipients, adjuvants, additives, preservatives, fillers, stabilizers and / or thickeners, and / or any components commonly found in the respective products. The selection of suitable ingredients and suitable methods of manufacture for formulating the composition are common knowledge to those skilled in the art.

[0148] The pharmaceutical composition can be in any form suitable for administration, such as solid, semi-solid, or liquid. Formulations can be selected from, but are not limited to, solutions, emulsions, suspensions, tablets, pills, and capsules. The compositions of this invention are not limited to any particular formulation, but can be formulated into any known pharmaceutically acceptable formulation. The pharmaceutical compositions can be prepared by any conventional method known in the art.

[0149] In one embodiment of the invention, a viral vector or pharmaceutical composition is used as an in situ medium to recruit T cells, enhance their therapeutic effects, and allow them to proliferate at the tumor site.

[0150] The pharmaceutical kit of the present invention may comprise an oncolytic adenovirus vector encoding a bispecific monoclonal antibody or an adoptive cell therapy composition and an oncolytic adenovirus vector encoding a bispecific monoclonal antibody. In one specific embodiment, the adoptive cell therapy composition is formulated in a first formulation, and the oncolytic adenovirus vector is formulated in a second formulation. In another embodiment of the invention, the first and second formulations are administered to the subject simultaneously or in any sequential order.

[0151] application

[0152] The adenovirus vector or pharmaceutical composition of the present invention can be administered to any eukaryotic subject selected from plants, animals, and humans. In a specific embodiment of the invention, the subject is a human or an animal. Animals can be selected from pets, livestock, and producing animals.

[0153] Any conventional method may be used to administer the carrier or composition to a subject. The route of administration depends on the formulation or form of the composition, the disease, the tumor location, the patient, comorbidities, and other factors.

[0154] In one embodiment of the invention, both the adenovirus vector and the adoptive cell therapy composition are administered to a subject. The administration of the adoptive cell therapy composition and the oncolytic adenovirus vector encoding at least one bispecific monoclonal antibody to the subject can be performed simultaneously or sequentially in any order. In one embodiment of the invention, the oncolytic virus vector and the adoptive cell therapy composition are administered separately. As used herein, "administered separately" or "separately" refers to a situation where the adoptive cell therapy composition and the oncolytic adenovirus vector are two different products or compositions.

[0155] The adenovirus vector of the present invention can have a therapeutic effect with a single application or a single application of the adoptive cell therapy composition and the oncolytic adenovirus vector. Any time period can exist between applications of the oncolytic adenovirus or between applications of the oncolytic adenovirus and the adoptive cell therapy composition, depending on, for example, the type, extent, or location of the patient and the cancer. In one embodiment of the invention, a time period of 1 minute to 4 weeks, particularly 1 to 10 days, more specifically 1 to 5 days, exists between consecutive applications of the adoptive cell therapy composition and the oncolytic adenovirus vector encoding a bispecific monoclonal antibody. Several applications of the adoptive cell therapy composition and the oncolytic adenovirus vector are also possible. The number of applications of the adoptive cell therapy composition and the oncolytic adenovirus vector can also vary during treatment. The oncolytic adenovirus vector or the drug or the adoptive cell composition can be applied, for example, 1 to 10 times during the first 2 weeks, 4 weeks, monthly, or during a treatment cycle. In one embodiment of the invention, the application of the vector or any composition is performed within the first 2 weeks, then 4 weeks, and then 3 to 7 times per month. In a specific embodiment of the invention, the application is performed 4 times during the first 2 weeks, then 4 weeks, and then 4 times per month. The length of treatment can vary, for example, it can last from 2 to 12 months or longer.

[0156] In one specific embodiment of the invention, the adoptive cell therapy composition and the oncolytic adenovirus vector are administered on the same day, and then the oncolytic adenovirus vector is administered weekly, bi-weekly, tri-weekly or monthly during the treatment period, which can continue for, for example, 1 to 6 to 12 months or more.

[0157] In one embodiment of the invention, the oncolytic virus is administered via intratumoral, intra-arterial, intravenous, intrapleural, intracystic, intracavitary, or intraperitoneal injection or oral administration. Any combination of administration is also possible. Although administered locally, this method can provide systemic efficacy. The adoptive cell therapy composition can be administered intravenously or intratumorally. In one embodiment, the adoptive cell therapy composition and / or the oncolytic virus vector encoding at least one bispecific monoclonal antibody is administered via intratumoral, intra-arterial, intravenous, intrapleural, intracystic, intracavitary, or intraperitoneal injection or oral administration. In a specific embodiment of the invention, TILs or T cells are administered intravenously and the viral vector is administered intratumorally and / or intravenously. Notably, the virus is delivered to the tumor separately from the administration of T cells; the virus is not used to modify T cell grafts in vitro. Essentially, the virus modifies the tumor in a manner that allows the T cell graft to function better.

[0158] The effective dose of the carrier depends at least on the subject requiring treatment, the type of tumor, the location of the tumor, and the stage of the tumor. The dose can range from, for example, approximately 1 × 10⁻⁶. 8 One viral particle (VP) to approximately 1 × 10 14 VP, especially about 5×10 9VP to approximately 1×10 13 VP, more specifically approximately 8 × 10 9 VP to approximately 1×10 12 VP changes. In one implementation, the oncolytic adenovirus vector encoding a bispecific monoclonal antibody is delivered at 1 × 10⁻⁶. 11 -1×10 14 The dosage is approximately 5 × 10⁶ virus particles. In another embodiment of the invention, the dosage is approximately 5 × 10⁶ virus particles. 10 -5×10 11 Within the range of VP.

