Virus encoding transgenes to supplement cell therapy

Abstract

An oncolytic group B adenovirus suitable for use in the treatment of solid tumors, such as sarcoma, cancer and / or lymphoma, 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 target sequence specific for a binding domain on a cell of a cell-based immunotherapy, a target sequence, such as a protein with a (exogenous) recombinant surface expression, such as a chimeric antigen receptor or NKG2D receptor, in particular wherein the target sequence specifically binds to the surface-expressed protein, more particularly the chimeric antigen receptor, on an immunotherapy cell; and the second transgene encodes a polypeptide comprising a molecule that promotes the cell-based immunotherapy to enter and transport within a tumor.

Description

[0001] The present disclosure relates to an oncolytic virus constructed to encode a transgene for specifically "coating" tissue cells with a target antigen for antigen-specific immune cells, a preparation comprising said virus, and the use of said virus and preparation in therapy, particularly in combination therapy, especially combination therapy comprising cell therapy (such as CAR-T). Background Art

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

[0003] CAR-T cells directed against B cell antigens have been tested clinically and often induce long-term remissions, even in heavily pretreated patients. There are currently approximately 5 CAR T cell therapies on the market for the treatment of hematological malignancies.

[0004] In contrast, CAR-T cell therapy has not achieved the same depth and reproducibility of response in patients with solid tumors.

[0005] CAR T cells targeting, for example, B7-H3, CEACAM5, CD133, CD171, claudin 6, EGFR, EGFRvIII, FRα, GD2, GPC3, HER2, IL-13Ra2, mesothelin, MUC1, PSMA, ROR1 and VEGF-R2 have been tested clinically but have shown limited ability to control disease.

[0006] Chimeric antigen receptor (CAR) T cells are T cells expressing a genetically modified receptor capable of recognizing surface antigens on target cells and triggering T cell activation and antigen-specific cytotoxicity. Many alternative cell technologies are currently being developed and are also suitable for use in combination with the viruses of the present disclosure.

[0007] Several factors have been hypothesized to contribute to this disappointing activity in solid tumors, including a) fewer solid tumor target antigens, b) heterogeneous target expression between tumor cells, c) poor trafficking of CAR-T cells to solid tumors, and d) the solid tumor microenvironment that is unfavorable for the function and survival of immune cells (Hou et al. Nat Rev Drug Discov. 2021). Specifically, the injected cell-based immunotherapy is widely dispersed in the blood and only a small fraction reaches the tumor microvascular system. Thus, insufficient trafficking of CAR-T cells to solid tumors (c, above) may be particularly important.

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

[0009] Ambrose et al. (Ambrose, Christine et al. “Anti-CD19 CAR T cells potently redirected to kill solid tumour cells.” PloS one volume 16, 3e0247701. March 18, 2021, doi:10.1371 / journal.pone.0247701) developed anti-CD19 CAR-T cells capable of secreting an anti-HER2-CD19 bridging protein, which, by binding to HER2-positive tumor cells, enables anti-CD19 CAR-T cells to recognize cells expressing both CD19 and HER-2. However, this secreted protein is expressed wherever the CAR-T cells are found after intravenous (IV) injection, and thus may cause extra-tumoral cytotoxicity.

[0010] The use of fusion proteins containing an antigen target for CAR-T therapy and a second binding domain that binds to certain entities in the tumor has been proposed, see for example WO2018 / 156791. The fusion protein can be expressed, for example, in the tumor, and the activation of CAR-T therapy occurs upon binding. However, this approach does not fundamentally solve the problem of delivering immunotherapy into the tumor.

[0011] More importantly, the stroma around solid tumors is like a city wall, difficult for immune cells to penetrate, and forms a hypoxic microenvironment that hinders the cytotoxic activity of immune cells. Thus, even after successful transport to the local microenvironment, the latter can impair the function of cell-based immunotherapy and / or promote its clearance through various different soluble molecules and immunosuppressive cells (such as TGFβ, adenosine, regulatory T cells, and myeloid-derived suppressor cells).

[0012] Some cells engineered for cell-based immunotherapy have been engineered to express “exogenous” cytokines to maintain their activation. However, this may lead to a problem because if the cytokine is constitutively expressed, it may cause off-target effects (side effects). Conversely, if the cytokine is inducibly expressed, for example, after the cell binds to its target antigen, then due to the tumor microenvironment, the cell may never reach the target.

