Transgene-encoding viruses for complementing cell therapy

Abstract

An oncolytic group B adenovirus suitable for the treatment of solid tumors (e.g., sarcomas, carcinomas, and / or lymphomas) comprising a sequence of formula (I): 5'ITR-B1-BA-B2-BX-BB-BY-B3-3'ITR(I), wherein a first transgene encodes a polypeptide comprising a targeting sequence specific for a binding domain on cells of a cell-based immunotherapy having an (exogenous) recombinant surface-expressed protein, such as a chimeric antigen receptor or an NKG2D receptor, in particular, the targeting sequence specifically binds to said surface-expressed protein (more specifically, a chimeric antigen receptor) on the immunotherapy cells, and a second transgene encodes a polypeptide comprising a molecule that facilitates the delivery of the cell-based immunotherapy into the tumor.

Description

[Technical Field]

[0001] The present disclosure relates to oncolytic viruses engineered to encode transgenes that specifically "paint" tissue cells with target antigens for antigen-specific immune cells, formulations comprising said viruses, and the use of said viruses and formulations in treatments, particularly combination therapies, especially combination therapies involving cell therapies such as CAR-T. [Background technology]

[0002] Cell-based immunotherapies, such as chimeric antigen receptor (CAR) T-cell therapy, have emerged as important treatments for hematological malignancies.

[0003] CAR T cells directed against B-cell antigens have been tested in the clinic and often induce long-term remissions, even in heavily pretreated patients. Currently, there are approximately five CAR T-cell therapies commercially available for hematologic malignancies.

[0004] In contrast, CAR-T cell therapy has yet to achieve similar depth and reproducibility of response in patients with solid tumors.

[0005] For example, CAR T cells targeting B7-H3, CEACAM5, CD133, CDI71, claudin-6, EGFR, EGFRvIII, FRa, GD2, GPC3, HER2, IL-13Ra2, mesothelin, MUCl, PSMA, RORI, and VEGF-R2 have been tested in the clinic but have demonstrated limited ability to control disease.

[0006] Chimeric antigen receptor (CAR) T cells are T cells that express a genetically engineered receptor that can recognize surface antigens on target cells and trigger T cell activation and antigen-specific cytotoxicity. Many alternative cell technologies are currently being developed that are also suitable for use in combination with the viruses of the present disclosure.

[0007] Several factors are thought to contribute to this overwhelming activity in solid tumors, including a) the paucity of target antigens in solid tumors, b) heterogeneous target expression among tumor cells, c) insufficient trafficking of CAR-T cells to solid tumors, and d) a solid tumor microenvironment that is hostile to immune cell function and survival (Non-Patent Document 1). In particular, injected cell-based immunotherapies are widely dispersed in the blood, with only a small proportion reaching the tumor microvasculature. Therefore, inadequate trafficking of CAR-T cells to solid tumors (item c above) may be particularly important.

[0008] As mentioned above, there is a lack of target antigens that are expressed only on tumor cells, but molecules can be designed to bind to tumor cell antigens that are not completely tumor-specific (i.e., can be expressed at some level in healthy tissue), which may lead to off-target effects.

[0009] Ambrose et al. (Non-Patent Document 2) developed anti-CD19 CAR-T cells capable of secreting an anti-HER2-CD19 cross-linking protein, which binds to HER2-positive tumor cells, enabling the anti-CD19 CAR-T cells to recognize both CD19- and HER-2-expressing cells. However, this secreted protein is expressed in the location where the CAR-T cells are found after intravenous (IV) injection, potentially causing extratumoral cytotoxicity.

[0010] It has been proposed to use a fusion protein containing an antigen target for CAR-T therapy and a second binding domain that binds to some entity within the tumor (see, for example, Patent Document 1). The fusion protein may be expressed, for example, within the tumor, and upon binding, activation of CAR-T therapy may occur. However, this approach does not fundamentally address the problem of delivering immunotherapy to the tumor.

[0011] Furthermore, the stroma surrounding solid tumors acts like a fortress wall, creating a hypoxic microenvironment that inhibits immune cell cytotoxic activity. Thus, even after successful delivery into the local microenvironment, the latter may impair the function of cell-based immunotherapies and / or promote their clearance through different soluble molecules and immune suppressor cells (e.g., TGFβ, adenosine, regulatory T cells, myeloid-derived suppressor cells).

[0012] Some cells engineered for use in cell-based immunotherapy are engineered to express "exogenous" cytokines to maintain activation. However, if the cytokine is constitutively expressed, problems can arise in that it can cause off-target effects (side effects). In contrast, if the cytokine is inducibly expressed, for example, after the cells bind to the target antigen, the cells may never reach the target due to the tumor microenvironment.

[0013] To successfully treat solid tumors with CAR-T cells, it will be important to find ways to enhance the homing of cell-based immunotherapies to tumors and / or reprogram the tumor microenvironment, e.g., from an immunosuppressive to an immunostimulatory microenvironment. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] International Publication No. 2018 / 156791 [Non-patent literature]

[0015] [Non-Patent Document 1] Hou et al.Nat Rev Drug Discov.2021 [Non-patent document 2] Ambrose, Christine et al. "Anti-CD19 CAR T cells potently redirected to kill solid tumor cells." PloS one vol.16,3 e0247701.18 Mar.2021, doi:10.1371 / journal.pone.0247701 Summary of the Invention [Problem to be solved by the invention]

[0016] The present inventors have engineered oncolytic viruses that preferentially infect cancer cells and are highly adapted to survive in the tumor microenvironment. These viruses not only pave the way to cancer by infecting, replicating, and lysing cancer cells, but in this disclosure they are also engineered to express transgenes that have two independent aspects: - locally expressing antigen targets (referred to herein as target sequences) of cell-based immunotherapy within tumors (e.g., to increase the local concentration of the target antigen); and Facilitating the recruitment and / or activity of cell-based immunotherapies from within tumors. [Means for solving the problem]

[0017] Thus, the present disclosure functions by increasing the local concentration of targets or activators to antigen-specific cells (particularly engineered antigen-specific cells). The virus also supports alterations in the tumor microenvironment to support the influx and / or survival of antigen-specific cells.

[0018] Advantageously, stimulating antigen-specific cells minimizes off-target effects and maximizes treatment by targeting only diseased tissue.

[0019] The local increase in target antigen cell surface expression of target sequences on infected cells; This can be increased by one or more (i.e., a combination) of secretion of fusion proteins from infected cells, which bind to entities on diseased cells (infected cells and / or surrounding cells) and present the target sequence.

[0020] This combination approach has not been used before, and the inventors have demonstrated that it increases the number of active immunotherapeutic cells recruited to the tumor.

[0021] To aid in the recruitment of cell-based immunotherapies to tumors, oncolytic viruses can be equipped with transgenes that reprogram the tumor microenvironment to promote the infiltration and activity of cell-based immunotherapies.

[0022] Once tumors are more accessible through "transport genes," the concentration of the target antigen increases, potentially acting like a magnet to mobilize and activate cell-based therapies, thereby increasing the local concentration of cell-based therapy where it is needed.

[0023] Cell lysis by oncolytic viruses also releases proinflammatory mediators, such as HMGB1, ATP, and type I interferons, which can induce antitumor immune responses and recruit innate immune cells from peripheral lymphoid organs.

[0024] Non-specific activation of immune cells can be used in combination with the techniques of the present disclosure, for example, a bispecific T cell activator that includes an agonist for CD3 can be encoded in a virus of the present disclosure.

[0025] Depending on the antigen target of cell-based immunotherapy, for example, if the target is a tumor antigen, therapeutic cells recruited to the tumor site directly activate tumor cells that express the antigen but are not necessarily infected with oncolytic viruses. Fusion proteins containing the tumor antigen targeted by cell therapy and a binding domain for a second antigen, such as a stromal antigen, can be secreted from infected cells. After binding to the stroma, the tumor antigen is presented for cell therapy, making it possible to target two different tissue types with a single cell therapy. Thus, the mechanisms of viral activation and cell therapy are complementary but independent.

[0026] Thus, in one embodiment, virally infected cells secrete a fusion protein that binds to antigens on surrounding cells, such as other tumor cells and / or stromal cells (infected or not), presenting a target sequence for the cell therapy to bind. This binding generally activates the cell therapy.

[0027] In one embodiment, the virus of the present disclosure encodes one or more, two or more, three or more, four or more fusion proteins comprising a binding domain and a targeting sequence for cell therapy.

[0028] If there are multiple fusion proteins encoded by the virus, the binding domains may bind to different entities and the targeting sequence may be the same, allowing one cell therapy to be used to target two or more different cells, tissues or therapeutic targets within cancer tissue.

[0029] In some patients, different types of cancer cells coexist, e.g., expressing different antigens. For these patients, it would be useful to be able to target these different cancer cells with a single cell therapy. This can be achieved by encoding two fusions, each with a binding domain specific for a different cancer cell, e.g., a single targeting sequence (or multiple targeting sequences, if desired).

[0030] In one embodiment, the oncolytic virus encodes a transgene that is expressed in the microenvironment, e.g., as a soluble protein, to protect / activate the engineered immune cells and / or immune cells in general. For example, IFNα, IL-12, and IL-15 are cytokines useful for protecting and / or activating immune cells in the tumor microenvironment, and chemokines such as CXCL9 and CCL21 are useful for directing immune cells to target cells within the tumor mass.

[0031] Thus, there are at least two or three mechanisms by which the oncolytic viruses and combination therapies of the present disclosure can improve the therapeutic outcome of solid tumors.

[0032] The present inventors have prepared tumor-specific group B adenoviruses, particularly EnAd, encoding a series of different bispecific proteins containing a moiety capable of binding to an antigen expressed on the surface of cells, specifically tumor cells or tumor-associated fibroblasts, and a moiety that binds to immune cells, such as T cells bearing an engineered CAR or TCR, or an endogenous receptor (e.g., TCR), or other surface molecule on the cell, thereby binding to immune cells, such as T cells bearing a CAR or engineered T cell receptor (TCR). The inventors discovered that in many cases, the tumor cell-binding moiety was functional, but the immune cell-binding moiety was dysfunctional. To solve this problem, different protein sequences and designs were screened until a functional bispecific protein was found. However, even when functional bispecific molecules were obtained, their expression levels were found to be lower than desired.

[0033] Surprisingly, when the bispecific protein was sequentially (tandemly) encoded with one or more different immunomodulatory molecules, including chemokines and cytokines, its expression levels were consistently increased.

[0034] According to the present disclosure, the tumor microenvironment (TME) is permissive to infiltration by adenovirus, thereby allowing sufficient levels of the bispecific protein to be delivered to the desired location. Furthermore, co-expression of a bispecific protein with an immunomodulatory molecule capable of recruiting T cells or promoting direct or indirect T cell activation may synergistically enhance the ability of CAR-T cells to recognize and kill target cells within the TME.

[0035] Surprisingly, the inventors found that adding two or more transgenes encoding bispecific proteins into the same transgene cassette clearly increased the expression of the bispecific proteins compared to the corresponding viral design encoding only the bispecific proteins.

[0036] The viruses of the present disclosure complement and / or improve cell therapy.