[0159] The number of metastatic cells will also depend on the patient, but the typical range is 1 × 10⁻⁶ per injection. 9 -1×10 12 The number of injections varies, but a typical implementation involves one or two rounds of treatment spaced several weeks apart (e.g., 2-4 weeks).

[0160] In addition to the treatments of this invention, any other treatments or combinations of treatments may be used. In specific embodiments, the methods or uses of this invention also include administering simultaneous or sequential radiotherapy, monoclonal antibodies, chemotherapy, or other anticancer drugs or interventions (including surgery) to a subject.

[0161] As used herein, the terms “treatment” or “increase” and the words derived therefrom do not necessarily mean 100% or complete treatment or increase. Rather, there are varying degrees of potential benefit or therapeutic effect that a person skilled in the art would consider to have. In this respect, the methods of the present invention can provide any amount of increase in the efficacy of T-cell therapy or any degree of treatment or prevention of disease.

[0162] Figure 1 and 7 The method and mechanism of the present invention are illustrated.

[0163] It will be apparent to those skilled in the art that the concept of this invention can be implemented in various ways as technology advances. The invention and its embodiments are not limited to the examples described above, but can be varied within the scope of the claims. Example

[0164] Materials and Methods

[0165] B16-OVA animal model: B16 cells expressing ovalbumin (B16-OVA) were maintained in RPMI, 10% FBS, 5 mg / ml G418, 20 mM L-glutamine, and 1×Pen / Strep solution (GIBCO). Immunized female C57BL / 6 mice aged 4-7 weeks were subcutaneously implanted with 50 μl of RPMI, 0% FBS, containing 2.5 × 10⁻⁶ cells per 10⁻⁶ cells. 5 One B16-OVA cell, one tumor per mouse. Approximately ten days after tumor implantation (when the tumor becomes injectable, with a minimum diameter of about 3 mm), mice are grouped and, in some experiments, administered 50 μl of PBS or 1×10⁻⁶ B16-OVA cells for six consecutive days. 9 Treatment involved intratumoral injection of viral particles (VP) of an oncolytic adenovirus. In other experiments, three injections were performed on days 0, 2, and 4. Because mouse cells do not tolerate human adenovirus, multiple intratumoral viral injections were used to mimic virus replication-induced inflammation (Blair et al., 1989).

[0166] Adoptive transfer: On day 1 of it treatment, mice were also adopted intraperitoneally via 100 μL RPMI, 0% FBS from 5 × 10⁶ C57BL / 6-Tg(TcraTcrb)1100Mjb / J(OT-1) mice (genetically engineered to have only ovalbumin (OVA)-specific CD8 T cell receptors) of 4–8 weeks old C57BL / 6-Tg(TcraTcrb)1100Mjb / J(OT-1) mice. 5 Up to 2×10 6 Overnight static CD8a-enriched and expanded spleen cells. CD8a enrichment was performed 5 days prior to transfer using mouse CD8a (Ly-2) MicroBeads, according to the manufacturer's instructions (Miltenyi Biotech, USA, catalog number 130-049-401). Enriched cells were expanded for 5 days in lymphocyte culture medium (RPMI, 10% FBS, 20 mM L-glutamine, 1×Pen / Strep solution, 15 mM HEPES, 50 μM 2-mercaptoethanol, 1 mM sodium pyruvate) in the presence of recombinant mouse IL-2 (160 ng / ml) and soluble anti-mouse CD3ε antibody (0.3 μg / ml, Abcam, clone 145-2C11).

[0167] Tissue preparation for flow cytometry: Mice were euthanized, and spleens, draining lymph nodes, and tumors were harvested in 1 to 10 ml RPMI, 10% FBS. Blood was collected into the pleural cavity via terminal cardiac pleolysis and transferred to microcentrifuge tubes containing EDTA using a disposable syringe for analysis. Solid tissue was roughly dissected with a scalpel and homogenized in 5 to 10 ml ACK lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH 7.2) in a 10 ml disposable sterile pipette tip. The mixture was incubated at room temperature (RT) for approximately 20 minutes. Cells were then precipitated at 1200 rpm for 5 minutes at +4°C. The cells were then resuspended in 1 to 10 ml RPMI, 10% FBS, depending on the estimated cell number, and passed through a 40-micron sterile filter to produce a single-cell solution. In some experiments, instead of surgical cutting (before adding ACK), tumor tissue was directly treated in a 1 ml total volume of a protease mixture (supplemented with 1 mg / ml collagenase A, H, or P type, Roche, and 125 units / ml final concentration of benzonase, Sigma, E1014-25KU RPMI) and incubated at 37°C, 5% CO2 for 1–2 hours. Then, 10 ml of ACK lysis buffer was added, and the cells were treated as above. Alternatively, 200 μl of whole blood was pipetted into 5 ml of ACK lysis buffer and treated as above. Cells were incubated overnight at 37°C, 5% CO2, or analyzed directly by immunostaining and flow cytometry.