[0013] To achieve successful treatment of solid tumors with CAR-T cells, it is important to find ways to promote homing of cell-based immunotherapy to tumors and / or reprogram the tumor microenvironment (e.g., from an immunosuppressive microenvironment to an immunostimulatory microenvironment). Summary of the Invention

[0014] The present inventors have designed oncolytic viruses that preferentially infect cancer cells and are highly adapted to survive in the tumor microenvironment. These viruses not only create an entry pathway into the cancer by infecting cancer cells, replicating, and lysing the cells, but are also designed in the present disclosure to express a transgene with two independent aspects, namely:

[0015] · Locally express an antigen target of cell-based immunotherapy (referred to herein as the target sequence) in the tumor (e.g., to increase the local concentration of the target antigen), and

[0016] · Promote the recruitment and / or activity of cell-based immunotherapy within the tumor.

[0017] Accordingly, the present disclosure acts by increasing the local concentration of a target or activator of antigen-specific cells (especially engineered antigen-specific cells). The virus also supports altering the tumor microenvironment to support the influx and / or survival of antigen-specific cells.

[0018] Advantageously, the stimulation of antigen-specific cells minimizes any off-target effects and maximizes the therapy by targeting only the diseased tissue.

[0019] The local increase in the target antigen can be increased by one or more of the following (i.e., in combination):

[0020] · Cell surface expression of the target sequence on infected cells;

[0021] · Secretion of a fusion protein from the infected cells, wherein the fusion protein binds to an entity on the diseased cells (infected cells and / or

[0022] surrounding cells) and presents the target sequence;

[0023] This combined approach has never been employed, and the present inventors have demonstrated that it results in a greater number of active immunotherapy cells being recruited to the tumor.

[0024] To help recruit cell-based immunotherapy to the tumor, the oncolytic virus can be equipped with transgenes that reprogram the tumor microenvironment to promote infiltration and activity of cell-based immunotherapy.

[0025] Once the "transport transgene" makes the tumor more accessible, higher concentrations of the target antigen may act like a magnet to recruit and activate cell-based therapy, thereby increasing the local concentration of cell-based therapy where it is needed.

[0026] The lysis of cells by oncolytic viruses also results in the release of pro-inflammatory mediators (such as HMGB1, ATP, type I interferon), which can then induce an anti-tumor immune response and recruit innate immune cells from peripheral lymphoid organs.

[0027] The non-specific activation of immune cells can be used in combination with the techniques of the present disclosure. For example, bispecific T cell activators containing agonists for CD3 can be encoded in the viruses of the present disclosure.

[0028] According to the antigen targets of cell-based immunotherapy, for example, if the target is a tumor antigen, the therapeutic cells recruited to the tumor site are directly active against tumor cells expressing the antigen but not necessarily infected by the oncolytic virus. A fusion protein can be secreted from the infected cells, and the fusion protein contains the binding domain of the tumor antigen targeted by the cell therapy and a second antigen (such as a stromal antigen). After binding to the stroma, the tumor antigen will be presented for cell therapy, enabling the targeting of two different tissue types by a single cell therapy. Therefore, the active mechanism of the virus and the cell therapy are complementary but also independent.

[0029] Thus, in one embodiment, the virus-infected cells secrete a fusion protein that binds to antigens on surrounding cells (such as other tumor cells and / or stromal cells (which may or may not be infected)) and presents target sequences for binding by cell therapy. Generally, such binding will activate cell therapy.

[0030] In one embodiment, the viruses of the present disclosure encode one or more, two or more; three or more, four or more fusion proteins, and the fusion proteins have binding domains and target sequences for cell therapy.

[0031] When the virus encodes multiple fusion proteins, the binding domains can bind to different entities, and the target sequences can be the same, so that one cell therapy can be used to target two or more different cells, tissues or therapeutic targets within the cancer tissue.

[0032] In some patients, different types of cancer cells coexist and, for example, express different antigens. In these patients, it would be useful to be able to target these different cancer cells with one cell therapy. This can be achieved by encoding two fusions, each with a binding domain specific for a different cancer cell, such as a single target sequence (or multiple target sequences if desired).

[0033] In one embodiment, the oncolytic virus encodes a transgene that is expressed, for example, as a soluble protein and expressed into the microenvironment to protect / activate engineered immune cells and / or general immune cells. For example, IFNα, IL-12, and IL-15 are useful cytokines for protecting and / or activating immune cells in the tumor microenvironment, and chemokines (such as CXCL9 and CCL21) can be used to direct immune cells to target cells within the tumor mass.