[0037] The present disclosure is summarized in the following paragraphs: 1. An oncolytic group B adenovirus suitable for treating solid tumors (e.g., sarcomas, carcinomas and / or lymphomas) comprising a sequence of formula (I), 5'ITR-B1-B A -B2-B X -B B -B Y -B3-3'ITR(I) During the ceremony, B1 is a bond or comprises E1A, E1B or E1A-E1B; B A contains E2B-L1-L2-L3-E2A-L4, B2 is a bond or contains E3; B X is a DNA sequence containing a junction or restriction site, one or more transgenes, or both, B B includes L5, B Yis a DNA sequence encoding at least two transgenes, i.e., a first transgene and a second transgene, for example under the control of a major late promoter, B3 is a bond or contains E4; During the ceremony, the first transgene encodes a polypeptide comprising a target sequence specific for a binding domain on a cell-based immunotherapy with an (exogenous) recombinant surface-expressed protein, such as a chimeric antigen receptor or an NKG2D receptor, in particular, the target sequence specifically binds to said surface-expressed protein (more particularly, a chimeric antigen receptor) on the immunotherapy cells; The second transgene is an oncolytic group B adenovirus encoding a polypeptide containing a molecule that facilitates delivery of the cell-based immunotherapy into the tumor. 2. The oncolytic group B adenovirus of paragraph 1, wherein expression of the first transgene increases the local concentration of the target sequence within the tumor. 3. The oncolytic group B adenovirus according to paragraph 1 or 2, wherein the target sequence is specific for a recombinant receptor on an immunotherapy cell. 4. The oncolytic group B adenovirus of any one of paragraphs 1 to 3, wherein the target sequence is a tumor antigen. 5. Target sequences are those expressed on cancer cells (CD20, CD19, CD22, CD33, CD34, CD37, CD38, CD47, CD52, CD56, CD70, CD74, CD133, CD138, CD147, CD152, CD221, CD254, CD261, CD262, CD309, CD340, BCMA, C-MYC, CAIX, claudin [claudin 1]). , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and / or 24, especially claudin-6 or claudin-18.2], EGFRvIII, EPHA3, folate receptor alpha [FRα], GPC3, WT1, CEA, MUC-1, EpCAM, MAGE, mesothelin, PRAME, NYESO, AFP, CA-125, ETA, tyrosinase, RAS, p53, HER receptor HER1 [EGFR], HER2, HER3, HER4, MCAM, PEM, A33, G250, carbohydrate antigen Le y , Le x , Le b , PSMA, TAG-72, STEAP1, CD166, CD24, CD44, E-cadherin, SPARC, ErbB2 and ErbB3, episialin, FOLR-1, 5T4, GPNMB, integrin αVβ3, integrin α5β1, Lewis Y antigen, MET [HGFR], mucin, PMSA, TAG-72, VEGFR, PDL1 [or an antigenic fragment of any one thereof], tumor antigens such as CD19, BCMA, CEA, claudin-6, claudin-18.2, EGFRvIII, FRα, GPC3, MCAM, mesothelin, MUC-1, EpCAM, MAGE, PRAME, AFP, CA-125, ETA, tyrosinase, RAS, p53, HER receptors HER1, HER2, HER3, HER4, PEM, A33, G250, carbohydrate antigen Le y , Le x , Le b 5. The oncolytic group B adenovirus of any one of paragraphs 1 to 4, wherein the oncolytic group B adenovirus is selected from the group consisting of PSMA, TAG-72, STEAP1, CD166, CD24, CD44, E-cadherin, SPARC, ErbB2, and ErbB3, or an antigenic fragment of any one thereof. 6. The oncolytic group B adenovirus according to any one of paragraphs 1 to 5, wherein the target sequence is CD19 or an antigenic fragment thereof. 7. The oncolytic group B adenovirus according to any one of paragraphs 1 to 6, wherein the targeting ligand is a non-human sequence, for example from yeast (which is beneficial in terms of reducing off-target effects). 8. The oncolytic virus of any one of paragraphs 1 to 7, wherein the target sequence is a ligand (including an antibody, such as an idiotypic antibody, or an antigen-binding fragment thereof). 9. The oncolytic group B adenovirus of any one of paragraphs 1 to 8, wherein the target sequence is a ligand (comprising an antibody binding domain) that interacts with a "native" receptor on immune cells, activates signaling through a "native" receptor on immunotherapy cells, such as an NKG2D ligand (the latter interacting with native NKG2D on NK cells and CD8 T cells to stimulate cytotoxic activity) or CD40L, OX40L, CD80, CD86, 4-1BBL (TNFSF9), CD70, LIGHT (TNFSF14), GITRL (TNFSF18), CD258 (HVEML, TNFRSF14), ICOSL (B7-H2), etc. 10. The oncolytic group B adenovirus according to any one of paragraphs 1 to 8, wherein the target sequence is a ligand (comprising an antibody binding domain) that inhibits signaling through a "native" receptor such as PD1, TIM3, LAG3, VISTA, TIGIT, B7-H3, B7-H4, HVEM, ILT-2, ILT-3, ILT-4, BTLA, CD160 on immunotherapy cells, e.g., PD1, TIM3, LAG3, VISTA, TIGIT on T cells. 11. The oncolytic group B adenovirus according to any one of paragraphs 1 to 10, wherein the first transgene encodes a targeting sequence in a membrane-anchored form suitable for expression on the surface of the infected cancer cell, in particular allowing the cell-based therapy to bind to the cancer cell directly (i.e., not via a fusion protein). 12. The oncolytic group B adenovirus according to paragraph 11, wherein the membrane-anchored form comprises a transmembrane domain or a GPI anchor. 13. The oncolytic group B adenovirus of any one of paragraphs 1 to 12, wherein the target sequence is a non-human, eg, murine, or yeast antigen such as GCN4. 14. The oncolytic group B adenovirus of any one of paragraphs 1 to 13, wherein the target sequence is a tag such as, for example, an HA tag (amino acids 98 to 106 of human influenza hemagglutinin), a His tag (e.g., containing at least six histidine residues), a FLAG tag, or a 2A peptide tag (such as P2A, T2A, E2A, and / or F2A). 15. The oncolytic group B adenovirus according to any one of paragraphs 1 to 13, wherein the target sequence is epitope E5B9 (derived from La-SS / B antigen), as disclosed, for example, in WO2015 / 181282, which is incorporated herein by reference. 16. The first transgene is a. a target sequence that binds to and activates cell-based immunotherapy, and b. A first binding protein specific for a protein expressed on cancer cells, stromal cells, or within stromal tissue (such as non-cellular matrix, particularly collagen), which specifically allows cell-based immunotherapies to indirectly bind to cancer cells and / or stromal cells or tissue via the fusion protein. 16. The oncolytic group B adenovirus of any one of paragraphs 1 to 15, encoding a fusion protein comprising: 17. The oncolytic group B adenovirus according to paragraph 16, wherein the virus encodes at least two fusion proteins and the first binding protein in part b) is different in each fusion protein, e.g., one fusion protein is encoded by a first transgene and a second fusion protein is encoded by a second transgene. 18. The oncolytic group B adenovirus according to paragraph 16, wherein the target sequence of part a) is the same for said at least two fusion proteins, i.e. both fusion proteins bind to the same entity on immunotherapy cells. 19. The oncolytic group B adenovirus according to paragraph 17, wherein the targeting sequences of part a) are different in the two fusion proteins (i.e., the fusion proteins bind to different entities on the same or different immune cells). 20. The oncolytic group B adenovirus according to any one of paragraphs 16 to 19, wherein the binding protein of part b) is, for example, a ligand for a protein or receptor found on cancer cells and / or stromal cells (e.g., if multiple fusion proteins are present, the binding domains are specific for different antigens / markers / targets). 21. The oncolytic group B adenovirus of paragraph 20, wherein the ligand is an antibody or an antigen-binding fragment thereof. 22. The oncolytic group B adenovirus according to any one of paragraphs 1 to 21, wherein the target sequence is expressed on the stroma or stromal cells. 23. The oncolytic group B adenovirus of paragraph 22, wherein the stromal cells are independently selected from the group comprising or consisting of cancer-associated fibroblasts, tumor-associated macrophages, or other suppressor cells, e.g., the target sequences are CD163, CD206, CD68, CD11c, CD11b, CD14, CSF1 receptor, CD15, CD33 and CD66b, fibroblast activation protein (FAP), TREM1, IGFBP7, FSP-1, platelet-derived growth factor-alpha receptor (PDGFR-α), platelet-derived growth factor-beta receptor (PDGFR-β), and vimentin or an antigenic fragment thereof. 24. An oncolytic group B adenovirus according to any one of paragraphs 1 to 23, encoding a second transgene thereby enhancing the efficacy (or broadening the spectrum of target tissues) of cell-based immunotherapy. 25. The oncolytic group B adenovirus of paragraph 24, wherein the further transgene (e.g., second transgene or third transgene) enhances the efficacy of cell-based immunotherapy by modulating the tumor microenvironment (e.g., blocking inhibitory properties of the tumor microenvironment). 26. The oncolytic group B adenovirus according to paragraph 25, wherein the microenvironment is modulated to make the microenvironment more amenable to cell-based therapy and / or to make the microenvironment more inflammatory. 27. The oncolytic group B adenovirus according to paragraph 25 or 26, wherein the further transgene is a matrix degrading or loosening agent that modulates the tumor microenvironment and allows for a more extensive (permissive) infiltration of immunotherapeutic cells and / or better promotes / supports the innate immune response, in particular when said immune cells remain activated in the microenvironment, e.g. aids cell migration within the tumor. 28. The oncolytic group B adenovirus according to paragraph 27, wherein the microenvironment is made more permissive by targeting stromal antigens such as fibroblast activation protein (FAP), LRRC15, CD10, GPR77, TREM1, IGFBP7, FSP-1, platelet-derived growth factor-alpha receptor (PDGFR-α), platelet-derived growth factor-beta receptor (PDGFR-β), and vimentin, and wherein the second gene may encode a bispecific T-cell activator, e.g., as disclosed in WO2018 / 041838 and WO2018 / 041827, both of which are incorporated by reference. 29. The oncolytic group B adenovirus according to any one of paragraphs 25 to 28, wherein the microenvironment is modulated by helping immune cells, including immunotherapeutic cells and natural immune cells, overcome inhibition. 30. The oncolytic group B adenovirus according to paragraph 29, wherein the ability to overcome inhibition is conferred by an immune checkpoint inhibitor, such as an anti-CTLA-4 inhibitor, an anti-PD-1 inhibitor, and an anti-PD-L1 inhibitor (in particular selected from ipilimumab, tremelimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, and cemiplimab). 31. The oncolytic group B adenovirus according to any one of paragraphs 25 to 30, wherein the microenvironment is modulated to become more inflammatory, for example through the production of transgene-encoded pro-inflammatory cytokines (e.g., IL-1, IL_6, TNFα, IFNγ) and / or chemokines (e.g., CXCL9, CCL3, CCL5) or the production induced by the encoded transgene (e.g., IL-12, IL-15). 32. The oncolytic group B adenovirus according to any one of paragraphs 24 to 31, wherein the microenvironment is modulated by enhancing the activation of immune cells (such as immunotherapeutic cells and / or natural immune cells), for example, wherein the second gene or further transgene encodes a cytokine. 33. Enhancement of immune cell activation can be achieved by e.g. IFNα, IFNβ, IFNγ, TNFα, TNFβ (LTα), IL-2, IL-7, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, IL- 22, IL-33, IL-35, VEGF-C, VEGF-D, IL-1α, IL-1β, IL-6, IL-9, IL-12, IL-13, IL-17, IL-18, IL-22, IL-23, IL-24, IL-25 33. The oncolytic group B adenovirus of paragraph 32, wherein the oncolytic group B adenovirus is mediated by a cytokine selected from the group consisting of IL-26, IL-27, IL-33, IL-35, IL-2, IL-4, IL-5, IL-7, IL-9, IL-10, IL-15, IL-21, IL-25, IL-1RA, IFNα, IFNβ, IFNγ, TNFα, VEGF-A, VEGF-C, VEGF-D, TGFβ, lymphotoxin alpha (LTA), and GM-CSF. 34. The oncolytic group B adenovirus according to paragraph 32 or 33, wherein the enhanced activation of immune cells is brought about by a membrane or soluble ligand comprising the extracellular domain of a protein involved in cell-to-cell interactions between immune cells, such as a costimulatory molecule expressed by antigen-presenting cells selected from CD40L, OX40L, CD80, CD86, 4-1BBL, TNFSF14 (LIGHT), GITRL (TNFSF18), CD70, CD258 (HVEML), and ICOSL. 35. The oncolytic group B adenovirus according to paragraph 32, wherein the enhanced activity is increased cytotoxic activity, for example by increasing the level of IFNα. 36. The oncolytic group B adenovirus according to any one of paragraphs 32 to 34, wherein the enhanced activity is the longevity (survival) of immune cells. 37. The oncolytic group B adenovirus of paragraph 36, wherein the extension of lifespan is mediated by cytokines such as IL-2, IL-7, IL-15, and / or IL-21. 38. The oncolytic group B adenovirus according to any one of paragraphs 1 to 37, wherein the second or further transgene encodes a chemokine that assists in the recruitment of immunotherapeutic cells to the tumor. 39. Additional transgenes encode synthetic proteins designed to bind to additional recombinant receptors expressed by the cells of the therapy to enhance their ability to enter and act in tumors and / or survival in the patient, for example, ortho-IL-2 as a synthetic ligand for ortho-IL-2Rβ (synthetic receptors—e.g., Zhang et al. 2021 Sci. Transl. Med. 13 (625) eabg6986, incorporated herein by reference), which can be engineered as a fusion with an antibody fragment to bind to tumor cells; TIM3 / CD28 switch receptors on CAR-T that interact with secreted TIM3 ligands, e.g., galectin-9, HMGB1 (Zhao et al. 2021 J. Immunotherapy Cancer 9, e003176, incorporated herein by reference); synNotch fusion receptors on CAR-T (e.g., when the CAR recognizes CD19 (CD19 binds to an anti-HER2 receptor); The oncolytic agent of any one of paragraphs 1 to 38, wherein the CAR is an anti-CD19 Scfv linked to the IC domain of Notch to signal a response in an engineered CAR (including when in a fusion protein such as an ScFv-CD19 fusion protein - Roybal et al 2016 Cell 167(2),419-432, incorporated herein by reference). 40. An oncolytic according to any one of paragraphs 1 to 39, wherein the further transgene encodes a polypeptide that enhances the recruitment and anti-tumor activity of the patient's endogenous immune cells. 41. The oncolytic group B adenovirus according to any one of paragraphs 1 to 38, wherein the cells expressing an (exogenous) recombinant antigen receptor comprise or are selected from the group consisting of T cells (T), macrophages (Mac), natural killer cells (NK), natural killer T cells (NKT) or innate lymphoid cells (ILC). 42. The oncolytic group B adenovirus according to any one of paragraphs 1 to 41, wherein the exogenous recombinant antigen receptor (or synthetic receptor) is a chimeric antigen receptor (CAR) or a T cell receptor (TCR). 43. An oncolytic group B adenovirus according to any one of paragraphs 1 to 41 for use in therapy, in particular for use in the treatment of cancer, such as solid tumors. 44. The use of an oncolytic group B adenovirus according to paragraph 43, wherein the cancer is a transformed epithelial cancer cell. 45. An oncolytic group B adenovirus according to any one of paragraphs 1 to 42 for use in the manufacture of a medicament for the treatment of cancer, such as solid tumors, in particular transformed epithelial cancer cells. 46. A method of treating a patient, particularly for treating cancer, more particularly solid tumors such as transformed epithelial cancer cells, comprising administering a therapeutically effective amount of an oncolytic group B adenovirus according to any one of paragraphs 1 to 42. 47. A combination therapy comprising an oncolytic group B adenovirus according to any one of claims 1 to 42 and engineered immunotherapeutic cells, for example to treat cancer. 48. A method for producing a virus according to any one of paragraphs 1 to 42, wherein the virus is replicated in a host cell, for example a mammalian cell such as an HEK cell. 49. Virus taken from paragraph 48.

[0038] Also provided according to the present disclosure is an oncolytic group B adenovirus, wherein the second transgene encodes, for example, a second binding protein different from the first binding protein and an antigen targeting sequence that enables the same or a different cell-based immunotherapy to bind, via the further fusion protein, to a second protein expressed on cancer cells, on stromal cells or in stromal tissue and that is a different entity than that bound by the first binding protein, and in particular enables the cell-based immunotherapy to indirectly bind to cancer cells and / or stroma via the fusion protein.

[0039] Combinations of embodiments may be used where technically feasible.

[0040] In one embodiment, however, the fusion protein does not comprise (or consist of) i) a tumor-associated or tumor-specific antigen, and ii) an anti-idiotypic antibody or fragment or anti-idiotypic peptide.

[0041] In one embodiment, the virus of the present disclosure does not encode a bispecific T cell activator.

[0042] In one embodiment, the second or further transgene encodes IL-15 or an active fragment thereof, and is optionally co-expressed, i.e., encoded adjacent to or linked to at least the sushi domain of IL-15Ralpha, such as the full-length 15Ralpha extracellular domain (e.g., via a linker or amide bond).

[0043] Thus, in one embodiment, the presently disclosed viral constructs are designed to deliver antigens within target tissues to activate an expansion of a class of antigen-specific immune cells (e.g., a local increase in the number of specific immune cells [especially antigen-specific T cells], including exogenously engineered immune cells, particularly CAR-Ts).

[0044] It is hypothesized that increasing the local concentration of antigen within the target tissue exceeds the threshold level of antigen required for immune cell activation, sending a large activation signal to the desired cell population.