[0168] Tissue preparation for cytokine analysis: Mice were euthanized, and approximately 2-10 mm of tissue was removed. 3 Tumor slides were frozen in 2 ml microcentrifuge tubes on dry ice and stored at -80°C. The slides were weighed and 200 μl of ice-cold PBS was added. The slides were homogenized using a Tissue Master 125 rotor, and 1x protease inhibitor mixture (Sigma) and 0.1% BSA were added to a final concentration. The tubes were kept on ice. The tumor homogenate was rotated at 2000 rpm for 10 minutes at +4°C, and the supernatant was analyzed on a BDFACSArray using CBA Flex Set cytokine beads (BD, USA) according to the manufacturer's instructions.

[0169] Experiments supporting this invention

[0170] Based on the Materials and Methods section of this disclosure, as well as disclosure WO2014170389(A1) and previously published articles The experiments described in the text were conducted.

[0171] Experiment 1 (Inducing danger signals in tumors with adenovirus treatment):

[0172] Treatment with Ad5 / 3-d24-GMCSF 5 / 3 chimeric adenovirus induced danger signals in B16.OVA tumors. The binding of the adenovirus pathogen-associated molecular pattern (PAMP) to the Toll-like receptor (TLR) on host cells induced the secretion of interferon-γ associated with immune cell activation and T cell stimulation, leading to rapid activation of both innate and adaptive immune responses. Therefore, adenoviruses can be used to generate immunogenic tumor phenotypes that are effectively recognized by the immune system. Figure 2 ).

[0173] Experiment 2 (Adenovirus has anti-immunosuppressive effects in the tumor microenvironment):

[0174] 5 / 3 chimeric adenovirus exhibits anti-immunosuppressive effects against the B16.OVA tumor microenvironment. Tumors are highly resistant to immune attack, and even large numbers of tumor-specific OT-1 T cells fail to overcome tumor immunosuppression. However, co-treatment of mice with 5 / 3 chimeric adenovirus downregulates immunosuppressive molecules (e.g., TIM-3) in the tumor. Figure 3 ).

[0175] Experiment 3 (Immunosuppression alone is insufficient to induce T cell transport to the tumor: BiTE is required):

[0176] The enhancement of immunosuppression was insufficient to induce T cell transport to B16.OVA tumors. Intratumoral injection of 5 / 3 chimeric adenovirus induced CD8+ T cells in peripheral blood, but these cells did not effectively penetrate the tumor. This poor tumor transport of T cells highlights the limitations of oncolytic adenoviruses and adoptive T cell therapy as single agents, supporting the present invention's enhancement of adoptive T cell transport via oncolytic adenovirus expressing BiTe. Figure 4 ).

[0177] Experiment 4 (Adenovirus outperforms vaccinia in inducing cellular anti-tumor immunity; enhancing key features of adoptive cell therapy)

[0178] Comparison between adenovirus (Ad) and vaccinia virus (VV) immunogenicity. Mice treated with 5 / 3 chimeric adenovirus showed higher levels of tumor-infiltrating CD8+ T cells in the spleen and B16.OVA compared to mice treated with double-deleted oncolytic Western reserve vaccinia virus (used by Yu et al., Mol Ther 2014). Therefore, oncolytic adenovirus appears to be an ideal expression platform for BiTe due to its inherent immunogenicity, particularly in the context of adoptive T-cell therapy. Figure 5 ).

[0179] Experiment 5 (Adenovirus is more effective than cowpox in inducing anti-tumor immunity)

[0180] Mice carrying tumors with the syngeneic B16.OVA gene were intratumorally injected with PBS, adenovirus, or vaccinia virus. Tumor cell samples were stained with pentamer-APC (which detects T-cell receptors specific to the SIINFEKL residues of ovalbumin) and evaluated by flow cytometry (n = 3). Data showed changes in antitumor T cells after adenovirus or vaccinia virus injection; adenovirus was much more effective in inducing antitumor immunity. Figure 6 ).

[0181] Experiment 6 (BiTE delivered by oncolytic adenovirus targets all types of tumor-targeting T cells, including antiviral T cells (which are generally considered to have a counteracting effect on cancer treatment))

[0182] Furthermore, this invention utilizes the widespread pre-existing Ad5 T cell immunity in the population, which typically limits the clinical utility of adenoviral vectors. Since the TILs of tumors treated with adenovirus contain both anti-tumor and antiviral T cells, the CD3-scFV of BiTe will activate these T cells regardless of their endogenous specificity (MHC I-independent). Therefore, tumor-specific killing by these T cells is achieved through scFV specific to tumor cell surface antigens (e.g., mesothelin, EpCAM1, MUC1), and off-tumor / off-target reactivity is not expected. Thus, this method redirects all CD8+ TILs (=anti-tumor and antiviral) to anti-tumor T cells by combining virally produced BiTe. Figure 7 ).