[0034] Thus, the oncolytic viruses and combination therapies of the present disclosure have at least two or three mechanisms to improve the treatment outcome of solid tumors.

[0035] The inventors prepared tumor-specific group B adenoviruses, particularly EnAd, which encode a series of different bispecific proteins that contain a portion capable of binding to an antigen expressed on the surface of cells (particularly tumor cells or tumor-associated fibroblasts), and a portion that binds to immune cells (such as T cells bearing CART or engineered T cell receptors (TCR)) by engaging with an engineered CAR or TCR or an endogenous receptor (such as TCR) or other surface molecules on the cell. They found that in many cases, although the tumor cell-binding portion was functional, the immune cell-binding portion was dysfunctional. To address this issue, different protein sequences and designs were screened until a functional bispecific protein was found. However, even when a functional bispecific molecule was obtained, the expression level was found to be lower than ideal.

[0036] Surprisingly, when the bispecific protein is encoded sequentially (in tandem) with one or more different immunomodulatory molecules, including chemokines and cytokines, its expression level continuously increases.

[0037] The tumor microenvironment (TME) allows infiltration of the adenoviruses according to the present disclosure, thereby delivering a large amount of the bispecific protein to the desired location. In addition, co-expression of the bispecific protein and immunomodulatory molecules that have the ability to recruit T cells or promote direct or indirect T cell activation has the potential to synergistically enhance the ability of CAR-T cells to recognize and kill target cells in the TME.

[0038] Surprisingly, we found that adding two or more transgenes to the same transgene cassette encoding the bispecific protein resulted in a significant increase in bispecific protein expression compared to the corresponding viral design encoding the bispecific protein alone.

[0039] The viruses of the present disclosure supplement and / or improve cell therapy.

[0040] The present disclosure is outlined in the following paragraphs:

[0041] 1. An oncolytic adenovirus type B suitable for treating solid tumors (such as sarcomas, carcinomas, and / or lymphomas), which comprises the sequence of formula (I):

[0042] 5’ITR-B1-B A -B2-B X -B B -B Y -B3-3’ITR (I)

[0043] Wherein:

[0044] B1 is a linker or comprises: E1A, E1B, or E1A-E1B;

[0045] B A comprises -E2B-L1-L2-L3-E2A-L4;

[0046] B2 is a linker or comprises: E3;

[0047] B X is a linker or a DNA sequence that comprises: a restriction site, one or more transgenes, or both;

[0048] B B comprises L5;

[0049] B Y is a DNA sequence that encodes at least two transgenes, namely a first transgene and a second transgene, for example under the control of a major late promoter; and

[0050] B3 is a linker or comprises: E4,

[0051] Wherein:

[0052] · The first transgene encodes a polypeptide that comprises a target sequence specific for a binding domain on a cell-based immunotherapy, such as a (heterologous) recombinantly surface-expressed protein, such as a chimeric antigen receptor, an NKG2D receptor, particularly wherein the target sequence specifically binds to the surface-expressed protein (more particularly the chimeric antigen receptor) on the immunotherapy cells, and

[0053] · The second transgene encodes a polypeptide that comprises a molecule that promotes entry of the cell-based immunotherapy into the tumor and trafficking within the tumor.

[0054] 2. The oncolytic adenovirus type B according to paragraph 1, wherein the expression of the first transgene increases the local concentration of the target sequence within the tumor.

[0055] 3. The oncolytic adenovirus type B according to paragraph 1 or 2, wherein the target sequence is specific for a recombinant receptor on the immunotherapy cells

[0056] 4. The oncolytic adenovirus group B according to any one of paragraphs 1 to 3, wherein the target sequence is a tumor antigen.

[0057] 5. The oncolytic adenovirus group B according to any one of paragraphs 1 to 4, wherein the target sequence is a sequence expressed on cancer cells (including 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 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 receptors 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 antigen fragment of any one of them], 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, p53, HER receptor 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 antigenic fragment of any one of them.

[0058] 6. The oncolytic adenovirus group B according to any one of paragraphs 1 to 5, wherein the target sequence is CD19 or an antigenic fragment thereof.