[0045] In one embodiment, the present disclosure does not relate to non-specific activation of immune cells such as T cells. Bispecific T cell activators are non-specific activators of T cells by combining CD3 with an agonist. This type of technology can be included as a complement to the present technology, but works by a different mechanism than the present technology, since the latter is involved in the trafficking and activation of antigen-specific cells. DETAILED DESCRIPTION OF THE INVENTION

[0046] As used herein, a target sequence refers to a target antigen sequence for an immune cell, e.g., a specific immune cell such as an antigen-specific immune cell, particularly an engineered immune cell (including a sequence that stimulates (particularly activates and / or protects) an existing immune cell or stimulates the production of said immune cell in a specific manner). In one embodiment, it does not refer to a binding domain of an antibody that circumvents specificity and nonspecifically stimulates immune cells such as T cells. Thus, an anti-CD3 agonist antibody that nonspecifically stimulates T cells is not included in the definition of a target sequence.

[0047] The target sequence may bind to a recombinant (engineered receptor) on an immune cell and / or may bind to a natural receptor on an immune cell, particularly a recombinant receptor on an immune cell, where the antigen-specific cell is functionally affected, e.g., activated, protected, proliferated.

[0048] In one embodiment, the target sequence is not an antibody sequence that non-specifically stimulates immune cells (such as an idiopathic antibody sequence).

[0049] As used herein, an antigenic fragment refers to a fragment of an antigen that is still capable of specifically binding to the binding domain, eg, an epitope, which may be at least 5 amino acids.

[0050] As used herein, cell-based immunotherapy refers to cell-based therapies that are prepared or created ex vivo and then administered to a patient, prepared using recombinant technology, e.g., vectors (such as viral vectors). In one embodiment, the cells in the therapy encode a transgene that specifically encodes a non-native protein. Cells used in cell therapy include, but are not limited to, T cells (including subclasses thereof), NK cells (including subclasses thereof, e.g., memory NK cells), NKT cells, macrophages, and the like.

[0051] As used herein, engineered cells are those that are modified using recombinant technology, including vectors such as viruses, and are particularly engineered ex vivo.However, the technology that includes vectors can also be used to target cells in vivo and modify them.If cells are modified (including transiently) at the genetic level by manipulation, they are considered as engineered cells in the context of this specification.

[0052] As used herein, a recombinant receptor or protein refers to a receptor or protein that has been produced or introduced by recombinant techniques.

[0053] In one embodiment, one or more constructs encoded by a virus according to the present disclosure are designed to bind to an engineered cell.

[0054] In one embodiment, one or more constructs encoded by a virus according to the present disclosure are designed to be specific for a moiety (e.g., an epitope or binding domain / ligand) that has been recombinantly introduced into a cell (an engineered cell), meaning that the original cell was recombinantly engineered, even though the engineered cell can be replicated to generate multiple cells containing the modification.

[0055] Thus, reference to cell-based immunotherapy does not simply refer to combining natural cells, such as naturally occurring T cells, that have not been modified ex vivo by recombinant techniques.

[0056] As used herein, recombinant technology refers to modifications to cells that are produced by human design and / or intervention, e.g., those that originate in a laboratory essentially, i.e., those that do not naturally occur in nature.

[0057] In one embodiment, the virus disclosed according to the present invention is isolated, ie, contained outside the body in a particularly purified form.

[0058] In one embodiment, the cell therapy used in the present disclosure is isolated, i.e., it is contained outside the body, particularly in a purified form.

[0059] In one embodiment, the constructs of the present disclosure do not exclusively engage the native TCR. In one embodiment, the constructs of the present disclosure do not engage the native TCR, although virally encoded constructs or additional elements may still engage the native TCR.

[0060] In one embodiment, the target sequence is not the binding domain of an antigen-specific T cell activator molecule. Accordingly, the present disclosure does not relate to bispecific T cell activators, which are disclosed in WO2018 / 041827 and WO2018 / 041838 and are incorporated herein by reference. However, bispecific T cell activators may be included as a "further" or additional transgene in the viruses of the present disclosure.

[0061] As used herein, "transport within a tumor" refers to when a gene product increases the number of active immunotherapeutic cells within a tumor. Therefore, "transport" in the context of the present disclosure relates to the concept of migration and / or activation of immunotherapeutic cells to reach their site of action within a tumor in an operable form, i.e., so that the cells reach their intended location without being neutralized. Therefore, transport agents include one or more of the following: agents that attract cells to tumors (e.g., chemokines); agents that disrupt the matrix / stromal barrier around tumors (e.g., enzymes or stromal antigens); agents that alter the microenvironment to be more tolerant (or less hostile) to immune cells (e.g., immunotherapeutic cells and / or natural immune cells), such as checkpoint inhibitors; agents that modulate the activity of immune cells (e.g., immunotherapeutic cells and / or natural immune cells); agents that recruit natural immune cells; a more inflammatory environment; lower hypoxic conditions; and combinations of two or more of these.

[0062] In one or more embodiments, the "second transgene" or transport component also assists in the transport of natural immune cells.

[0063] As used herein, modulation of immune cell activity refers to "activating an immune cell in any form, including, for example, directing the activity of an immune cell to a target, increasing the cytotoxic activity of an immune cell, increasing the proliferation of an immune cell, enhancing the production of soluble mediators (e.g., cytokines and chemokines) by an immune cell, extending the survival and / or lifespan of an immune cell, and protecting an immune cell from inhibition and / or anergy.

[0064] As used herein, naturally occurring immune cells are intended to refer to immune cells found in the body, i.e., modified cells that have been introduced or engineered as a therapy.

[0065] As used herein, "activating" an immune cell refers to inducing or enhancing one or more functions of the immune cell and / or providing a signal that enables the cell to functionally respond to other signals.

[0066] For example, IFNα can directly induce the production of cytokines and chemokines (e.g., IL-6, BAFF, April) and, when triggered by ligand interaction with the T cell receptor or CAR on CAR-T cells, can enhance the killing function of T cells.

[0067] As used herein, "increasing the local concentration of a target sequence" includes an increase from a starting concentration of zero or undetectable, and does not necessarily include an increase in the number of immune cells, or an increase in the activity of immune cells as well as an increase in their number.

[0068] As used herein, alterations in the tumor microenvironment refer to changes that allow immune cells to migrate into or out of the environment, including changes in the vasculature, hypoxia, inflammatory conditions, and / or immunosuppressive conditions.

[0069] As used herein, "specific" refers to the fact that a binding domain recognizes a target antigen with higher affinity and / or avidity (e.g., greater than 10, 20, 50, 10, or 1000) than other antigens for which it is not specific. This does not necessarily mean that a specific binding region will not bind to non-target antigens, but rather that it can be used to purify a (specific) target antigen from a complex antigen mixture containing antigens from the same protein family, by interacting with the target.

[0070] As used herein, a bispecific protein refers to a natural or synthetic protein, such as a fusion protein, that comprises at least two different binding domains.

[0071] As used herein, a binding protein refers to a polypeptide that comprises a binding domain.

[0072] As used herein, a binding domain refers to a protein domain that binds to a specific atom or molecule (generally a molecule, particularly a specific epitope (linear or conformational), often a specific amino acid sequence), and includes, for example, a ligand, receptor, antibody, or binding fragment of any one of these.

[0073] As used herein, binding fragment or antigen-binding fragment refers to the ability of the entity to specifically bind to a target antigen, and generally does not include the ability of regions such as the Fc to bind to a receptor.

[0074] As used herein, an antibody binding domain (or antibody fragment) refers to a molecule comprising a variable region having a hypervariable domain, e.g., a VH, a VHH, a VL, or a VH and a VL, comprising an epitope-binding fragment of any of the above, including sc-Fv, Fab, modified Fab, Fab', modified Fab', F(ab'), Fv, single-domain antibodies (e.g., VH or VL or VHH), scFv, bivalent, trivalent or tetravalent antibodies, Bis-scFv, diabodies, triabodies, tetrabodies, and disulfide-linked forms thereof (see, e.g., Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217).

[0075] As used herein, a ligand generally is a substance that forms a complex with a biomolecule (including those displayed on a cell surface) and serves a biological purpose, e.g., signaling, blocking, occupying, neutralizing, activating, etc. In one embodiment, the ligand is a binding domain from an antibody.

[0076] Soluble ligands involved in cell-cell interactions are essentially any cell surface molecule that can be bound by T cells or other immune cells, and that, when used as a soluble EC domain (with or without fusion to a second molecule), can signal to T cells or other immune cells. In one embodiment, anti-FAP or anti-collagen antibody fragments are fused to signaling cytokines or other proteins so that they remain in the local microenvironment where they can concentrate to better activate immune cells.

[0077] Soluble in the context of this specification generally means not membrane anchored.

[0078] As used herein, a stromal antigen is an "antigen" that is found only on stromal tissue or stromal cells, i.e., does not include an "antigen" that is also present on cancer cells. Thus, antigens expressed on cancer cells and stromal cells are considered to be cancer antigens in the context of this specification. Thus, stromal antigens may be presented on the surface of stromal cells (cells located in the stroma) and / or on soluble molecules located in the stromal matrix, particularly targetable molecules.

[0079] Examples of stromal antigens include CD163, CD206, CD68, CD11c, CD11b, CD14, CSF1 receptor, CD15, CD33 and CD66b, CD10, fibroblast activation protein (FAP), GPR77, LRRC15, TREM1, IGFBP7, FSP-1, platelet-derived growth factor-α receptor (PDGFR-α), platelet-derived growth factor-β receptor (PDGFR-β), and vimentin, such as CD163, CD206, CD68, CD11c, CD11b, CD14, CSF1 receptor, CD15, CD33 and CD66b, fibroblast activation protein (FAP), TREM1, IGFBP7, FSP-1, platelet-derived growth factor-α receptor (PDGFR-α), platelet-derived growth factor-β receptor (PDGFR-β), and vimentin.

[0080] Examples of cancer targets include PARP, CD20, CD19, CD22, CD33, CD34, CD37, CD38, CD47, CD52, CD56, CD70, CD74, CD133, CD138, CD147, CD152, CD221, CD254, CD261, CD262, CD309, CD340, BCMA, C-MYC, CAIX, claudins (such as claudins 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and / or 24), EGFR (such as EGFRvIII), VEGFR (such as VEGFR-1 and VEGFR-2), EPHA3, and folate receptor alpha [FRα]. GPC3, WT1, CEA, MUC-1, EpCAM, MAGE, mesothelin, PRAME, NYESO AFP, CA-125, ETA, tyrosinase, RAS, p53, HER receptor HER1 [EGFR], HER2, HER3, HER4, MCAM, PEM, A33, G250, carbohydrate antigen Le y , Le x , Le b , PSMA, TAG-72, STEAP1, CD166, CD24, CD44, E-cadherin, SPARC, ErbB2 and ErbB3, episialin, FOLR-1, 5T4, GPNMB, integrin αVβ3, integrin α5β1, Lewis Y antigen, MET [HGFR], mucin, PMSA, TAG-72, PDL1, mTOR, BRAF, VISTA, PI3Kγ, Bcr-AbL, ROS1, ALK, PDGF, PDGFR, RAF, p38 MAPK, Hsp90, MEK, MET, MKK1, calcineurin [or any one of its antigenic fragments] and tumor antigens, such as CD19, BCMA, CEA, claudin-6, claudin-18.2, EGFRvIII, FRα, GPC3, MCAM, mesothelin, MUC-1, EpCAM, MAGE, PRAME, AFP, CA-125, ETA, tyrosinase, RAS, p53, HER receptors HER1, HER2, HER3, HER4, PEM, A33, G250, carbohydrate antigen Le y , Le x , Le b, PSMA, TAG-72, STEAP1, CD166, CD24, CD44, E-cadherin, SPARC, ErbB2 and ErbB3, or an antigen fragment of any one of them.

[0081] In one embodiment, the cancer target is PARP, WT1, VEGF, EGFR, mTOR, BRAF, VEGFR-1, VISTA, PI3Kγ, Bcr-AbL, ROS1, ALK, PDGF, PDGFR, RAF, p38 MAPK, Hsp90, MEK, MET, MKK1, calcineurin, CD33, CD19, CD52, and tumor antigens (e.g., CEA, AFP, CA-125, ETA, tyrosinase, MAGE, PRAME, ras, p53, MUC-1, EpCAM, HER receptors HER1, HER2, HER3, HER4, PEM, A33, G250, carbohydrate antigen Le y , Le x , Le b , PSMA, TAG-72, STEAP1, CD166, CD24, CD44, E-cadherin, SPARC, ErbB2 and ErbB3).

[0082] As used herein, cancer antigens (also referred to herein as tumor antigens) include tumor-specific antigens (TSAs) and cancer-associated antigens (TAAs). Tumor-specific antigens are present only in tumor cells and not in other cells, while tumor-associated antigens are present in some tumor cells and some normal cells. Tumor antigens include: Mutated oncogene and tumor suppressor gene products, Other mutant gene products Overexpressed or aberrantly expressed cellular proteins, Tumor antigens produced by oncogenic viruses, Carcinoembryonic antigen, Altered cell surface glycolipids and glycoproteins, and  Contains cell type specific differentiation antigens.

[0083] Cancer antigens (tumor antigens) are antigens that are found specifically on cancer cells (i.e., not normally found on healthy cells or highly upregulated on cancer cells), such as CEA, MUC-1, EpCAM, HER receptors HER1, HER2, HER3, HER4, PEM, A33, G250, carbohydrate antigen Le, etc. y , Le x , Le b , PSMA, TAG-72, STEAP1, CD166, CD24, CD44, E-cadherin, SPARC, ErbB2, ErbB3, WT1, MUC1, LMP2, idiotype, HPV E6 and E7, EGFRvIII, HER-2 / neu, MAGE A3, NY-ESO-1, p53 non-mutant, p53 mutant, NY-ESO-1, GD2, PSMA, PCSA, PSA, MelanA / MART1, Ras mutant, proteinase 3 (PR1), bcr-abl, tyrosinase, survivin, PSA, hTERT, in particular WT1, MUC1, HER-2 / neu, NY-ESO-1, survivin, and hTERT. Cancer antigens as used herein include tumor-specific antigens and cancer-associated antigens. In one embodiment, the CAR binding domain is specific for a cancer antigen (including any of the specific target sequences disclosed herein).

[0084] Other cancer antigens include epithelial tumor antigen, CA-125, and alphafectoprotein.