[0183] Experiment 7 (Oncolytic adenovirus encoding functional antibodies, rather than non-replicating adenovirus, led to effective antibody production and release from cancer cells)

[0184] SKOV-3, BT-474, and 293 cells were infected with the adenoviruses shown, at 100 viral particles (VP) / cell, and antibody expression was analyzed by human IgG ELISA (A) or Western blot (B) several days later. At each specified time point post-infection, (A) oncolytic virus Ad5 / 3-d24-trastuzumab (grey and black columns) showed a significant production of functional antibodies from ovarian cancer SKOV-3 cells: antibody levels decreased in cell lysates (LYS) during progressive infection and cancer cell killing, and showed significant accumulation in the supernatant (SN). (OV refers to Ad5 / 3-d24, and Ab refers to the antibody trastuzumab). In contrast, non-replicating virus Ad5 / 3-Ab did not produce detectable antibodies in the supernatant, even though cell lysates showed evidence of antibodies after day 7 post-infection (white column). Notably, cells treated with non-replicating Ad5 / 3-Ab virus survived throughout the experiment, indicating a lack of active antibody secretion in cancer cells. (B) Western blot analysis of the supernatants of breast cancer BT-474 cells (left) and human embryonic 293 cells (right) 6 days post-infection with the specified virus. Under reducing conditions, heavy chain (HC), light chain (LC), and full-length antibodies produced by the oncolytic virus Ad5 / 3-OV-Ab were observed in the supernatants of both cell lines. Non-replicating Ad5-Ab and Ad5 / 3-Ab viruses failed to show antibody release from BTB-474 cells, which are not permitted to replicate. To confirm antibody expression by the non-replicating virus, we used human embryonic 293 cells (right), which also allow replication of E1A-deficient adenovirus, followed by cell lysis and antibody release, which was readily detected by Western blot. The non-replicating control virus Ad5 / 3-Luc, encoding luciferase, was used as a negative control. HC and LC were detected using polyclonal goat anti-human IgG and donkey anti-goat IgG-HRP antibodies, respectively. Antibody affinity was lower for LC than for HC, resulting in a weaker signal. Columns represent mean ± SEM. **, P<0.01; *, P<0.05; both are Student's T-tests ( Figure 8 ).

[0185] Experiment 8 (Oncolytic adenovirus encoding antibodies showed higher intratumoral levels and lower systemic antibody levels compared to systemic antibody treatment)

[0186] Intratumoral injection of oncolytic Ad5 / 3-OV-Ab virus (2×10⁻⁶) was administered on days 0, 4, 8, and 15. 8Nude / NMRI mice carrying subcutaneous N87 gastric cancer (Park et al. 1990) xenografts (n=5 per group) were treated with either VP / tumor or intraperitoneal injection of commercial antibodies (Ab; 0.3 μg / g). Animal health was monitored, and tumor and blood samples were collected from mice sacrificed at days 32 and 40 (systemic Ab), day 46 (systemic Ab and Ad5 / 3-OV-Ab virus), and day 50 (Ad5 / 3-OV-Ab virus). A) End-point tumor and blood samples were measured by human IgG ELISA to assess antibody concentrations: Ad5 / 3-OV-Ab treated mice sacrificed earlier at days 32, 40, and 46 showed significantly higher antibody concentrations still in tumors (P<0.001, left) compared to mice treated with systemic Ab at days 32, 40, and 46 (P<0.001, right), while exhibiting much lower circulating levels (P<0.001, right). B) Antibody levels in tumor and blood samples were compared for each individual animal to assess antibody distribution. In mice treated with Ad5 / 3-OV-Ab virus, the mean ratio of tumor to blood antibodies was greater than 1.0, while systemic Ab treatment resulted in a very low ratio of less than 0.01. Therefore, treatment with an antibody-expressing oncolytic virus can improve intratumoral antibody concentrations while significantly reducing systemic exposure in animals. Notably, most virus-treated mice survived longer (up to 50 days), thus showing evidence of sustained local antibody production. Error bars represent mean + SEM. **, P < 0.01, Student's T-test ( Figure 9 ).

[0187] Experiment 9 (The expression of T cell exhaustion markers and the immunosuppressive receptor TIM3 was reduced after oncolytic adenovirus treatment and was associated with improved survival)

[0188] In the context of an advanced treatment access program (Taipale et al., 2016), 15 patients with advanced solid tumors were treated with oncolytic adenovirus. Baseline and post-treatment tumor biopsies were analyzed using an RNA microarray (HumanHT-12 v4 Expression BeadChips array, Illumina), and gene expression levels were compared to identify differentially expressed genes. T-cell immunoglobulin mucin-3 (TIM3) (a marker of exhaustion and negative regulator of both innate and adaptive immune responses in tumors) was one of the top differentially expressed genes: TIM3 showed major downregulation (a change of 1.0 relative to Δ[log2]) in 5 patients and minor downregulation (a mean change of 0.38 Δ[log2]) in 4 patients. Meanwhile, TIM3 downregulation was not observed in 6 patients, with 2 showing post-treatment upregulation. When comparing overall survival between these groups, patients with downregulated TIM3 (n=9) showed a significant improvement in survival compared to patients with no change / upregulated TIM3 (n=6) (P=0.004, time test). The median survival in the TIM3 downregulated and upregulated groups was 204 days and 64 days, respectively. Therefore, two-thirds of the oncolytic adenovirus treatment appears to lead to a reduction in the immunosuppressive receptor and exhaustion marker TIM3, which is strongly associated with prolonged overall survival. Figure 10 ).