[0059] 7. The oncolytic adenovirus group B according to any one of paragraphs 1 to 6, wherein the target ligand is a non-human sequence, such as from yeast (advantageously reducing off-target effects).

[0060] 8. The oncolytic virus according to any one of paragraphs 1 to 7, wherein the target sequence is a ligand (including an antibody or an antigen-binding fragment thereof, such as an idiotypic antibody).

[0061] 9. The oncolytic adenovirus group B according to any one of paragraphs 1 to 8, wherein the target sequence is a ligand (including an antibody-binding domain) that activates signal transduction through the "natural" receptor on the immunotherapy cell, such as the NKG2D ligand that interacts with its "natural" receptor on immune cells (the latter interacts with natural NKG2D on NK and CD8 T cells to stimulate cytotoxic activity), or CD40L, OX40L, CD80, CD86, 4-1BBL (TNFSF9), CD70, LIGHT (TNFSF14), GITRL (TNFSF18), CD258 (HVEML, TNFRSF14), ICOS-L (B7-H2).

[0062] 10. The oncolytic adenovirus group B according to any one of paragraphs 1 to 8, wherein the target sequence is a ligand (including an antibody-binding domain) that inhibits signal transduction through the "natural" receptor on the immunotherapy cell, such as PD1, TIM3, LAG3, VISTA, TIGIT, B7-H3, B7-H4, HVEM, ILT-2, ILT-3, ILT-4, BTLA, CD160 on the immunotherapy cell, for example PD1, TIM3, LAG3, VISTA, TIGIT on T cells.

[0063] 11. The oncolytic adenovirus group B according to any one of paragraphs 1 to 10, wherein the first transgene encoding the target sequence is suitable for expression on the surface of infected cancer cells, particularly a membrane-anchored form that allows cell-based therapy to directly bind to the cancer cells (i.e., without passing through a fusion protein).

[0064] 12. The oncolytic adenovirus group B according to paragraph 11, wherein the membrane-anchored form comprises a transmembrane domain or a GPI anchor.

[0065] 13. An oncolytic adenovirus type B according to any one of paragraphs 1 to 12, wherein the target sequence is non-human, such as a murine or yeast antigen, such as GCN4.

[0066] 14. An oncolytic adenovirus type B according to any one of paragraphs 1 to 13, wherein the target sequence is a marker, such as an HA tag (amino acids 98 to 106 of human influenza hemagglutinin), a His tag (e.g., comprising at least 6 histidine residues), a FLAG tag or a 2A peptide tag (such as P2A, T2A, E2A and / or F2A).

[0067] 15. An oncolytic adenovirus type B according to any one of paragraphs 1 to 13, wherein the target sequence is epitope E5B9 (from the La-SS / B antigen).

[0068] For example, as disclosed in WO2015 / 181282, which is incorporated herein by reference.

[0069] 16. An oncolytic adenovirus type B according to any one of paragraphs 1 to 15, wherein the first transgene encodes a fusion protein comprising:

[0070] a. a target sequence that binds to and activates the cell-based immunotherapy, and

[0071] b. a first binding protein that is specific for a protein expressed on cancer cells, stromal cells or stromal tissue (such as an acellular matrix stroma, especially collagen), and in particular allows the cell-based immunotherapy to indirectly bind to the cancer cells and / or stromal cells or stromal tissue through the fusion protein

[0072] 17. An oncolytic adenovirus type B 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. For example, one fusion protein is encoded by the first transgene and a second fusion protein is encoded by the second transgene.

[0073] 18. An oncolytic adenovirus type B according to paragraph 16, 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.

[0074] 19. An oncolytic adenovirus type B according to paragraph 17, wherein the target sequence of part a) is different in the two fusion proteins (i.e., the fusion proteins bind to different entities on the same or different immune cells).

[0075] 20. The oncolytic group B adenovirus according to any one of paragraphs 16 to 19, wherein the binding protein in part b) is a ligand for a protein or receptor found on cancer and / or stromal cells (e.g., when there are multiple fusion proteins, the binding domain is specific for different antigens / markers / targets).

[0076] 21. The oncolytic group B adenovirus according to paragraph 20, wherein the ligand is an antibody or an antigen-binding fragment thereof.

[0077] 22. The oncolytic group B adenovirus according to any one of paragraphs 1 to 21, wherein the target sequence is a sequence expressed in the stroma or on stromal cells.