[0085] Cancer-targeting antibodies include avelumab, bevacizumab, brentuximab, cemiplimab, cetuximab, daratumumab, dinutuximab, elotuzumab, enfortumab, gemtuzumab, ibritumomab, inotuzumab, ipilimumab, isaximab, mogamulizumab, moxetumomab, necitumumab, nivolumab, obinutuzumab, ofatumumab, olaratumumab, panitumumab, pembrolizumab, pertuzumab, polatuzumab, ramucirumab, rituximab, sacituzumab, tositumomab, trastuzumab, Fab-G8 and Fab-Hyb3 (MAGE A1 (targeting EADPTGHSY), G2D12, and G3G4 (targeting KTWGQYWQV in GP100), 1A9, 1C8, 1A11, 1A7, and G1 (targeting IMDQVPFSV in GP100), 2F1, 2B2, 2C5, and 2D1 (targeting YLEPGPVTV / A in GP100), GPA7 (targeting ITDQVPFSV), 4A9 and 4G9 (targeting ILAFLHWL in hTERT), 3H2 and 3G3 (targeting RLVDDFLLV in hTERT), 3M4E5 (targeting SLIMWITQC of NY-ESO-1), 7D4, 8A11, 2G12 and 9E6 (targeting FLWGPRALV of MAGE3), RL4B / 3.2G1 and 1B10 (targeting GVLPALPQV of hCGβ), 3F9 (targeting TMTRVLQGV of hCGβ), 1B8 (targeting KIFGSLAFL of Her2 / Neu), CAG10 and CLA12 (EAAGIGILTV of MelanA / MARR-1), Fab-D2 (targeting FLRNFSLML of TARP), and I3.These include M3-2A6 (targeting p53 LLGRNSFEV), T1-116C, T1-29D, and T1-84C (targeting p53 RMPEAAPPV), T2-108A, T2-2A, and T2-116A (targeting p53 GLAPPQHLIRV), T2A (specific for tyrosinase YMDGTMSQV), RL6A (specific for p68 YLLPAIVHI), RL21A (specific for MIF FLSELTQQL), 8FA (specific for proteinase 3 VLQELNVTV), ESK1 F2, F3, and clone 45 (specific for WT1 RMFPNAPYL), #131 (specific for HA-1H VLHDDLLEA), and Pr20 (specific for ALYVDSLFFL).

[0086] In one embodiment, the constructs encoded by the viruses according to the present disclosure, such as antibodies or binding fragments thereof, are human or humanized.

[0087] In one embodiment, an "immune checkpoint inhibitor antibody" or fragment, such as anti-PD1, PDL1, CTLA4, LAG3, GITR, TIGIT, CD40, is encoded within a virus of the disclosure.

[0088] In one embodiment, immunosuppression of the microenvironment is inhibited by inhibiting endogenous checkpoint blockade, for example, E3 ubiquitin protein ligase (such as CBL-B) and / or cytokine-inducible SH2-containing protein (such as CISH).

[0089] Thus, in one embodiment, a virus according to the present disclosure encodes a checkpoint kinase inhibitor selected from, for example, checkpoint kinase inhibitor 1 (CHEK1 / CHK1), checkpoint kinase inhibitor 2 (CHEK2 / CHK2), an ataxia telangiectasia and Rad3 related (ATR) inhibitor, an ataxia telangiectasia mutated (ATM) inhibitor, a Wee1 dual specificity protein kinase (Wee1) inhibitor, a poly ADP-ribose polymerase (PARP) inhibitor, and a Myt1 inhibitor, and combinations of two or more thereof, e.g., a checkpoint kinase inhibitor, in particular a CHK1 inhibitor.

[0090] As used herein, a GPI anchor refers to a glycolipid that can be attached to the C-terminus of a protein during post-translational modification. It consists of a phosphatidylinositol group linked to the C-terminal amino acid of a mature protein via a carbohydrate-containing linker (glucosamine and mannose glycosidically linked to an inositol residue) and an ethanolamine phosphate (EtNP) bridge. Two fatty acids within the hydrophobic phosphatidylinositol group anchor the protein to the cell membrane.

[0091] Glypiylated (GPI-linked) proteins contain a signal peptide, which directs the protein to the endoplasmic reticulum (ER). The C-terminus consists of hydrophobic amino acids that remain inserted into the ER membrane. The hydrophobic tail is then cleaved and replaced with a GPI anchor. As proteins are processed through the secretory pathway, they are transported via vesicles to the Golgi apparatus and ultimately to the extracellular space, where they remain attached to the outer leaflet of the plasma membrane. Because glypiation is the only means by which such proteins are membrane-bound, cleavage of the group by phospholipases allows the protein to be released from the membrane in a controlled manner. The latter mechanism can be used in vitro; i.e., membrane proteins released from the membrane in enzymatic assays are glypiylated proteins.

[0092] Phospholipase C (PLC) is an enzyme known to cleave the phosphoglycerol bond found in GPI-anchored proteins. Treatment with PLC releases GPI-linked proteins from the outer membrane. The T cell markers Thy-1 and acetylcholinesterase, as well as both intestinal and placental alkaline phosphatase, are known to be GPI-linked and are released by treatment with PLC. GPI-linked proteins are thought to preferentially locate in lipid rafts, suggesting a high level of organization within plasma membrane microdomains.

[0093] A review of GPI anchors written by Ferguson, Kinoshita, and Hart appears in Chapter 11 of the second edition of Essentials of Glycobiology.

[0094] In one embodiment, a combination of a transmembrane domain and a secretory signal sequence is used to express a virally encoded protein (e.g., as described herein) on the surface of infected cancer cells. The inventors have shown that the encoded protein is expressed only on cells permissive for infection by the virus, i.e., cancer cells.

[0095] In one embodiment, the fragment (such as a transmembrane fragment) used to express the protein on the surface of the infected cancer cell is selected from the group comprising the TM domain sequence (minimal portion) shown in the table below.

number

[0096] The use of non-human sequences may be advantageous as it may increase specificity for cancer, thereby widening the therapeutic window and effectively reducing off-target effects.

[0097] In one embodiment, the cellular immunotherapy is a transgenic cell, particularly a CAR-T, an engineered NK cell, and / or an engineered NKT cell, more particularly a CAR-T.

[0098] As used herein, a transgenic cell refers to an engineered cell, e.g., a cell that has been engineered using recombinant techniques to contain a non-naturally occurring polynucleotide(s) that alters the function of the cell, i.e., the cell has been modified to express a synthetic receptor on its surface.

[0099] As used herein, CAR refers to a chimeric antigen receptor, i.e., a synthetic receptor such as an antibody binding domain coupled to a signaling function, such as an intracellular signaling function. CARs are most commonly created by combining the heavy and light chain variable regions of a monoclonal antibody, but may also be created using other antibody formats (e.g., single-chain VHH antibodies from camelids) or other antigen / ligand binding proteins. Receptors bind to the antigen or ligand for which they are specific and stimulate a signaling pathway within the transgenic cell.

[0100] In CAR, signal transduction is inherent in receptor or integrated with receptor.However, alternative technologies have been developed in which signal transduction is not physically connected with synthetic receptor.All of these types are suitable for use in the virus of the present disclosure.

[0101] The examples provided below for CAR-T cells may also be applied to other immunotherapeutic cells, including NK cells and NKT cells, where technically appropriate.

[0102] First-generation CAR-T cells often had a CD3-zeta-based intracellular signaling unit. However, second-generation CARs typically incorporate costimulatory elements such as CD28, 4-1BB, CD136, CD137, or CD27 and ICOS into the intracellular signaling domain, while third-generation CARs can incorporate multiple costimulatory elements, such as CD28 and 4-1BB (see, e.g., US 7,446,190, Dotti et al. 2009 Human Gene Therapy 20:1229-1239; Finney et al. J Immuno. 1998, 161(6):2791-2797; Finney et al. 2004 J Immunol 172(1)104-113; Milone et al. Mol Ther. 2009 17(8):1453-1464).

[0103] In one embodiment, engineered cells, such as T cells, are provided that are engineered to express a recombinant (also referred to herein as synthetic) TCR specific for a tumor or other target antigen, where the TCR recognizes an MHC / antigen peptide complex. These cells can be engineered in a manner similar to that described herein for CAR-T.

[0104] Immunotherapeutic cells may be engineered by techniques such as CRISPR / Cas9 to, for example, ablate expression of inhibitory proteins (e.g., PD1) and / or ablate endogenous receptors such as endogenous TCRs (to increase target specificity).

[0105] Companies such as ProMab Biotechnologies, Inc. market these products.

[0106] Thus, in one embodiment, the CAR comprises a CD3 zeta signaling unit. First generation CARs are disclosed in Irving and Weiss, Cell 1991 March 8; 64(5):891-901. Letourmeur 1991 October 15; 88(20)8905-8909. Romeo, Cell, Vol 68, issue 5, p889-897, March 06, 1992.

[0107] In one embodiment, the CAR comprises a CD28 signaling unit, see e.g., Maher et al., Nat Biotechnol 2002 Jan;20(1);70-75 and Carpenito et al., PNAS Mar 3, 2009 106(9)3360-3365. *In one embodiment, the CAR comprises a CD27 signaling unit, the latter of which plays an essential role in the function of mature CD4+ and CD8+ T cells. In one embodiment, the CAR comprises an ICOS signaling unit, where ICOS stands for inducible T cell costimulatory factor. In one embodiment, the CAR comprises 4-1BB, see e.g., Imai 2004, Leukemia 18, 676-684. *In one embodiment, the CAR therapy comprises one costimulatory factor. In one embodiment, the CAR therapy comprises a combination of costimulatory factors, e.g., a combination of two, three, or four, such as CD28 and 4-1BB, CD28 and ICOS, CD27 and 4-1BB, or CD27 and ICOS. Guedan et al Blood 2014,124(7):1070-1080, incorporated herein by reference, discloses a chimeric antigen receptor based on ICOS. *Duong PLoS 2013 May 7;8(5) discloses the manipulation of T cell function using chimeric antigen receptors. *Signaling unit, as used herein, is an element that contributes to cell signaling of a CAR. Chimeric T cell receptors are disclosed in US2004043401. *

[0108] *The structural features of CARs disclosed herein (signaling aspects, not specificity) are incorporated by reference and may be used as the basis for amending the claims.

[0109] The binding domain of the CAR can be antibody-like and comprise, for example, an scFv, see, e.g., Kuwana et al Biochem Biophys Res Commun. 1987 Dec 31, 149(3); and Eshhar et al Proc Natl Acad Sci USA 1993 Jan 15;90(2):270-724.* See second generation CARs.

[0110] In one embodiment, the binding domain of the CAR is specific for a blood cell antigen, such as CD19, CD30, CD123, or FLT (including combinations of CD19 and CD20 or CD22, etc.), and is particularly useful for treating blood cancers such as ALL, AML, CLL, DLBCL, BCMA, leukemia, and multiple myeloma. Porter et al. N Engl J Med 2011;365:1937-1939 discloses CAR-modified T cells in CLL. Grupp et al. N Engl J Med 2013 April 18;368(16)1509-1518 discloses CAR-modified T cells for ALL. Maude et al. N Engl J Med 2014,371:1507-1517 disclosed CAR-T cells for sustained remission of leukemia. Garfall et al N Engl J Med 2015;373:1040-1047 disclosed CAR T cells against CD19 for multiple myeloma.

[0111] In one embodiment, the recombinant receptor, such as a CAR, is specific for solid tumors and / or supporting tissue.

[0112] In one embodiment, the recombinant receptor, such as a CAR, is specific for a cancer antigen, which are described above.

[0113] In one embodiment, the binding domain of a recombinant receptor, such as a CAR, targets abnormal sugars on the surface of cancer cells.

[0114] In one embodiment, the recombinant receptor, such as a CAR, is specific for a stromal antigen, for example, as defined herein.

[0115] In one embodiment, the binding domain of a recombinant receptor such as a CAR is selected from the group consisting of CD19, HER-3, HER-4, CEA, EGFR, EpCAM, EGFRvIII, PSMA, CD20, VEGFR-1, VEGFR-3, c-Met, Lewis A, ROR-1, CD326, CD133, NKG2d, MUC-1, PSCA, PSA, CA-125, Notch, FLT-3 CD20, CD22, CD33, CD34, CD37, CD38, CD47, CD52, CD56, CD70, CD74, CD133, CD138, CD147, CD152, CD221, CD254, CD261, CD262, CD309, CD340, BCMA, C-MYC, CAIX, claudins [such as claudins 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and / or 24], EGFRvIII, EPHA3, folate receptor alpha [FRα], GPC3, WT1, CEA, MUC-1, EpCAM, MAGE, mesothelin, PRAME, and NYESO The specific antigen is selected from the group including AFP, CA-125, ETA, tyrosinase, RAS, p53, HER receptors HER1 [EGFR], HER2, HER3, HER4, MCAM, PEM, A33, G250, carbohydrate antigens Ley, Lex, Leb, PSMA, TAG-72, STEAP1, CD166, CD24, CD44, E-cadherin, SPARC, ErbB2 and ErbB3, episialin, FOLR-1, 5T4, GPNMB, integrin αVβ3, integrin α5β1, Lewis Y antigen, MET [HGFR], mucin, PMSA, TAG-72, VEGFR, and PDL1.

[0116] In one embodiment, the engineered cells encode at least two entities, for example, a CD19 CAR and PD-1 siRNA, CD19 TIGIT siRNA, BCMA-CS1, or BCMA-CD33.