[0189] Experiment 10 (Improving in vitro cell killing with a combination of TIL and oncolytic adenovirus)

[0190] HapT1 cells were infected with oncolytic adenovirus Ad5 / 3-d24 (100 VP / cell) for 3 days before the addition of HapT1 TIL. Target cell viability was measured 24 hours after TIL addition. Error bars, SE. **** P<0.0001. Optimal killing was observed when T cells were stimulated with oncolytic adenovirus (TIL). Figure 11 ).

[0191] Experiment 11 (In the absence of BiTe molecules, TIL extracted from HapT1 tumors did not exhibit a cumulative effect on target cell killing when combined with oncolytic adenovirus)

[0192] HapT1 cells were seeded in 96-well plates and incubated for 5 days with either oncolytic adenovirus Ad5 / 3-E2F-d24 alone or oncolytic adenovirus Ad5 / 3-E2F-d24 armored with human IL-2. Cell viability was measured by MTS assay after 24 hours after the addition of TIL extracted from established HapT1 tumors. No synergistic effect was observed between the virus and TIL. Figure 12 ).

[0193] Experiment 12 (Superior lytic activity of combined virus + BiTE + PBMCs)

[0194] SW480 tumor cells were seeded at 10,000 cells / well in 96-well plates and incubated for 24 hours. Cells were infected with Ad5 / 3-E2F-d24-E3 virus at doses of 0.01, 0.1, 1, 10, 100, and 1000 viral particles per cell and 10 ng of human CD3-specific EpCAM-targeting BiTE (Antihuman EpCam, catalog number CABT-33295MH) at least three replicates, in assay medium (L-15, 2% FBS, 2 mM L-glutamine, 100 U / ml penicillin, and 100 μg / ml streptomycin) 50 μl / well. Effector cells (PBMCs) were added at a 5:1 effector:target ratio. The following day, 50 μl of 10% L-15 was added to the cells. Forty-eight hours post-infection, the infection medium was replaced with 100 μl of growth medium containing 10% CellTiter 96AQueous One Solution (Promega, Madison, WI, USA) and incubated for 2 hours. Absorbance was read at 490 nm. Error bars represent triplicate SEMs. Virus + cells vs. virus + PBMCs * P = 0.0184, Virus + cells vs. virus + PBMCs + BiTE*** P = 0.001 ( Figure 13A ). Figure 13B SW480 tumor cells were infected with 1000 VPs of Ad5 / 3-E2F-d24-E3 virus and 10 ng BiTE. Effector cells (PBMCs) were added at a 5:1 effector:target ratio. Cell viability was assessed at 48 hours post-infection using the MTS assay. Error bars represent SEM measurements in triplicate. *P = 0.0184 for virus + cells versus virus + PBMCs, and ***P = 0.001 for virus + cells versus virus + BiTE + PBMCs.

[0195] Fraction product method:

[0196] The fractional product method is used to evaluate synergies, a method derived from one originally developed by Webb (Webb J, 1963).

[0197]

[0198] 0, 1 VP = 1.245 = synergistic effect

[0199] 1 VP = 1.32 = synergistic effect.

[0200] 10 VPs = 1.2 = synergistic effect

[0201] 100 VPs = 1.1 = synergistic effect

[0202] 1000 VPs = 1.1 = synergistic effect

[0203] illustrate:

[0204] Synergistic effect = ratio greater than 1

[0205] Cumulative effect = equal to 1

[0206] Antagonism = less than 1

[0207] result:

[0208] These findings suggest that BiTE and TILT oncolytic adenovirus have a synergistic effect.

[0209] Experiment 13 (In vitro cell viability assay of combined Ad-BiTE and OT1 T cells on B16-OVA target cells)

[0210] B16-OVA cells were seeded at 1×10e4 cells / well in 96-well plates and infected with Ad-BiTE at 100 VP / cell, T cells (2:1 effector:target ratio), or both. Cell viability was assessed by MTS assay after 24 hours.

[0211] Experiment 14 (Adenovirus or adenovirus armored with IL2 is insufficient to accumulate T cells at the tumor site)

[0212] Adenovirus therapy was combined with adoptive T-cell transfer, resulting in suboptimal T-cell infiltration into B16.OVA melanoma tumors. Tumors collected 18 days after treatment initiation were analyzed by flow cytometry for ovalbumin-specific CD8+ T cells (OVA) and gp100-specific CD8+ T cells. OVA and gp100 are epitopes expressed on melanoma cells. Differences between treatment groups were not statistically significant and were not significant compared to T-cell therapy alone (virus-free). (Horizontal line, mean) Figure 14 ).

[0213] Experiment 15 (Oncolytic adenovirus cannot recruit cytotoxic CD8+ T cells to tumors)

[0214] Subcutaneous hamster pancreatic tumors (HapT1) were treated a total of five times over 19 days with either oncolytic adenovirus Ad5 / 3-E2F-d24 alone or oncolytic adenovirus Ad5 / 3-E2F-d24 armored with human IL-2. On day 25, animals were sacrificed, and tumor cells were labeled with a cross-reactive anti-rat CD8b PE antibody. (Sample number: simulated and unarmed n = 5, IL2 n = 1). Oncolytic adenovirus alone failed to recruit Cd8 cells to the tumor. IL2 appeared more promising, but the increase was not significant. Figure 15 ).