[0078] 23. The oncolytic group B adenovirus according to paragraph 22, wherein the stromal cells are independently selected from the group consisting of or comprising cancer-associated fibroblasts, tumor-associated macrophages, or other inhibitory cells, e.g., wherein the target sequence is 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 or an antigenic fragment thereof.

[0079] 24. The oncolytic group B adenovirus according to any one of paragraphs 1 to 23, wherein encoding the second transgene increases the effectiveness of the cell-based immunotherapy (or broadens the range of targeted tissues)

[0080] 25. The oncolytic group B adenovirus according to paragraph 24, wherein the additional transgene (e.g., the second transgene or the third transgene) increases the effectiveness of the cell-based immunotherapy by modulating the tumor microenvironment (e.g., blocking the inhibitory characteristics of the tumor microenvironment).

[0081] 26. The oncolytic group B adenovirus according to paragraph 25, wherein the microenvironment is modulated to be more permissive for the cell-based therapy and / or to make the microenvironment more inflammatory.

[0082] 27. The oncolytic group B adenovirus according to paragraph 25 or 26, wherein the additional transgene modulates the tumor microenvironment to be more permissive (tolerant) for infiltration by immunotherapy cells and / or to more promote / support the innate immune response, especially when the immune cells remain activated in the microenvironment, e.g., a matrix degrading agent or loosening agent to assist cell migration within the tumor

[0083] 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-α receptor (PDGFR-α), platelet-derived growth factor-β receptor (PDGFR-β), and vimentin. For example, the second gene may encode a bispecific T cell activator as disclosed in, for example, WO2018 / 041838 and WO2018 / 041827, both of which are incorporated by reference.

[0084] 29. The oncolytic group B adenovirus according to any one of paragraphs 25 to 28, wherein the microenvironment is regulated by helping immune cells (including the immunotherapy cells and natural immune cells) overcome inhibition.

[0085] 30. The oncolytic group B adenovirus according to claim 29, wherein the ability to overcome inhibition is provided by immune checkpoint inhibitors, such as anti-CTLA-4 inhibitors, anti-PD-1 inhibitors, and anti-PD-L1 inhibitors, particularly selected from ipilimumab, tremelimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, and cemiplimab.

[0086] 31. The oncolytic group B adenovirus according to any one of paragraphs 25 to 30, wherein the microenvironment is regulated to be more inflammatory, for example, by producing pro-inflammatory cytokines (such as IL-1, IL_6, TNFα, IFNγ) and / or chemokines (such as CXCL9, CCL3, CCL5) - encoded as transgenes or induced by encoded transgenes (such as IL-12, IL-15).

[0087] 32. The oncolytic group B adenovirus according to any one of paragraphs 24 to 31, wherein the microenvironment is regulated by enhancing the activation of immune cells (such as immunotherapy cells and / or natural immune cells), for example, the second gene or additional transgenes encode cytokines.

[0088] 33. The oncolytic group B adenovirus according to paragraph 32, wherein the enhanced activation of immune cells is provided by cytokines, such as selected from: 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, 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 α (LTA), and GM-CSF.

[0089] 34. The oncolytic group B adenovirus according to paragraph 32 or 33, wherein the enhanced activation of immune cells is provided by a membrane or soluble ligand that comprises an extracellular domain of a protein involved in cell-cell interactions between immune cells, such as co-stimulatory molecules expressed by antigen-presenting cells, for example selected from CD40L, OX40L, CD80, CD86, 4-1BBL, TNFSF14 (LIGHT), GITRL (TNFSF18), CD70, CD258 (HVEML), ICOS-L.

[0090] 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.

[0091] 36. The oncolytic group B adenovirus according to any one of paragraphs 32 to 34, wherein the enhanced activity is an increase in the lifespan (survival) of the immune cells.

[0092] 37. The oncolytic group B adenovirus according to paragraph 36, wherein the increased lifespan is provided by cytokines, such as IL-2, IL-7, IL-15, and / or IL-21.

[0093] 38. The oncolytic group B adenovirus according to any one of paragraphs 1 to 37, wherein the second transgene or additional transgene encodes a chemokine to assist in the recruitment of immunotherapy cells to the tumor.