[0117] In one embodiment, the recombinant receptor, such as a CAR, CD19 (e.g., CD19-CD28, CD19scFv-CD28-CD3ζ, CD19scFv-4-1BB-CD3ζ, CD19scfv-CD28-4-1BB, CD19scFv-CD28-4-1BB-CD3ζ, or iCas9-T2A-antiCD19scFv-CD28-CD3ζ, CD19FLAG-CD28-CD3ζ, or iCas9 HA-T2A-antiCD19scFv-CD28-CD3ζ-GGGS-FLAG, humanized CD19 scFv-TM28-CD28-CD3ζ, CD19 scFv-Beam-TM28-CD28-CD3ζ, or humanized CD19 scFv-Beam-TM28-CD28-CD3ζ, CD19 scFv-CD22 scFv-4-1BB-CD3-T2A-tEGFR or CD19 scFv-TM28-GITR-CD3ζ or CD19 scFv-TM8-GITR-CD3ζ), mesothelin (e.g., mesothelin scFv-CD28-CD3ζ, mesothelin scFv-4-1BB-CD3ζ, mesothelin scFv-CD28-4-1BB-CD3ζ, mesothelin scFv FLAG-4-1BB-CD3ζ, mesothelin scFV-TM28-CD28-4-1BB-CD3ζ, mesothelin scFv-Beam-TM28-4-1BB-CD3ζ, mesothelin scFv-Beam-CD28-CD3ζ, mesothelin scFv-TM8-4-1BB-CD3ζ, mesothelin scFv-TM28-CD28-CD3ζ), VGFR2 (e.g., VGFR2 scFv-CD28 CD3ζ), GPC3 (e.g., GPC3 scFv-CD28-CD3ζ), CD133 (CD133 scFv-CD28-CD3ζ), EpCAM (e.g., EpCAMscFv-CD28-CD3ζ, such as a version with an Nhel restriction site introduced at the N-terminus of the scFv amino acid sequence), EGFR (e.g., EGFR scFv-CD28-CD3ζ, EGFR scFv-4-1BB-CD3ζ, EGFR scFv-TM28-GITR-CD3ζ, scFv-TM28-CD3ζ-GITR, CD33 (e.g., CD33 scFv-TM28-CD28-CD3ζ or CD33 scFv-Beam2-TM28-CD28-CD3ζ), CD38 (e.g., CD38 scFv-TM28-CD28-CD3ζ), CD138 (e.g., CD138scFv-Beam-TM28-CD28-CD3ζ), CD22 (e.g., CD22 scFv-TM28-CD28 CD3ζ, -CD22 scFv-TM28-4-1BB CD3ζ, or CD22 scFV-Beam-TM28-CD28-CD3ζ), BCMA (e.g., BCMA-4-CD28 CD3ζ or humanized BCMA-4scFv-TM8-4-1BB-CD3ζ or BCMA-2scFv-Tm-CD28-CD3ζ), HER2 (e.g., HER2 scFv-CD28-CD3ζ, HER2 scFv-4-1-BB-CD3Z-EGFRt, or HER2 scFV-4-1BB-CD3ζ-GFP), CD4 (e.g., CD4 scFv-Beam-TM28-CD28-CD3ζ), ROR-1 (e.g., ROR-1 scFv TM28-CD28-CD3ζ, ROR-1 scFv TM28-4-1BB-CD3ζ, or humanized ROR-1 scFv TM28-4-1BB-CD3ζ), CD19 and CD22 (e.g., CD19 scFv CD22 scFv-4-1BB-CD3ζ, or CD19 scFv-CD22 scFv-4-1BB-CD3-T2A-RQR8), CEA (e.g., CEA scFv-TM28-CD28 CD3ζ or humanized CEA scFv-TM28-CD28 CD3ζ), NGFR (e.g., NGFR scFv-TM28-CD28-CD3ζ), MCAM (e.g., MCAM scFv-TM28-CD28-CD3ζ), CD47 (e.g., CD47 scFv-TM28-CD28-CD3ζ or humanized CD47 scFv-TM28-CD28-CD3ζ), PDL-1 (e.g., PDL-1 scFv-TM28-CD28-CD3ζ), CD123 (e.g., CD123 scFv-TM28-CD28-CD3ζ), CD37 (e.g., CD37 scFv-TM28-CD28-CD3ζ, CD37 scFv-TM28-4-1-BB-CD3ζ, CS1 (e.g., CS1 scFv-TM28-CD28-CD3ζ), B7H4 (e.g., B7H4 scFv-TM28-CD28-CD3ζ), CD24 (e.g., CD24 scFv-TM28-CD28-CD3ζ) and CD20 (e.g., CD20 scFv-TM28-CD28-CD3ζ), NKG2D such as CYAD-01, AMG553 and other FLT3 and DLL3 specific.

[0118] In one embodiment, the recombinant receptor, such as a CAR, is specific for HER-2, for example, having the specificity of the CAR used in the examples disclosed herein.

[0119] In one embodiment, the CAR is provided in a T cell (such as an autologous T cell or an allogeneic T cell, more particularly an HLA-matched T cell). In one embodiment, the CAR-T cell is selected from tisagenlecleucel, axicabtagene ciloleucel, lysocabtagene maralecel, idecabtagene biculeucel, brexcabtagene autoleucel, JCAR015 (CD19 CAR T from Juno), Descartes-08 (BCMA), and AMG119.

[0120] In one embodiment, the immune cell is a phagocyte encoding a recombinant receptor (e.g., a CAR) enumerated herein, such as, for example, a CD19 scFv-CAR or a mesothelin scFv CAR. In one embodiment, the phagocyte is a macrophage.

[0121] In one embodiment, recombinant receptors such as CARs are provided on NKT cells. The advantage of NKT cell CARs is that they do not require HLA matching with patients. Therefore, they can be used, for example, to provide "off-the-shelf" products with the specificities listed herein. WO2013 / 040371 discloses NKTs engineered with CARs and is incorporated herein by reference. In one embodiment, the NKT cells encode cytokines.

[0122] In one embodiment, the immune cells are NK cells, see, eg, Tran et al, J Immunol 1995 Jul, 155(2);1000-1009, incorporated herein by reference.

[0123] In one embodiment, the engineered cells are derived from pluripotent stem cells, such as iPSCs.

[0124] Thus, in one embodiment, the immune cell therapy further comprises a transgene (i.e., an engineered gene) encoding a cytokine selected from, for example, IL-2, IL-5, IL-7, IL-12, and IL-15.

[0125] In one embodiment, the engineered cells do not contain a transgene encoding a cytokine.

[0126] In one embodiment, the engineered cells contain only a transgene expressing a recombinant receptor (such as a CAR), although this does not prevent deleting a particular wild-type gene if this is beneficial (e.g., to improve specificity).

[0127] In one embodiment, the immune cell therapy does not include a transgene (i.e., an engineered gene) encoding a cytokine selected from, for example, IL-2, IL-5, IL-7, IL-12, and IL-15.

[0128] Immune cells such as T cells and NKT cells can be activated or maintained active by IL-15 expressed by the virus of the present invention. This may help counteract the anergic / hypoxic microenvironment of tumors. A hypoxic microenvironment may have the ability to neutralize the killing power of natural cells and even engineered cell therapies used in combination with the present invention. Therefore, the use of the virus of the present disclosure may trigger several mechanisms for killing cancer, especially when used in combination with cell therapy.

[0129] As used herein, a therapeutic dose refers to an amount of a virus, such as an oncolytic adenovirus, that, when used in an appropriate treatment regimen, is adequate to achieve the intended therapeutic effect, e.g., ameliorate disease symptoms or conditions, particularly without dose-limiting toxicity. A dose may be considered a therapeutic dose in the treatment of cancer or metastasis if the number of viral particles is sufficient to slow or stop tumor or metastasis growth, or to reduce the size of tumors or metastases, and / or result in an increased patient lifespan. Infection of cancer cells after systemic delivery of a virus of the present disclosure is indicative of a therapeutic dose, i.e., indicates that a therapeutic dose has been delivered to the target cells. An appropriate therapeutic dose generally balances therapeutic efficacy with acceptable toxicity, e.g., when side effects and toxicity are acceptable given the benefits achieved by the treatment.

[0130] In one embodiment, a virus or therapeutic construct according to the present disclosure (including a formulation comprising same) is administered every 3-4 weeks, e.g., in week 1, doses are administered on days 1, 3, and 5, followed, e.g., by multiple additional doses 3-4 weeks later.

[0131] In one embodiment, a virus or therapeutic construct according to the present disclosure (including a formulation comprising same) is administered every two weeks or every three weeks, for example, on days 1, 3, and 5 in week 1, and again on days 1, 3, and 5 in week 3 or 4. This dosing regimen can be repeated as often as necessary.

[0132] In one embodiment, the initial dose is lower than the subsequent doses, e.g., the initial dose is 1 x 10 11 ~1×10 12 and subsequent doses are in the range of 1 x 10 viral particles. 12 ~1×10 13 The range is 100 virus particles.

[0133] In one embodiment, six doses are given over a two week period, e.g., on days 1, 3, 5, 8, 10 and 12, and each dose may be given ±1 day, including, for example, where the dose on day 1 is lower than the other doses.

[0134] In one embodiment, a virus or therapeutic construct according to the present disclosure (including a formulation comprising same) is administered monthly.

[0135] In one embodiment, the first dose of virus is administered prior to treatment with immune cell therapy, for example, 7 to 28 days prior to cell therapy.

[0136] In one embodiment, the viruses and constructs of the present disclosure are prepared by recombinant techniques. Those skilled in the art will understand that the armed adenoviral genome can be produced by other technical means, including completely synthesizing the genome, or a plasmid containing a portion of the entire genome. Those skilled in the art will understand that if the genome is synthesized, the inserted region may not contain restriction site nucleotides, as these are artifacts of gene insertion using cloning methods.

[0137] The disclosure herein further extends to adenoviruses of formula (I) or subformulas thereof which are obtained or obtainable by inserting a transgene or transgene cassette.

[0138] As used herein, "is" means to include.

[0139] In the context of this specification, "comprising" should be interpreted as "including."

[0140] Embodiments of the invention comprising particular features / elements are also intended to extend to alternative embodiments that "consist of" or "consist essentially of" the associated elements / features.

[0141] Where technically appropriate, embodiments of the invention may be combined.

[0142] Technical references, such as patents and applications, are incorporated herein by reference.

[0143] Any embodiment specifically and explicitly described herein may form the basis of a disclaimer, either alone or in combination with one or more further embodiments.

[0144] The background section contains technically relevant details that can be used as a basis for modifications. [Brief explanation of the drawings]

[0145] [Figure 1] FIG. 1 is a schematic diagram of a series of transgene cassettes encoding bispecific proteins and membrane-anchored antigens, with or without additional transgenes. [Figure 2] Figure 1 shows the design of bispecific proteins and how they enable tumor cell recognition by cell therapy (e.g., CAR-T cells) and enhanced cell therapy activity by delivery agents. [Figure 3]Screening of NG-1100-1126 designs for their ability to bind to A549 tumor cells and present cell therapy ligands in vitro. A. Expression of transmembrane CD19 on A549 cells after transfection with the NG-1108 pUC57 plasmid. B. Supernatants from A549 cells transfected with the NG-1100, NG-1101, NG-1102, NG-1106, and NG-1107 pUC57 plasmids or a control pUC57 vector were incubated with fresh A549 cells, and binding of the bispecific proteins was measured by flow cytometry to detect the C-terminal 2A peptide tag. C-E. Supernatants from A549 cells transfected with the NG-1109 through NG-1118 pUC57 plasmids or a control plasmid were incubated with fresh A549 cells, and binding of the bispecific proteins to EpCAM was measured by flow cytometry as a reduction in binding of AF647-labeled anti-EpCAM antibody. F. Supernatants from A549 cells transfected with pUC57 plasmids NG-1106, NG-1107, NG-1109, NG-1110, NG-1113 to NG-1121, or NG-1123 to NG-1126 or control plasmids were incubated with fresh A549 cells, and binding of the bispecific proteins was measured by detecting CD19 by flow cytometry. [Figure 4] A and B. Binding of anti-HER2ScFv-CD19 fusion protein to A549 and SKOV3 tumor cells after incubation with infected supernatant. A549 tumor cells were infected with EnAd or NG-1124, and the infected supernatant was collected and incubated with uninfected A549 (A) or SKOV3 (B) cells, after which CD19 expression was analyzed by flow cytometry. C. Expression of transmembrane CD19 after infection with NG-1108. A549 tumor cells were infected with EnAd or NG-1108 virus, and the culture supernatant was added to uninfected A549 cells. CD19 expression in both cell sets was then analyzed by flow cytometry. [Figure 5]Binding of virally encoded bispecific proteins secreted by infected A549 tumor cells to uninfected SKOV3 cells in a coculture system. A and B. A549 cell cultures, SKOV3 cell cultures, and A549 + SKOV3 cell cocultures were infected with EnAd or NG-1100, and binding of anti-EpCAM-RAE1TG3 fusion proteins to the SKOV3 (A) or A549 (B) cell surface was assessed by flow cytometric detection of the C-terminal 2A peptide tag peptide. C and D. A549 cell cultures, SKOV3 cell cultures, and A549 + SKOV3 cell cocultures were infected with EnAd or NG-1124, and binding of anti-HER2-CD19 fusion proteins to the SKOV3 (C) or A549 (D) cell surface was assessed by flow cytometric detection of the CD19 antigen. [Figure 6] Anti-CD19 CAR-T cell-mediated cytotoxicity against SKOV3 tumor cells in the presence of cell culture supernatant (SN) from A549 cells infected with NG-1124. SKOV3 cells were incubated with cell culture supernatant from A549 cells infected with EnAd (A) or NG-1124 (B), followed by addition of anti-CD19 CAR or control (Ctrl) T cells, and tumor cell death was monitored in real time. [Figure 7] Anti-CD19 CAR-T-mediated cytotoxicity against A549 tumor cells in the presence of cell culture supernatant from NG-1124-infected cells. A549 cells were infected with either NG-1124 or EnAd, and viral particles were removed from the culture supernatant by exclusion column filtration prior to testing. A. Levels of anti-HER2-CD19 bispecific protein were shown to be similar before and after viral particle removal. B-D. A549 cells were incubated in the presence of these virally cleared culture supernatants (VR SN), followed by addition of anti-CD19 CAR or control (Ctrl) T cells, and tumor cell death was monitored in real time. [Figure 8]A. Effect of encoding multiple transgenes on the level of bispecific protein expression and binding to tumor cells. B. Expression of CXCL9, CCL21, IFNα, and IL-15 transgene proteins by A549 tumor cells infected with NG-1101, NG-1104, and NG-1125. [Figure 9] In vivo expression and tumor cell binding of the anti-HER2-CD19 fusion protein encoded by NG-1125. A. Flow cytometry gating after intracellular staining of the virus separates cells into negative (uninfected), low (low VP), or high (high VP) levels of infection. B. Frequency of tumor cells that remain uninfected or have low or high VP after IV administration of either EnAd or NG-1125. C. Levels of cell surface-bound anti-HER2-CD19 on uninfected low VP or high VP subsets of A549 tumor cells in the tumor mass after IV administration of EnAd or NG-1125. [Figure 10] In vivo demonstration of enhanced CAR-T cell activity in A549 xenograft tumors after IV administration of NG-1125, which expresses the anti-HER2-CD19 bispecific protein and two additional transgenes, compared to that observed with NG-1124 or EnAd, which express only the anti-HER2-CD19 protein. Resected tumors were processed into single-cell suspensions and analyzed by flow cytometry for the presence of total human CD45+ cells (A), activated cytotoxic human CD8+CD107a+ cells (B), or activated cytotoxic human CD4+CD107a+ cells (C). [Figure 11] The general structure of a chimeric antigen receptor is shown. [Figure 12]In vivo demonstration of enhanced CAR-T cell recruitment and activation in A549 xenograft tumors after intravenous administration of NG-1125 (anti-HER2-CD19 bispecific protein expressing CXCL9 and IFNα), NG-641 (expressing CXCL9, CXCL10, IFNα, and an unrelated bispecific protein), or EnAd. Resected tumors were processed into single-cell suspensions and analyzed by flow cytometry for the presence of total human CD45+ cells (corresponding to engrafted T cells) (A), activated cytotoxic human CD45+CD107a+ cells (B), or activated human CD45+CD25+ cells (C). Blood was also processed to assess the presence of circulating human CD45+ cells (D).