[0215] Experiment 16 (Reattack in immunized hamsters)

[0216] Hamsters previously cured with unarmored oncolytic adenovirus Ad5 / 3-E2F-d24 or with armored adenovirus treated with cytokines (TNFα, IL-2, or both) were resistant to the same tumor type (HapT1) but not to a different tumor type (DDT1-MF2). Untreated (naïve) animals that had not previously encountered either cell line served as controls. Armored viruses with molecules capable of inducing antitumor immunity (e.g., BITE) are essential for inducing protective immunity (i.e., signs of a memory response against tumor epitopes). Figure 16 ).

[0217] Experiment 17 (In vivo efficacy of armored or unarmored oncolytic adenovirus with or without T-cell therapy)

[0218] On days 1 and 8, oncolytic adenovirus Ad5 / 3-d25 (1×10⁻⁶) was administered. 7 HapT1-infiltrated tumors were established through intratumoral injection of VP / tumor. On day 2, cultured HapT1 tumor-infiltrating lymphocytes (1.5 × 10⁻⁶) were administered intratumorally. 6 TIL / tumor). Error bars, SE. * p < 0.05, ** p < 0.01. The best antitumor efficacy was observed when tumors were treated with oncolytic viruses (e.g., BiTe-encoded viruses) and TILs were also administered. Figure 17 ).

[0219] Experiment 18 (Hypothetical results from an in vivo antitumor efficacy study of combined Ad-BiTE and OT1T cell metastasis in immunocompetent mice carrying B16-OVA tumors)

[0220] Subcutaneously implanted B16-OVA tumors (0.25 × 10e6 cells / tumor) will be treated with a single intraperitoneal injection of CD8-rich OT1T cells, intratumoral injection of Ad-BiTE (1 × 10e9 VP / tumor), or both. Viral injections will be repeated every 7 days. Figure 18 ).

[0221] Experiment 19 (Adenoviral delivery of cytokines IL2 and TNFα enhances the efficacy of adoptive cell therapy: the mechanism of including cytokines in an oncolytic adenovirus encoding BiTE)

[0222] On day 1, C57 mice carrying B16-OVA tumors were intratumorally treated with 1 × 10e9 viral particles of armored adenovirus and intraperitoneally treated with 1.5 × 10e6 CD8-rich OT-1 T cells. Viral treatment was continued every 7 days. Figure 19 ).

[0223] Experiment 20 (Novel Viral Construct)

[0224] We generated a novel oncolytic Ad5 / 3 adenovirus carrying the following backbone: Ad5 / 3-E2F-D24-transfer. The transgene is located in the deleted E3 gp19k / 6.7K region. The following transgene was used as a vector:

[0225] aMesothelin-aCD3

[0226] aEpCAM-aCD3

[0227] aMUC1-aCD3

[0228] aMesothelin-aCD3-IRES-IL2

[0229] aMesothelin-aCD3-IRES-TNFα

[0230] aEpCAM-aCD3-IRES-IL2

[0231] aEpCAM-aCD3-IRES-TNFα

[0232] aMUC1-aCD3-IRES-IL2

[0233] aMUC1-aCD3-IRES-TNFα

[0234] ( Figure 20 and 21 )

[0235] Figure 20 adenovirus vector or Figure 21The construct map contains nucleotide sequences of, for example, the transgenic aMesothelin-aCD3 (SEQ ID NO: 9), aEpCAM-aCD3 (SEQ ID NO: 4), or aMUC1-aCD3 (SEQ ID NO: 8) listed in Table 2. The nucleotide sequences of the viral vectors of the present invention comprise or consist of the following: for example, SEQ ID NO: 1, 2, 3, 5, 6, 9 (aMesothelin-aCD3-IRES-IL2); SEQ ID NO: 1, 2, 3, 5, 6, 7 (aMesothelin-aCD3-IRES-TNFα); SEQ ID NO: 1, 2, 3, 4, 5, 6 (aEpCAM-aCD3-IRES-IL2); SEQ ID NO: 1, 2, 3, 4, 5, 7 (aEpCAM-aCD3-IRES-TNFα); SEQ ID NO: 1, 2, 3, 5, 6, 8 (aMUC1-aCD3-IRES-IL2); SEQ ID NO: 1, 2, 3, 5, 7, 8 (aMUC1-aCD3-IRES-TNFα). Adenovirus vectors are constructed according to the sequences listed in Table 2. The general methods for constructing adenovirus vectors also used in this invention are well known to those skilled in the art and described, for example, in Koski et al. 2010 and Hemminki et al. 2015.

[0236] Table 2. Sequence List.

[0237]

[0238] References

[0239] Blair GE, Dixon SC, Griffiths SA, Zajdel ME. Restricted replication of human adenovirus type 5 in mouse cell lines. Virus Res. 1989 Dec;14(4):339-46.

[0240] Ekkens MJ, Shedlock DJ, Jung E, Troy A, Pearce EL, Shen H, Pearce EJ. Th1 and Th2 cells help CD8 T-cell responses. Infect Immun. 2007 May;75(5):2291-6.