[0094] 39. The oncolytic according to any one of claims 1 to 38, wherein the additional transgene encodes a synthetic protein that is designed to engage an additional recombinant receptor expressed by the cells of the therapy to enhance the ability of the additional recombinant receptor to enter the tumor and function within the tumor and / or its survival in the patient, such as an orthogonal IL-2 as a synthetic ligand for an orthogonal IL-2Rβ (synthetic receptor – for example, Zhang et al., Sci. Transl. Med. 13(625) eabg6986, 2021, which is incorporated herein by reference;

[0095] the TIM3 / CD28 switch receptor in CAR-T that interacts with secreted TIM3 ligands such as galectin 9, HMGB1), which can be engineered to be fused with an antibody fragment so that the antibody fragment binds to tumor cells (Zhao et al., J. Immunotherapy Cancer 9, e003176, 2021, which is incorporated herein by reference); the synNotch fusion receptor on CAR-T (for example, the CAR is an anti-CD19 Scfv that is coupled to the IC domain of Notch to signal a response in the engineered CAR when they recognize CD19 (including where the CD19 is in a fusion protein such as an anti-HER2 ScFv-CD19 fusion protein) – Roybal et al., Cell 167(2), 419 - 432, 2016, which is incorporated herein by reference.

[0096] 40. The oncolytic according to any one of paragraphs 1 to 39, wherein the additional transgene encodes a polypeptide that enhances the recruitment and anti-tumor activity of the patient's endogenous immune cells

[0097] 41. The oncolytic group B adenovirus according to any one of paragraphs 1 to 38, wherein the cells expressing the (exogenous) recombinant antigen receptor are selected from the group consisting of or comprising: T cells (T), macrophages (Mac), natural killer cells (NK), natural killer T cells (NKT) or innate lymphoid cells (ILC)

[0098] 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).

[0099] 43. The oncolytic group B adenovirus according to any one of paragraphs 1 to 41, which is used in therapy, particularly for the treatment of cancer such as solid tumors.

[0100] 44. Use of the oncolytic group B adenovirus according to paragraph 43, wherein the cancer is a transformed epithelial cancer cell.

[0101] 45. An oncolytic adenovirus serotype B 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.

[0102] 46. A method of treating a patient, the method comprising administering a therapeutically effective amount of an oncolytic adenovirus serotype B as defined in any one of paragraphs 1 to 42, in particular for the treatment of cancer, more specifically solid tumors, such as transformed epithelial cancer cells.

[0103] 47. A combination therapy comprising an oncolytic adenovirus serotype B according to any one of claims 1 to 42 and engineered immunotherapy cells, for example for the treatment of cancer.

[0104] 48. A method of producing a virus according to any one of paragraphs 1 to 42, wherein the virus replicates in a host cell, such as a mammalian cell, such as a HEK cell.

[0105] 49. A virus obtainable from paragraph 48.

[0106] There is also provided an oncolytic adenovirus serotype B according to the present disclosure, wherein the second transgene encodes an additional fusion protein, the fusion protein comprising, for example, a second binding protein different from the first binding protein and an antigen target sequence, to allow binding to a second protein (different from the entity bound by the first binding protein) expressed on cancer cells, on stromal cells or in stromal tissue via the additional fusion protein, the same or different cell-based immunotherapies, in particular allowing the cell-based immunotherapy to indirectly bind to the cancer cells and / or stroma via the fusion protein.

[0107] Where technically feasible, combinations of the examples can be used.

[0108] In one embodiment, provided that the fusion protein does not comprise (or consist of): i) a tumor-associated antigen or tumor-specific antigen and ii) an anti-idiotypic antibody or fragment or anti-idiotypic peptide.

[0109] In one embodiment, the virus of the present disclosure does not encode a bispecific T cell activator.

[0110] In one embodiment, the second or additional transgene encodes IL-15 or an active fragment thereof, optionally co-expressed, i.e., encoding at least immediately adjacent to or linked to (e.g., via a linker or amide bond) the sushi domain of IL-15Rα (such as the full-length extracellular domain of 15Rα).

[0111] Thus, in one embodiment, the presently disclosed viral constructs are designed to provide an antigen in a target tissue for activating an increase in a class of antigen-specific immune cells (such as a local increase in the number of specific immune cells including exogenously engineered immune cells, especially CAR-T [especially antigen-specific T cells]).

[0112] By increasing the local concentration of the antigen in the target tissue, it is assumed that the antigen threshold level required for activating immune cells is exceeded and a large activation signal is sent to the necessary cell population.