[0146] Sequence Listing SEQ ID NO: 1 Short splice acceptor (SSA) sequence (CAGG) SEQ ID NO: 2 Splice acceptor sequence SEQ ID NO: 3 Splice acceptor sequence SEQ ID NO: 4 Polyadenylation (PA) sequence (SV40 late polyA sequence) SEQ ID NO: 5 Highly efficient self-cleavable T2A peptide sequence SEQ ID NO: 6 Highly efficient self-cleavable E2A peptide sequence SEQ ID NO: 7 Highly efficient self-cleavable F2A peptide sequence SEQ ID NO: 8 Highly efficient self-cleavable P2A peptide sequence SEQ ID NO: 9 Flexible Linker (FL) SEQ ID NO: 10 Rigid Linker (RL) SEQ ID NO: 11 Myc peptide tag SEQ ID NO: 12 His tag SEQ ID NO: 13 Human CXCL9 SEQ ID NO: 14 Human CXCL9-T2A SEQ ID NO: 15 Human CXCL9-P2A SEQ ID NO: 16 Human CXCL9-E2A SEQ ID NO: 17 Human CCL21 with N-terminal signal peptide SEQ ID NO: 18 Human CCL21 with N-terminal signal peptide and C-terminal E2A peptide SEQ ID NO: 19 Human IL-15Ra sushi domain with signal sequence SEQ ID NO: 20 Human IL-15Ra sushi domain with signal sequence and C-terminal P2A peptide SEQ ID NO: 21 Human IL-15 with immunoglobulin leader sequence SEQ ID NO: 22 (Gly4Ser)3 linker (G4S) SEQ ID NO: 23 Short Gly4Ser liner (sG4S) SEQ ID NO: 24 Long (Gly4Ser)4 linker (G4S4) SEQ ID NO: 25 Human CD19 (extracellular domain) SEQ ID NO: 26 Human CD19m1 (extracellular domain, mutated) SEQ ID NO: 27 Human CD19m2 (extracellular domain, mutated) SEQ ID NO: 28 Protein sequence of anti-EpCAM-ScFv-OKT3-ScFv fusion protein (without C-terminal tag) including leader sequence, as encoded by NG-611 SEQ ID NO: 29 Protein sequence of anti-EpCAM-ScFv-RAET1G3 fusion protein with F2A C-terminal tag, including leader sequence, encoded within NG-1100, NG-1101 SEQ ID NO: 30 Protein sequence of RAET1G3-anti-EpCAM-ScFv fusion protein with F2A C-terminal tag, including leader sequence, encoded within NG-1102 SEQ ID NO: 31 Protein sequence of anti-EpCAM-ScFv-OKT3-ScFv fusion protein with F2A C-terminal tag, including leader sequence, encoded by NG-1104 SEQ ID NO: 32: Protein sequence of anti-EpCAM-ScFv-human CD19 EC domain fusion protein with P2A C-terminal tag, including leader sequence, encoded by NG-1106, NG-1119 SEQ ID NO: 33 Protein sequence of human CD19 EC domain-anti-EpCAM-ScFv fusion protein with P2A C-terminal tag, including leader sequence, encoded by NG-1107 SEQ ID NO: 34 Protein sequence of transmembrane human CD19 with a short cytoplasmic tail and a P2A C-terminal tag, including the leader sequence, encoded by NG-1108 SEQ ID NO: 35 Protein sequence of anti-EpCAM-ScFv-human CD19 EC domain fusion protein (without C-terminal tag) including leader sequence, as encoded by NG-1109 SEQ ID NO: 36: Protein sequence of human CD19 EC domain-anti-EpCAM-ScFv fusion protein (without C-terminal tag) including leader sequence, as encoded by NG-1110 SEQ ID NO: 37 Protein sequence of anti-EpCAM-ScFv-RAET1G3 fusion protein (without C-terminal tag), including leader sequence, encoded within NG-1111 SEQ ID NO: 38 Protein sequence of RAET1G3-anti-EpCAM-ScFv fusion protein (without C-terminal tag), including leader sequence, encoded in NG-1112 SEQ ID NO: 39: Protein sequence of anti-EpCAM-ScFv-flexible linker-human CD19 EC domain fusion protein (without C-terminal tag), including leader sequence, encoded by NG-1113 and NG-1115 SEQ ID NO: 40 Protein sequence of anti-EpCAM-ScFv-rigid linker-human CD19 EC domain fusion protein (without C-terminal tag) including leader sequence, encoded by NG-1114, NG-1116 SEQ ID NO: 41 Protein sequence of anti-EpCAM-ScFv-flexible linker-RAET1G3 fusion protein (without C-terminal tag), including leader sequence, encoded within NG-1117 SEQ ID NO: 42 Protein sequence of anti-EpCAM-ScFv-rigid linker-RAET1G3 fusion protein (without C-terminal tag), including leader sequence, encoded within NG-1118 SEQ ID NO: 43 Protein sequence of anti-EpCAM-ScFv-human CD19m1 EC domain fusion protein with P2A C-terminal tag, including leader sequence, encoded by NG-1120 SEQ ID NO: 44 Protein sequence of human CD19 EC domain-anti-HER2-ScFv fusion protein with a C-terminal P2A tag, including the leader sequence, encoded by NG-1121 SEQ ID NO: 45 Protein sequence of human CD19 EC domain-anti-HER2-ScFv fusion protein with a C-terminal Myc tag, including the leader sequence, encoded by NG-1122 SEQ ID NO: 46 Protein sequence of human CD19m2 EC domain-anti-HER2-ScFv fusion protein with a C-terminal P2A tag, including the leader sequence, encoded by NG-1123 SEQ ID NO: 47 Protein sequence of anti-HER2-ScFv-CD19m1 fusion protein with C-terminal P2A tag, including leader sequence, encoded by NG-1124, NG-1125 SEQ ID NO: 48 Protein sequence of anti-HER2-ScFv-CD19 fusion protein with C-terminal P2A tag, including leader sequence, encoded by NG-1126 SEQ ID NO: 49 Protein sequence encoded by the NG-611 transgene cassette SEQ ID NO: 50 Protein sequence encoded by the NG-1100 transgene cassette SEQ ID NO: 51 Protein sequence encoded by the NG-1101 transgene cassette SEQ ID NO: 52 Protein sequence encoded by the NG-1102 transgene cassette SEQ ID NO: 53 Protein sequence encoded by the NG-1104 transgene cassette SEQ ID NO: 54 Protein sequence encoded by the NG-1106 transgene cassette SEQ ID NO: 55 Protein sequence encoded by the NG-1107 transgene cassette SEQ ID NO: 56 Protein sequence encoded by the NG-1108 transgene cassette SEQ ID NO: 57 Protein sequence encoded by the NG-1109 transgene cassette SEQ ID NO: 58 Protein sequence encoded by the NG-1110 transgene cassette SEQ ID NO: 59 Protein sequence encoded by the NG-1111 transgene cassette SEQ ID NO: 60 Protein sequence encoded by the NG-1112 transgene cassette SEQ ID NO: 61 Protein sequence encoded by the NG-1113 transgene cassette SEQ ID NO: 62 Protein sequence encoded by the NG-1114 transgene cassette SEQ ID NO: 63 Protein sequence encoded by the NG-1115 transgene cassette SEQ ID NO: 64 Protein sequence encoded by the NG-1116 transgene cassette SEQ ID NO: 65 Protein sequence encoded by the NG-1117 transgene cassette SEQ ID NO: 66 Protein sequence encoded by the NG-1118 transgene cassette SEQ ID NO: 67 Protein sequence encoded by the NG-1119 transgene cassette SEQ ID NO: 68 Protein sequence encoded by the NG-1120 transgene cassette SEQ ID NO: 69 Protein sequence encoded by the NG-1121 transgene cassette SEQ ID NO: 70 Protein sequence encoded by the NG-1122 transgene cassette SEQ ID NO: 71 Protein sequence encoded by the NG-1123 transgene cassette SEQ ID NO: 72 Protein sequence encoded by the NG-1124 transgene cassette SEQ ID NO: 73 Protein sequence encoded by the NG-1125 transgene cassette SEQ ID NO: 74 Protein sequence encoded by the NG-1126 transgene cassette SEQ ID NO: 75 Genome sequence of NG-1100 virus SEQ ID NO: 76 Genome sequence of NG-1101 virus SEQ ID NO: 77 Genome sequence of NG-1102 virus SEQ ID NO: 78 Genome sequence of NG-1104 virus SEQ ID NO: 79 Genome sequence of NG-1106 virus SEQ ID NO: 80 Genome sequence of NG-1107 virus SEQ ID NO: 81 Genome sequence of NG-1108 virus SEQ ID NO: 82 DNA sequence of pUC57 CMV expression plasmid containing the transgene cassette insert of NG-1109 SEQ ID NO: 83 DNA sequence of pUC57 CMV expression plasmid containing the transgene cassette insert of NG-1110 SEQ ID NO: 84 DNA sequence of pUC57 CMV expression plasmid containing the transgene cassette insert of NG-1111 SEQ ID NO: 85 DNA sequence of pUC57 CMV expression plasmid containing the transgene cassette insert of NG-1112 SEQ ID NO: 86 DNA sequence of pUC57 CMV expression plasmid containing the transgene cassette insert of NG-1113 SEQ ID NO: 87 DNA sequence of pUC57 CMV expression plasmid containing the transgene cassette insert of NG-1114 SEQ ID NO: 88 DNA sequence of pUC57 CMV expression plasmid containing the transgene cassette insert of NG-1115 SEQ ID NO: 89 DNA sequence of pUC57 CMV expression plasmid containing the transgene cassette insert of NG-1116 SEQ ID NO: 90 DNA sequence of pUC57 CMV expression plasmid containing the transgene cassette insert of NG-1117 SEQ ID NO: 91 DNA sequence of pUC57 CMV expression plasmid containing the transgene cassette insert of NG-1118 SEQ ID NO: 92 DNA sequence of pUC57 CMV expression plasmid containing the transgene cassette insert of NG-1119 SEQ ID NO: 93 DNA sequence of pUC57 CMV expression plasmid containing the transgene cassette insert of NG-1120 SEQ ID NO: 94 DNA sequence of pUC57 CMV expression plasmid containing the transgene cassette insert of NG-1121 SEQ ID NO: 95 DNA sequence of pUC57 CMV expression plasmid containing the transgene cassette insert of NG-1122 SEQ ID NO: 96 DNA sequence of pUC57 CMV expression plasmid containing the transgene cassette insert of NG-1123 SEQ ID NO: 97 Genome sequence of NG-1124 virus SEQ ID NO: 98 Genome sequence of NG-1125 virus SEQ ID NO: 99 DNA sequence of pUC57 CMV expression plasmid containing the transgene cassette insert of NG-1126 SEQ ID NO: 100 Transmembrane protein sequence from human PDGFR receptor A SEQ ID NO: 101 Transmembrane protein sequence from human PDGFR receptor B SEQ ID NO: 102 Transmembrane protein sequence from human insulin-like growth factor 1 SEQ ID NO: 103 Transmembrane protein sequence from human IL-6R SEQ ID NO: 104 Transmembrane protein sequence from human CD28 [Example]

[0147] Example 1: Production of expression plasmids and viruses encoding bispecific proteins comprising tumor cell-binding antibodies fused to CAR / TCR or other cell therapy targeting ligands A series of transgene cassette designs were initially synthesized as pUC57 plasmids in which transgene protein expression was under the control of a CMV promoter, allowing initial studies of different bispecific protein and transgene combinations to be evaluated through transient transfection experiments. In each transgene cassette, the 5' end of the cDNA encoding the protein sequence was flanked by a short splice acceptor sequence (SSA, SEQ ID NO: 1). The 3' end of the protein sequence encoded an SV40 late poly(A) sequence (PA, SEQ ID NO: 4). In viruses with multiple encoded transgenes, each sequence was linked to a 2A ribosomal skipping sequence (T2A, E2A, F2A, or P2A) (SEQ ID NOs: 5-8), allowing each individual protein to be translated and produced as a separate chain. In some designs, a 2A sequence was also added to the final transgene within the cassette to serve as an epitope tag for analytical purposes. A schematic diagram of the different transgene cassettes is shown in Figure 1 and listed in Table 1. [Table 1] TIFF2025526409000003.tif63170 1 SEQ ID NO:1; 2 SEQ ID NO:28; 3 SEQ ID NO: 12; 4 SEQ ID NO:4; 5 SEQ ID NO:29; 6 SEQ ID NO:7; 7 SEQ ID NO: 13; 8 SEQ ID NO:5; 9 SEQ ID NO: 17; 10 SEQ ID NO:6; 11 SEQ ID NO: 19; 12 SEQ ID NO:8; 13 SEQ ID NO:21; 14 SEQ ID NO:30; 15 SEQ ID NO:32; 16 SEQ ID NO:33; 17 SEQ ID NO:34; 18 SEQ ID NO:39; 19 SEQ ID NO:40; 20 SEQ ID NO:41; 21 SEQ ID NO:42; 22SEQ ID NO:43; 23 SEQ ID NO:44; 24 SEQ ID NO:46; 25 SEQ ID NO:47; 26 SEQ ID NO:48;

[0148] Virus production Different viral vectors were generated by directly inserting transgene cassette sequences obtained from the pU57 plasmid by restriction enzyme digestion using the pColoAd2.4 plasmid. The pColoAd2.4 plasmid was digested with AsiSI and SbfI restriction enzymes, and each excised transgene cassette was directly ligated into the digested pColoAd2.4 plasmid. The construction of the plasmid DNA of each viral vector was confirmed by restriction analysis and Sanger sequencing.

[0149] To generate the virus, the plasmid was linearized by restriction digestion with the enzyme AscI to generate the viral genome. Virus amplification and purification were performed according to the following method.