[0241] Hemminki, O., S. Parviainen, J. Juhila, R. Turkki, N. Linder, J. Lundin, M. Kankainen, A. Ristimaki, A. Koski, I. Liikanen, M. Oksanen, D. M. Nettelbeck, K. Kairemo, K. Partanen, T. Joensuu, A. Kanerva and A. Hemminki(2015). Immunological data from cancer patients treated with Ad5 / 3-E2F-Delta24-GMCSF suggests utility for tumor immunotherapy. Oncotarget 6(6):4467-4481.

[0242] Kanerva A et al. 2005, Gene Therapy 12, 87-94.

[0243] Kanerva A et al. Clin Cancer Res. 2013 May 15;19(10):2734-44.

[0244] Koski, A., L. Kangasniemi, S. Escutenaire, S. Pesonen, V. Cerullo, I. Diaconu, P. Nokisalmi, M. Raki, M. Rajecki, K. Guse, T. Ranki, M. Oksanen, S. L. Holm, E. Haavisto, A. Karioja-Kallio, L. Laasonen, K. Partanen, M. Ugolini, A. Helminen, E. Karli, P. Hannuksela, S. Pesonen, T. Joensuu, A. Kanerva and A. Hemminki (2010). Treatment of cancer patients with a serotype5 / 3 chimeric oncolytic adenovirus expressing GMCSF. Mol Ther 18(10): 1874-1884.

[0245] Kratky W, Reis e Sousa C, Oxenius A, Spörri R. Direct activation of antigen - presenting cells is required for CD8+ T - cell priming and tumor vaccination. Proc Natl Acad Sci U S A. 2011 Oct 18;108(42):17414 - 9.

[0246] Lugade AA, Sorensen EW, Gerber SA, Moran JP, Frelinger JG, Lord EM. Radiation - induced IFN - gamma production within the tumor microenvironment influences antitumor immunity. J Immunol. 2008 Mar 1;180(5):3132 - 9.

[0247] Park, J. G., H. Frucht, R. V. LaRocca, D. P. Bliss, Jr., Y. Kurita, T. R. Chen, J. G. Henslee, J. B. Trepel, R. T. Jensen, B. E. Johnson and etc. (1990). Characteristics of cell lines established from human gastric carcinoma. Cancer Res 50(9): 2773 - 2780.

[0248] Parviainen, S., M. Ahonen, I. Diaconu, M. Hirvinen, A. Karttunen, M. Vaha - Koskela, A. Hemminki and V. Cerullo (2014). CD40 ligand and tdTomato - armed vaccinia virus for induction of antitumor immune response and tumor imaging. Gene Ther 21(2): 195 - 204.

[0249] Propper DJ, Chao D, Braybrooke JP, Bahl P, Thavasu P, Balkwill F,Turley H, Dobbs N, Gatter K, Talbot DC, Harris AL, Ganesan TS. Low-dose IFN-gamma induces tumor MHC expression in metastatic malignant melanoma. ClinCancer Res. 2003 Jan;9(1):84-92.

[0250] Schroder K, Hertzog PJ, Ravasi T, Hume DA. Interferon-gamma: anoverview of signals, mechanisms and functions. J Leukoc Biol. 2004 Feb;75(2):163-89.

[0251] Street D, Kaufmann AM, Vaughan A, Fisher SG, Hunter M,Schreckenberger C, Potkul RK, Gissmann L, Qiao L. Interferon-gamma enhancessusceptibility of cervical cancer cells to lysis by tumor-specific cytotoxicT cells. Gynecol Oncol. 1997 May;65(2):265-72.

[0252] Tahtinen, S., S. Gronberg-Vaha-Koskela, D. Lumen, M. Merisalo-Soikkeli, M. Siurala, AJ Airaksinen, M. Vaha-Koskela and A. Hemminki(2015). Adenovirus Improves the Efficacy of Adoptive T-cell Therapy by Recruiting Immune Cells to and Promoting Their Activity at the Tumor. CancerImmunol Res 3(8): 915-925.

[0253] Tahtinen, S., S. Kaikkonen, M. Merisalo-Soikkeli, S. Gronberg-Vaha-Koskela, A. Kanerva, S. Parviainen, M. Vaha-Koskela and A. Hemminki (2015). Favorable alteration of tumor microenvironment by immunomodulatory cytokines for efficient T-cell therapy in solid tumors. PLoS ONE 10(6): e0131242.

[0254] Taipale, K., I. Liikanen, J. Juhila, R. Turkki, S. Tahtinen, M.Kankainen, L. Vassilev, A. Ristimaki, A. Koski, A. Kanerva, I. Diaconu, V.Cerullo, M. Vaha-Koskela, M. Oksanen, N. Linder, T. Joensuu, J. Lundin and A.Hemminki (2016). Chronic Activation of Innate Immunity Correlates With PoorPrognosis in Cancer Patients Treated With Oncolytic Adenovirus. Mol Ther 24(1): 175-183.

[0255] Webb J. Effect of more than one inhibitor, antagonism, summation, and synergism. In: Webb J, ed. Enzyme and metabolic inhibitors. New York: Academic Press, 1963. 488–512.

[0256] Yu et al 2014, Mol Ther 22(1):102-11.