[0113] In one embodiment, the present disclosure is not about the non-specific activation of immune cells (such as T cells). Bispecific T cell activators are non-specific activators of T cells that engage CD3 with an agonist. This type of technology can be included as a supplement to the present technology, but acts through a mechanism different from the present technology, as the latter involves the trafficking and activation of antigen-specific cells. DETAILED DESCRIPTION

[0114] A target sequence, as used herein, refers to a target antigen sequence of an immune cell (such as a specific immune cell, such as an antigen-specific immune cell, especially an engineered immune cell (including a sequence that stimulates (especially activates and / or protects) existing immune cells or stimulates the production of said immune cells in a specific manner)). In one embodiment, it does not refer to the binding domain of an antibody that bypasses specificity and non-specifically stimulates immune cells (such as T cells). Thus, anti-CD3 agonistic antibodies that non-specifically stimulate T cells are not within the definition of a target sequence.

[0115] A target sequence can bind to a recombinant (engineered receptor) on an immune cell and / or can bind to a native receptor on an immune cell (especially a recombinant receptor on an immune cell), provided that the antigen-specific cell is functionally affected, such as being activated, protected, or proliferated.

[0116] In one embodiment, a target sequence is not an antibody sequence that non-specifically stimulates immune cells (such as an idiotypic antibody sequence).

[0117] An antigen fragment, as used herein, refers to a fragment of an antigen that is still capable of specifically binding to a binding domain, such as an epitope that can be at least 5 amino acids.

[0118] As used herein, cell-based immunotherapy refers to cell-based therapies that are prepared or generated ex vivo and then administered to a patient, such as those prepared using recombinant techniques, such as vectors (e.g., viral vectors). In one embodiment, the cells in the therapy encode a transgene, particularly one that encodes a non-native protein. Cells used in cell therapy include, but are not limited to, T cells (including their subtypes), NK cells (including their subtypes, such as memory NK cells), NKT cells, macrophages, and the like.

[0119] As used herein, engineered cells are cells that have been modified using recombinant techniques (including vectors, such as viruses, particularly ex vivo manipulation). Nevertheless, techniques (including vectors) can be used to target cells in vivo and modify them. If a cell is modified at the genetic level by manipulation, including transient modification, it will be considered an engineered cell in the context of this specification.

[0120] As used herein, a recombinant receptor or protein refers to a receptor or protein that is prepared or introduced by recombinant techniques.

[0121] In one embodiment, one or more constructs encoded by a virus according to the present disclosure are designed to engage with engineered cells.

[0122] 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 a binding domain / ligand) introduced into a cell (an engineered cell) by recombinant techniques. This means that the original cell is produced by recombinant techniques, even if the resulting cells can then be replicated to produce multiple cells containing the modification.

[0123] Thus, a reference to cell-based immunotherapy does not merely refer to engaging with, for example, native cells (such as native T cells) that have not been modified by ex vivo recombinant techniques.

[0124] As used herein, recombinant techniques refer to modifications of cells that are produced by human design and / or intervention, such as being produced substantially in the laboratory, i.e., not naturally occurring in nature.

[0125] In one embodiment, the virus according to the present invention is isolated, i.e., contained in vitro, particularly in purified form.

[0126] In one embodiment, the cell therapy used in the present disclosure is isolated. I.e., contained in vitro, particularly in purified form.

[0127] In one embodiment, the constructs of the present disclosure do not merely engage with native TCRs. In one embodiment, the constructs of the present disclosure do not engage with native TCRs. Nevertheless, the constructs or additional elements encoded in the virus can additionally engage with native TCRs.

[0128] In one embodiment, the target sequence is not the binding domain of an antigen-specific T cell activator molecule. Thus, the present disclosure does not relate to bispecific T cell activators and is disclosed in WO2018 / 041827 and WO2018 / 041838, which are incorporated herein by reference. However, bispecific T cell activators can be included as "additional" or extra transgenes in the viruses of the present disclosure.

[0129] As used herein, "delivery in a tumor" refers to an increase in the number of active immunotherapy cells within the tumor by a gene product. Thus, in the context of the present disclosure, "delivery" is related to the concept of the movement and / or activation of immunotherapy cells to reach the site of action within the tumor in a surgical form, such that they reach the intended location without being neutralized. Thus, delivery agents include one or more of the following: reagents that attract cells to the tumor (such as chemokines), reagents that disrupt the stromal / stroma barrier around the tumor (such as enzymes or stromal antigens); reagents that alter the microenvironment to be more permissive (or less hostile) to immune cells (such as immunotherapy cells and / or innate immune cells), such as checkpoint inhibitors; reagents that regulate the activity of immune cells (such as immunotherapy cells and / or innate immune cells); reagents that recruit innate immune cells; a more inflammatory environment, less hypoxia, and combinations of two or more thereof.