[0150] The digested DNA was purified by phenol / chloroform extraction and precipitated in 600 μl of >95% molecular biology-grade ethanol and 15 μl of 3M sodium acetate at -20°C for 16 ± 2 hours. The precipitated DNA was pelleted by centrifugation at 13,000 rpm for 5 minutes and washed twice with 500 μl of 70% ethanol. The clean DNA pellet was air-dried, resuspended in 500 μl of OptiMEM containing 15 μl of Lipofectamine transfection reagent, and incubated at room temperature for 30 minutes. The transfection mixture was then added dropwise to a T-25 flask containing HEK-293 cells grown to 70% confluency. After incubating the cells with the transfection mix at 37°C and 5% CO2 for approximately 2 hours, 4 ml of cell culture medium (DMEM high glucose with glutamine supplemented with 2% FBS) was added to the cells, and the flask was incubated at 37°C and 5% CO2.

[0151] Transfected HEK-293 cells were monitored every 24 hours, and additional medium was replenished as needed. Virus production was monitored by observing significant cytopathic effect (CPE) in the cell monolayer. Once extensive CPE was observed, the virus was harvested from the HEK-293 cells by three freeze-thaw cycles. The harvested virus was used to reinfect HEK-293 cells and amplify the viral stock. Viable virus production during amplification was confirmed by observing significant CPE in the cell monolayer. Once CPE was observed, the virus was harvested from the HEK-293 cells by three freeze-thaw cycles. The amplified viral stock was used for further amplification, after which the virus was purified by density gradient centrifugation to produce purified viral stocks.

[0152] Example 2: Screening of bispecific protein designs for their ability to bind to tumor cells in vitro. To verify the correct folding of the bispecific proteins prior to generating viral vectors encoding them, transgene cassettes NG-1100 to NG-1126 were constructed as pUC57 plasmid DNA vectors. These plasmids were transfected into A549 human lung tumor cells, and the cells were cultured for 72 hours. The cell culture supernatant (SN) containing the secreted bispecific proteins was collected and added to fresh, untransfected A549 cells in suspension, followed by incubation at room temperature for 1.5 hours. During this time, the bispecific proteins present in the SN were able to bind to A549 cells via the anti-EpCAM ScFv or anti-HER2 ScFv moiety. Binding to A549 cells was detected by flow cytometry via a) a decrease in the mean fluorescence intensity (MFI) of EpCAM staining (due to competitive binding between the anti-EpCAM ScFv and the anti-EpCAM antibody clone 9C4) and b) detection of the 2A peptide tag using an anti-P2A antibody, for designs containing the 2A peptide tag. For designs with CD19 as the CAR-T target, the CD19 extracellular domain portion of the protein was detected by flow cytometry using an anti-CD19 antibody. For all of these readouts, background levels of signal intensity were established using SN from cells transfected with a control plasmid encoding an unrelated protein.

[0153] First, we tested a design encoding the transmembrane CD19 targeting protein NG-1108. To verify the expression of transmembrane CD19, A549 cells were transfected with the pUC57 plasmid encoding the NG-1108 transgene cassette (SEQ ID NO: 56) or a negative control plasmid, and the transfected cells were stained with an anti-CD19 antibody by flow cytometry. Compared to the control, high levels of transmembrane CD19 expression were detected in A549 cells transfected with pUC-1108 (Figure 3A).

[0154] Data from a series of experiments using different plasmid sets (Figure 3B–F) indicate that different secreted bispecific protein designs can bind to tumor cell surfaces. The data showed that bispecific protein designs using anti-EpCAM ScFv as the tumor cell-binding component only functioned when the ScFv antibody moiety was placed at the N-terminus of the antigen (RAET1G3 or CD19), but not at the C-terminus, whereas anti-Her2 ScFv antibody could function in either the N- or C-terminal position.

[0155] Example 3: Spread of virally encoded bispecific proteins from infected to uninfected tumor cells. We hypothesized that if a secreted bispecific protein were encoded by a viral vector, the secreted protein would bind to both infected and nearby uninfected tumor cells, thereby enabling the target antigen to spread to the microenvironment. To demonstrate this in vitro, two different cell culture systems were used. In the first model, A549 cells were infected with EnAd or NG-1124 (SEQ ID NO: 97) viral vectors at 0.1 or 1 ppc for 7 days or at 10 or 50 ppc for 3 days. The infected supernatants were collected and incubated with uninfected A549 or SKOV3 cells for 1.5 hours, after which CD19 expression was analyzed by flow cytometry. The presence of the anti-HER2 ScFc-CD19m1 bispecific protein (SEQ ID NO: 47) in the SN was demonstrated by the presence of positive CD19 staining in both SKOV3 and A549 cells in all conditions tested (Figures 4A and B). In contrast, if CD19 is encoded intracellularly as a transmembrane protein rather than as part of a bispecific soluble molecule (as in NG-1108), CD19 expression would be expected to be detected only in infected cells and not transmitted to uninfected cells. Consistently, we found that CD19 was expressed on the membrane of A549 cells infected with NG-1108 (SEQ ID NO: 81) 3 days after infection with 1 ppc. However, when infection supernatants from these cells were transferred and incubated with uninfected A549 cells, CD19 was not detected in the latter (Figure 4C).

[0156] In the second model, a cell type that readily allows viral replication and transgene expression over a short culture period, specifically A549 lung tumor cells, and a cell line that is less permissive for viral replication and transgene expression over this period, specifically SKOV3 ovarian cancer cells, were cultured and infected separately or in cocultures containing both cell types. Both cell lines were shown to express EpCAM and HER2 cell surface target antigens prior to use in the assay. SKOV3 cells were labeled with Cell Trace Violet dye so that they could be distinguished from A549 cells using flow cytometry. Cells were infected with 10 ppc of either EnAd, NG-1100 (SEQ ID NO: 75), or NG-1124 (SEQ ID NO: 97) viral vectors. Three days later, cells were harvested and analyzed by flow cytometry to quantify bispecific protein binding to each cell type based on P2A tag or CD19 detection. After infection with NG-1100, SKOV3 cells cultured alone did not exhibit P2A signals above background (EnAd infection) because the virus was unable to produce sufficient transgene protein in this cell type at the time of assay (Figure 5A). However, when cultured with A549, approximately 13% of SKOV3 cells stained positive for P2A, indicating the migration and binding of the bispecific protein produced by the A549 cells present in the culture (Figure 5A). As a control, A549 cells, due to their high viral replication capacity, were able to produce the bispecific protein and exhibit positive P2A staining even when cultured without SKOV3 (Figure 5B). In a similar experiment, SKOV3 cells were shown not to produce or secrete any bispecific protein after infection with NG-1124 (SEQ ID NO: 97). However, when co-cultured with A549 cells, SKOV3 cells were able to bind to the bispecific construct (SEQ ID NO: 47) secreted by A549 cells, resulting in a CD19 positivity rate of approximately 100% (Figure 5C). As a control, A549 cells were able to produce and bind the bispecific protein and display a positive CD19 signal both when cultured alone and when co-cultured with SKOV3 cells (Figure 5D).

[0157] Example 4: Anti-CD19 CAR-T mediated cytotoxicity against SKOV3 and A549 tumor cells in the presence of cell culture supernatant from NG-1124 infected cells. The ability of the anti-HER2 ScFv-CD19m1 fusion protein (SEQ ID NO: 47) encoded by NG-1124 (SEQ ID NO: 97) to bind to anti-CD19 CAR-T cells and induce cytotoxic activity against HER2-expressing tumor cells was assessed by real-time cytotoxicity assay (RTCA) using SKOV3 or A549 cell lines as target cells. For assays using SKOV3 cell lines, cells were incubated for 3 days in the presence of cell culture supernatant (diluted 1:10 in culture medium) from A549 cells infected with NG-1124 or EnAd at 10 ppc. After 1.5 hours of incubation, anti-CD19 or control (Ctrl) T cells were added to SKOV3 tumor cells at a T cell:tumor cell ratio of 3:1, and tumor cell death was monitored by RTCA using an xCELLigence instrument. Complete tumor cell death was determined using a control condition with medium containing 4% Tween 20 (100% lysis control). Addition of anti-CD19 CAR-T cells to SKOV3 target cells in the presence of NG-1124-infected SN resulted in complete target cell death within 24 hours of CAR-T addition, but not EnAd-infected SN (Fig. 6A and B). As a control, Ctrl T cells nonspecifically preactivated with anti-CD3 / anti-CD28 antibodies showed no detectable cytotoxicity in the presence of NG-1124-infected SN (Fig. 6A and B).

[0158] Because A549 tumor cells are highly susceptible to adenovirus replication and adenovirus-induced oncolysis, to assess CAR-T-specific cytotoxicity in these cells without perturbing virus-associated cell death, we generated cell culture supernatants with reduced virus content (VR SNs) and tested them in the CAR-T cell A549 RTCA assay. To generate VR SNs, A549 cells were infected with NG-1124 or EnAd at 0.1 ppc for 7 days. The cell culture SNs were collected and filtered through a 300 kDa size-exclusion column to separate viral particles from the remaining SNs containing the bispecific protein. SNs before and after virus removal were incubated with A549 cells and assessed by detecting CD19 expression. The filtration process had minimal impact on anti-HER2-CD19 protein content in the SNs (Figure 7A). To evaluate the ability of secreted anti-HER2-CD19 bispecific proteins to bind to anti-CD19 CAR-T cells against tumor cells, A549 cells were incubated with VR SN for 1.5 hours, followed by the addition of CAR-T cells or Ctrl T cells at a T cell:tumor cell ratio of 3:1. Tumor cell cytotoxicity was monitored via RTCA. Complete tumor cell death was defined as 100% lysis in the presence of 4% Tween 20. Complete A549 cell death was observed within 24 hours after CAR-T addition when cells were incubated in the presence of NG-1124 VR SN containing anti-Her2 ScFv-CD19 protein, but not in the presence of EnAd VR SN or normal cell culture medium (Figure 7B-D). Control activated T cells (generated as described in Figure 5) showed low levels of nonspecific killing activity, regardless of the presence or type of viral SN added.

[0159] These RTCA-based assays demonstrated that the anti-Her2 ScFv-CD19m1 bispecific construct (SEQ ID NO: 47) encoded by the NG-1124 adenovirus (SEQ ID NO: 97) is functional and can redirect the cytotoxic activity of anti-CD19 CAR-T cells against HER2+ tumor cells that do not endogenously express CD19.

[0160] Example 5: Effect of encoding multiple transgenes on bispecific protein activity. To maximize the ability of adenoviruses to synergistically enhance the efficacy of cell therapies, a series of viruses were generated that encode bispecific proteins that target CAR T cells and additional transgenes (e.g., chemokines, cytokines, and other immunomodulators) that can provide additional signals to promote the activity of cell therapies, such as CAR- or TCR T-cell therapy. To verify that these more complex designs can also effectively express bispecific proteins, A549 cells were infected with NG viruses encoding various transgenes or 0.1 ppc of EnAd for 7 days. SNs from cell cultures infected with NG-1100 (SEQ ID NO: 75), NG-1101 (SEQ ID NO: 76), NG-1124 (SEQ ID NO: 97), NG-1125 (SEQ ID NO: 98), NG-611 (SEQ ID NO: 77 in patent application WO2019 / 043020), NG-1104 (SEQ ID NO: 78), or EnAd were collected and incubated with uninfected A549 cells for 1.5 hours. The presence of bispecific protein binding to the cells was determined by flow cytometry detection of either the P2A tag, CD19 antigen, or OKT3 ScFv, depending on the virus design. For each staining, either an anti-2A peptide antibody, anti-CD19 antibody, or anti-OKT3 ScFv antibody was used. Fluorescence signals were considered positive if they exceeded the background fluorescence measured in cells incubated with EnAd-infected SNs. Surprisingly, the inventors found that adding two or more transgenes encoding bispecific proteins into the same transgene cassette clearly increased the expression of the bispecific proteins compared to the corresponding viral design encoding only the bispecific proteins.Specifically, I) approximately 45% of A549 cells incubated with SNs from NG-1101 (SEQ ID NO: 76) infection were positive for the 2A tag peptide (indicating binding of the anti-EpCAM ScFv-RAET1G3 protein), compared with approximately 15% of cells incubated with NG-1100 SNs; II) approximately 12% of A549 cells incubated with SNs from NG-1104 (SEQ ID NO: 78) infection showed a positive signal for anti-OKT3 ScFv (indicating binding of the anti-EpCAM-OKT3 ScFv protein), compared with approximately 7.5% of cells incubated with NG-611 SNs; and III) approximately 55% of A549 cells incubated with SNs from NG-1125 (SEQ ID NO: 98) infection were positive for CD19 staining (indicating binding of the anti-HER2-CD19m1 protein), compared with approximately 20% of cells incubated with NG-1124 SNs (Figure 8A). The production of other encoded transgenes (CXCL9, CCL21, IFNa, and IL-15) after treatment with NG-1101, NG-1104, or NG-1125 was demonstrated by specific ELISA assays (Fig. 8B).

[0161] Example 6: In vivo binding of NG-1125-encoded anti-HER2 ScFv-CD19 bispecific protein in A549 tumors. To test the efficiency of anti-HER2 ScFv-CD19m1 bispecific protein production and tumor cell binding in vivo, 5 × 10 9 A549 lung tumor cells were inoculated subcutaneously to generate xenografts, with tumors growing to approximately 100–200 mm 3 When the mice reached 5 × 10 9 Viral particles (VP) were administered intravenously (days 0 and 3). Mice were euthanized 9 days after the first viral administration, and tumors were processed into single cell suspensions and analyzed by flow cytometry. Tumor cells were classified as viable cells (i.e., negative for LIVE / DEAD™ Fixable Aqua Dead Cell viability dye staining), CD45 - EpCAM+ Based on nuclear staining of adenovirus capsid proteins, uninfected tumor cells (no positive adenovirus staining above the negative isotype antibody control) could be distinguished from infected tumor cells with low VP load (dark adenovirus staining fluorescence intensity) and infected tumor cells with high VP load (bright adenovirus staining fluorescence intensity) (Figure 9A). Infected tumor cells with low and high VP loads accounted for an average of 20% and 5% of all tumor cells, respectively (Figure 9B). Within each tumor cell subset, we used surface CD19 staining to quantify the proportion of tumor cells that bound the anti-HER2-ScFv-CD19m1 protein (SEQ ID NO: 47) encoded by NG-1125. In both uninfected and infected tumor cell populations, we found that CD19 + We found a subset of tumor cells that were resistant to HER2 ScFv-CD19 (Figure 9C). These data indicated that the anti-HER2 ScFv-CD19 bispecific protein could be effectively secreted in vivo by tumor cells infected with the NG-1125 virus, diffuse into the TME, and bind to both infected and non-infected tumor cells, potentially enabling the recognition and destruction of these cells by anti-CD19 CAR-T cells.