Claims

1. Oncolytic adenovirus vector, which contains 1) The E2F1 promoter for tumor-specific expression of E1A; 2) A 24bp deletion (D24) in the Rb binding constant region 2 of adenovirus E1; 3) The viral gp19k and 6.7k reading frames in the E3 region are missing nucleic acid sequences; and 4) The nucleic acid sequence encoding the bipartite molecule, replacing the missing nucleic acid sequence as defined in point 3), wherein the bipartite molecule comprises a single-stranded variable fragment (scFv) specific to the cell surface molecule CD3 and an scFv specific to the tumor antigen MUC1. The vector has an Ad5 nucleic acid backbone and also contains Ad3 fibrous knots.

2. The oncolytic adenovirus vector according to claim 1, further comprising IL-2 transgene.

3. The oncolytic adenovirus vector of claim 2, wherein the vector comprises an internal ribosome entry site (IRES) located between the bigenotype molecule and the IL-2 transgene.

4. A pharmaceutical composition comprising an oncolytic adenovirus vector according to any one of claims 1-3.

5. The oncolytic adenovirus vector according to any one of claims 1-3, for the treatment of cancer.

6. The oncolytic adenovirus vector used in claim 5, wherein the cancer is ovarian cancer.

7. The oncolytic adenovirus vector used in claim 5, wherein the cancer is lung cancer.

8. The oncolytic adenovirus vector used in any one of claims 5-7, used in conjunction with an adoptive cell therapy composition.

9. The oncolytic adenovirus vector according to claim 8, used to enhance the efficacy of adoptive cell therapy in subjects.

10. Use of the oncolytic adenovirus vector of any one of claims 1-3 in the preparation of a composition for treating cancer in a subject, wherein the method comprises administering the oncolytic adenovirus vector of any one of claims 1-3 to the subject.

11. The use according to claim 10, wherein the method further comprises administering the adoptive cell therapy composition to the subject.

12. The use according to claim 11, wherein the adoptive cell therapy composition comprises a cell type selected from the group consisting of tumor-infiltrating lymphocytes (TILs), T-cell receptor-modified lymphocytes, and chimeric antigen receptor-modified lymphocytes.

13. The use according to claim 11 or claim 12, wherein the adoptive cell therapy composition comprises a cell type selected from the group consisting of T cells, CD8+ cells, CD4+ cells, NK cells, delta-gamma T cells, regulatory T cells, and peripheral blood mononuclear cells.

14. The use according to any one of claims 11-13, wherein the adoptive cell therapy composition comprises T cells.

15. The use according to any one of claims 9-14, wherein the cancer is selected from the group consisting of: nasopharyngeal cancer, synovial cancer, hepatocellular carcinoma, renal cancer, cancer of connective tissues, melanoma, lung cancer, bowel cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, throat cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, von Hippel-Lindau disease. Hippel-Lindau disease, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, ureter cancer, brain cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, bonecancer, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, gastrointestinal carcinoid.tract, fibrosarcoma, breast cancer, Paget's disease, cervical cancer, colorectal cancer, rectal cancer, esophagus cancer, gallbladder cancer, head cancer, eye cancer, neck cancer, kidney cancer, Wilms' tumor, liver cancer, Kaposi's sarcoma, prostate cancer, lung cancer, testicular cancer, Hodgkin's disease, non-Hodgkin's lymphoma, oral cancer, skin cancer, mesothelioma, multiple myeloma, ovarian cancer, endocrine pancreatic cancer. Cancer, glucagonoma, pancreatic cancer, parathyroid cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestinal cancer, stomach cancer, thymus cancer, thyroid cancer, trophoblastic cancer, hydatidiform mole, uterine cancer, endometrial cancer, vaginal cancer, vulva cancer, acoustic neuroma, mycosisThe following cancers are associated with cancer: fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gum cancer, heart cancer, lip cancer, meninges cancer, mouth cancer, nerve cancer, palate cancer, parotid gland cancer, peritoneum cancer, pharynx cancer, pleural cancer, salivary gland cancer, tongue cancer, and tonsil cancer.

16. The use according to any one of claims 10-15, wherein the cancer is lung cancer or ovarian cancer.

17. The use according to claim 16, wherein the oncolytic adenovirus vector comprises the characteristic Ad5 / 3-E2F-d24-aMUC1aCD3 and the cancer being treated is lung cancer or ovarian cancer.

18. The use according to claim 16, wherein the oncolytic adenovirus vector comprises the characteristic Ad5 / 3-E2F-d24-aMUC1aCD3-IRES-IL2 and the cancer to be treated is ovarian cancer.

19. The use according to any one of claims 10-18, wherein the oncolytic virus vector and adoptive cell therapy composition are administered to the subject simultaneously or sequentially in any order during the method.

20. The use according to any one of claims 10-19, wherein the method further comprises administering to the subject simultaneous or sequential radiotherapy, monoclonal antibody, chemotherapy or other anticancer drugs or interventions.

21. Use of the oncolytic adenovirus vector according to any one of claims 1-3 in the preparation of a composition for a method of increasing the efficacy of adoptive cell therapy in a subject, the method being carried out by administering the oncolytic adenovirus vector according to any one of claims 1-3 to a subject in need, wherein adoptive cell therapy has been or will be administered to the subject.

22. The use according to claim 21, wherein the oncolytic adenovirus vector comprises characteristic Ad5 / 3-E2F-d24-aMUC1aCD3, the adoptive cell therapy composition comprises T cells, and the cancer being treated is lung cancer or ovarian cancer.

Images