[0162] Further in vivo studies involved administering 5 × 10 6 A549 lung tumor cells were inoculated subcutaneously to generate xenografts, with tumors growing to approximately 100–200 mm 3 When the mice reached 5 × 10 6 1.7 × 10 viral particles (VP) were administered intravenously (days 0 and 3). On day 6, mice received 1.7 × 10 7CD19-specific human CAR-T cells (generated and provided by ProMab Biotechnologies Inc., Richmond, CA, USA) were intravenously administered. Mice were then re-administered with the same virus twice more intravenously on days 30 and 34, after which intratumoral accumulation of adoptively transferred human T cells was assessed on day 50. Tumors were excised and gently disrupted to form cell suspensions, and flow cytometry was used to count total human CD45+ cells and activated human CD8+ cells expressing the CD107a degranulation marker, a biomarker of functional cytotoxicity. + and CD4 + Figure 10 shows that NG-1125 (SEQ ID NO: 98), expressing the anti-HER2 ScFv-CD19m1 bispecific protein (SEQ ID NO: 47) together with human CXCL9 and human IFNa, significantly increased the number of total human T cells (A) and cytotoxic activated CD8 T cells (B) within human tumor xenografts compared to NG-1124 (SEQ ID NO: 97), expressing the CD19 bispecific protein (SEQ ID NO: 47) alone or empty vector (EnAd). + (B) and CD4 + (C) Higher T cell density.

[0163] Example 7: In vivo enhancement of T cell recruitment to tumors by NG-641 and NG-1125 In further in vivo studies, NSG immunodeficient mice were inoculated subcutaneously with A549 tumor cells as described in Example 6, and tumors grew to approximately 100-200 mm 3 When the mice reached 400 mg / mL, they were injected with 5 × 10 ng-641 (SEQ ID NO: 84 in WO2019 / 043020), NG-1125 (SEQ ID NO: 98), or empty vector (EnAd). 910 viral particles (VP) were administered intravenously, all on days 0, 3, and 5, or no viral treatment was given. NG-641 expresses the CXCL9 and CXCL10 chemokines, IFNα, and FAP-TAC, a bispecific molecule composed of an antibody fragment targeting the human FAP fibroblast activation protein (FAP), which does not bind to mouse FAP conjugated to an anti-CD3 agonist antibody fragment (human FAP is not expressed in the tumor microenvironment of A549 xenografts). Because the anti-FAP-anti-CD3 construct is expected to be inactive in this model, the NG-641 virus was used as a "no antigen" control to consider the effects of CXCL9 and IFNα on CAR-T cell recruitment and activation, independent of the presence of the CD19 antigen. On day 12, mice received 2.5 x 10 7 CD19-specific human CAR-T cells (generated and provided by ProMab Biotechnologies Inc., Richmond, CA, USA) were administered intravenously. Ten days after CAR-T cell injection, tumor-derived single cell suspensions and blood samples were analyzed by flow cytometry to detect human CD45 + The frequency of T cells (representing all transferred T cells) and expression of the CD25 activation marker and CD107a degranulation marker, the latter used to identify active cytotoxic T cells, were measured. Figure 12 shows that administration of both NG-641 and NG-1125 (SEQ ID NO: 98) significantly increased the expression of total human CD45 T cells compared to the no EnAd and no virus condition. + cells (Figure 12A) and activated human CD45 + CD25 + (Fig. 12B) and CD45 + CD107 + (Figure 12C) showed increased intratumoral cell density, indicating that CXCL9 and IFNα (expressed by both of these viruses) are effective in recruiting and activating CAR-T cells within the tumor. NG-1125, which also expresses the anti-HER2 ScFv-CD19m1 bispecific protein (SEQ ID NO: 47) along with CXCL9 and IFNα, increased the intratumoral density of total and activated human CD45+ cells. +This increased the overall frequency of human CD45 cells, indicating an additive effect of tumor-specific CD19 antigen expression on CXCL9- and IFNα-mediated CAR-T cell boosting, likely as a result of antigen-specific engagement of the anti-CD19 CAR-T receptor (Figures 12A, B, and C). + T cell frequencies were higher in mice that did not receive the viral vector compared to mice that received NG-641, NG-1125, and EnAd (Figure 12D), suggesting that viral infection alone can promote the recruitment of circulating CAR-T cells to solid tumors and that this may be further enhanced by selected transgene expression (as shown in Figure 12A-C).

Claims

1. 1. An oncolytic group B adenovirus suitable for treating solid tumors (e.g., sarcomas, carcinomas and / or lymphomas) comprising a sequence of formula (I): 5’ITR-B １ -B Ａ -B ２ -B Ｘ -B Ｂ -B Ｙ -B ３ -3’ITR(I) During the ceremony, B １ is a bond or comprises E1A, E1B or E1A-E1B, B Ａ comprises E2B-L1-L2-L3-E2A-L4, B ２ is a bond or includes E3, B Ｘ is a junction or a DNA sequence containing a restriction site, one or more transgenes, or both; B Ｂ includes L5, B Ｙ is a DNA sequence encoding at least two transgenes, i.e., a first transgene and a second transgene, e.g., under the control of a major late promoter, B ３ is a bond or contains E4, During the ceremony, the first transgene encodes a polypeptide comprising a target sequence specific for a binding domain on cells of a cell-based immunotherapy having an (exogenous) recombinant surface-expressed protein, such as a chimeric antigen receptor or an NKG2D receptor, in particular said target sequence specifically binding to said surface-expressed protein (more particularly said chimeric antigen receptor) on said immunotherapy cells; - An oncolytic group B adenovirus, wherein the second transgene encodes a polypeptide comprising a molecule that facilitates delivery of said cell-based immunotherapy into the tumor.

2. 2. The oncolytic group B adenovirus of claim 1, wherein expression of the first transgene increases the local concentration of the target sequence within the tumor.

3. 3. The oncolytic group B adenovirus of claim 1, wherein the target sequence is specific for a recombinant receptor on the immunotherapy cells.

4. The target sequence may be a tumor antigen, such as CD20, CD19, CD22, CD33, CD34, CD37, CD38, CD47, CD52, CD56, CD70, CD74, CD133, CD138, CD147, CD152, CD221, CD254, CD261, CD262, CD309, CD340, BCMA, C-MYC, CAIX, Claude [such as claudins 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and / or 24], EGFRvIII, EPHA3, folate receptor alpha [FRα], GPC3, WT1, CEA, MUC-1, EpCAM, MAGE, mesothelin, PRAME, NYES AFP, CA-125, ETA, tyrosinase, RAS, p53, HER receptor HER1 [EGFR], HER2, HER3, HER4, MCAM, PEM, A33, G250, carbohydrate antigen Le ｙ , Le ｘ , Le ｂ , PSMA, TAG-72, STEAP1, CD166, CD24, CD44, E-cadherin, SPARC, ErbB2 and ErbB3, episialin, FOLR-1, 5T4, GPNMB, integrin αVβ3, integrin α5β1, Lewis Y antigen, MET [HGFR], mucin, PMSA, TAG-72, VEGFR, PDL1 [or an antigenic fragment of any one thereof], e.g. For example, tumor antigens such as CD19, BCMA, CEA, claudin-6, claudin-18.2, EGFRvIII, FRα, GPC3, MCAM, mesothelin, MUC-1, EpCAM, MAGE, PRAME, AFP, CA-125, ETA, tyrosinase, RAS, and p53; HER receptors HER1, HER2, HER3, HER4, PEM, A33, and G250; and carbohydrate antigens Le. ｙ , Le ｘ , Le ｂ 4. The oncolytic group B adenovirus according to claim 1, wherein the oncolytic group B adenovirus is selected from the group consisting of PSMA, TAG-72, STEAP1, CD166, CD24, CD44, E-cadherin, SPARC, ErbB2, and ErbB3, and an antigenic fragment of any one of them.

5. The oncolytic group B adenovirus according to any one of claims 1 to 4, wherein the target sequence is CD19 or an antigen fragment thereof.

6. The targeting sequence is a ligand (including an antibody, such as an idiotypic antibody, or an antigen-binding fragment thereof) that interacts with a "native" receptor on an immune cell, for example, an NKG2D ligand (the latter interacting with native NKG2D on NK cells and CD8 T cells to stimulate cytotoxic activity) or a ligand (including an antibody binding domain) that activates signaling through a "native" receptor on the immunotherapy cell, such as CD40L, OX40L, CD80, CD86, 4-1BBL (TNFSF9), CD70, LIGHT (TNFSF14), GITRL (TNFSF18), CD258 (HVEML, TNFRSF14), ICOSL (B7-H2), or The oncolytic virus of any one of claims 1 to 5, wherein the target sequence is a ligand (comprising an antibody binding domain) that inhibits signaling via a "native" receptor such as PD1, TIM3, LAG3, VISTA, TIGIT, B7-H3, B7-H4, HVEM, ILT-2, ILT-3, ILT-4, BTLA, CD160 on the immunotherapy cells, e.g., PD1, TIM3, LAG3, VISTA, TIGIT on T cells.

7. 7. The oncolytic group B adenovirus according to claim 1, wherein the first transgene encodes the target sequence in a membrane-anchored form that is suitable for expression on the surface of infected cancer cells and that allows the cell-based therapy to directly bind to the cancer cells, e.g., the membrane-anchored form comprises a transmembrane domain or a GPI anchor.

8. 8. The oncolytic group B adenovirus according to any one of claims 1 to 7, wherein the target sequence is a non-human, e.g. mouse, or yeast antigen such as GCN4, or a completely synthetic molecule (particularly of non-human origin) comprising an amino acid sequence selected to be recognized by a specific receptor embodied by the cell-based immunotherapy.

9. The oncolytic group B adenovirus according to any one of claims 1 to 8, wherein the target sequence is a tag such as an HA tag (amino acids 98 to 106 of human influenza hemagglutinin), a His tag (e.g., containing at least six histidine residues), a FLAG tag, or a 2A peptide tag (such as P2A, T2A, E2A, and / or F2A).

10. The first transgene is a. the target sequence that binds to and activates the cell-based immunotherapy, and b. A first binding protein specific for a protein expressed on cancer cells, stromal cells, or within stromal tissue, which specifically enables the cell-based immunotherapy to indirectly bind to the cancer cells and / or stromal cells or tissue via the fusion protein. The oncolytic group B adenovirus according to any one of claims 1 to 9, encoding a fusion protein comprising:

11. 11. The oncolytic group B adenovirus of claim 10, wherein the virus encodes at least two fusion proteins, and the first binding protein in part b) is different in each fusion protein, e.g., one fusion protein is encoded by the first transgene and a second fusion protein is encoded by the second transgene.

12. 12. The oncolytic group B adenovirus of claim 11, wherein the target sequence of part a) is the same for the at least two fusion proteins, i.e. both fusion proteins bind to the same entity on the immunotherapy cells.

13. 13. The oncolytic group B adenovirus according to claim 11 or 12, wherein the targeting sequences of part a) are different in the two fusion proteins (i.e., the fusion proteins bind to different entities on the same or different immune cells).

14. The oncolytic group B adenovirus according to any one of claims 10 to 13, wherein the binding protein of part b) is, for example, a ligand for a protein or receptor found on the cancer and / or stromal cells.

15. The oncolytic group B adenovirus of any one of claims 1 to 14, encoding said second transgene, thereby improving the efficacy of said cell-based immunotherapy.

16. 16. The oncolytic group B adenovirus of claim 15, wherein an additional transgene (e.g., the second transgene or a third transgene) enhances the efficacy of the cell-based immunotherapy by modulating the tumor microenvironment (e.g., by blocking an inhibitory property of the tumor microenvironment), e.g., modulating the microenvironment to make the microenvironment more inclusive to the cell-based therapy and / or to make the microenvironment more inflammatory.

17. 37. The oncolytic group B adenovirus according to any one of claims 1 to 36, wherein the second or further transgene encodes a chemokine that helps recruit immunotherapeutic cells to the tumor.

18. Further transgenes encode synthetic proteins designed to bind to additional recombinant receptors expressed by the cells of said therapy to enhance their ability to enter and act in tumors and / or survival in the patient, for example, ortho-IL-2 as a synthetic ligand of ortho-IL-2Rβ (synthetic receptors—e.g., Zhang et al 2021 Sci. Transl. Med. 13 (625) eabg6986, incorporated herein by reference), which can be engineered as a fusion with an antibody fragment to bind to tumor cells, or the TIM3 / CD28 switch receptor of CAR-T that interacts with secreted TIM3 ligands, e.g., galectin-9, HMGB1 (Zhao et al 2021 J. Immunotherapy Cancer 13 (625) eabg6986, incorporated herein by reference). 9, e003176), a synNotch fusion receptor on the CAR-T (e.g., where the CAR recognizes CD19 (including where CD19 is in a fusion protein such as an anti-HER2 ScFv-CD19 fusion protein), an anti-CD19 scFv bound to the IC domain of Notch to signal a response in the engineered CAR - Roybal et al 2016 Cell 167(2), 419-432, incorporated herein by reference).

19. 19. The oncolytic group B adenovirus according to any one of claims 1 to 18, wherein the cells expressing the (exogenous) recombinant antigen receptor comprise or are selected from the group consisting of T cells (T), macrophages (Mac), natural killer cells (NK), natural killer T cells (NKT) or innate lymphoid cells (ILC).

20. The oncolytic group B adenovirus according to any one of claims 1 to 19, wherein the exogenous recombinant antigen receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

21. An oncolytic group B adenovirus according to any one of claims 1 to 42 for use in therapy, in particular for use in the treatment of cancer, such as solid tumors, for example where the cancer is a transformed epithelial cancer cell.

22. An oncolytic group B adenovirus according to any one of claims 1 to 20 for use in the manufacture of a medicament for the treatment of cancers, such as solid tumors, in particular transformed epithelial cancer cells.

23. 21. A method of treating a patient, in particular for treating cancer, more particularly solid tumors such as transformed epithelial cancer cells, comprising administering a therapeutically effective amount of an oncolytic group B adenovirus according to any one of claims 1 to 20.

24. A combination therapy comprising an oncolytic group B adenovirus according to any one of claims 1 to 20 and engineered immunotherapeutic cells, for example to treat cancer.

25. 21. A method for producing a virus according to any one of claims 1 to 20, wherein the virus is replicated in a host cell, such as a mammalian cell, such as an HEK cell.

26. 26. A virus obtained from claim 25.

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