[0130] In one or more embodiments, the "second transgene" or delivery component also aids in the delivery of innate immune cells.

[0131] As used herein, the regulation of immune cell activity refers to "activating immune cells in some form, such as activation, including directing the activity of immune cells to a target, increasing the cytotoxic activity of immune cells, increasing the proliferation of immune cells, enhancing the production of soluble mediators (such as cytokines and chemokines) by immune cells, increasing the survival and / or lifespan of immune cells; and protecting immune cells from inhibition and / or non-responsiveness.

[0132] As used herein, innate immune cells are intended to refer to immune cells found in the body, i.e., cells that are not introduced or designed as a therapy.

[0133] As used herein, "activation" of an immune cell refers to triggering or enhancing one or more functions of the immune cell and / or providing a signal that enables the cell to respond functionally to other signals.

[0134] For example, IFNα can directly trigger the production of cytokines and chemokines (such as IL-6, BAFF, April), and once triggered by interaction of a ligand with the T cell receptor or the CAR on a CAR-T cell, can enhance the killing function of T cells

[0135] "Increasing the local concentration of a target sequence" as used herein includes increasing from a starting concentration of zero or an undetectable concentration and includes an increase in activity, and not necessarily or solely an increase in the number of immune cells.

[0136] A change in the tumor microenvironment as used herein refers to a change that permits the trafficking of immune cells within or into the environment, including changes in the vasculature, hypoxia, inflammatory state, and / or immunosuppressive state, among others.

[0137] "Specificity" as used herein refers to the fact that the binding domain recognizes the target antigen with an affinity and / or avidity greater than that for other antigens to which it is not specific (e.g., 10, 20, 50, 100, or 1000 times greater). This does not necessarily mean that the specific binding region does not bind to any non-target antigens, but rather that the interaction with the target enables it to be used to purify the target antigen (to which it is specific) from a complex mixture of antigens, including antigens within the same protein family.

[0138] A bispecific protein as used herein refers to a natural or synthetic protein, such as a fusion protein, that contains at least two different binding domains.

[0139] A binding protein as used herein refers to a polypeptide that contains a binding domain.

[0140] A binding domain as used herein refers to a protein domain that binds to a specific atom or molecule (usually a molecule, particularly a specific epitope - linear or conformational, usually a specific amino acid sequence), and includes, for example, a ligand, receptor, or antibody, or a binding fragment of any of them.

[0141] A binding fragment or antigen-binding fragment as used herein refers to the ability of an entity to specifically bind to a target antigen. Generally, it does not include the ability of regions such as Fc to bind to receptors.

[0142] An antibody binding domain (or antibody fragment) as used herein refers to a variable region having hypervariable domains, such as a molecule containing VH, VHH, VL, or VH and VL, including sc-Fv, Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, single-domain antibodies (e.g., VH or VL or VHH), scFv, bivalent, trivalent, or tetravalent antibodies, bis-scFv, diabodies, triabodies, tetra-bodies, and epitope-binding fragments of any of the foregoing, including their disulfide-linked forms (see, for example, Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews - Online 2(3), 209-217).

[0143] Ligands as used herein are generally substances that form complexes with biomolecules, including molecules present on the cell surface, and serve a biological purpose (such as signal transduction, blocking, occupancy, neutralization, activation, etc.). In one embodiment, the ligand is a binding domain from an antibody.

[0144] Soluble ligands involved in cell - cell interactions are essentially any cell - surface molecules that can be bound by T cells or other immune cells and, when used as soluble EC domains (fused or not fused 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 a signaling cytokine or other protein such that they remain in the local microenvironment where they can be concentrated to better activate immune cells.

[0145] In the context of this specification, soluble generally means not membrane - anchored.

[0146] A stromal antigen as used herein is an "antigen" found only in stromal tissue or on stromal cells, i.e., it does not include "antigens" that are also present on cancer cells. Thus, in the context of this specification, antigens expressed on both cancer cells and stromal cells are considered cancer antigens. Accordingly, stromal antigens can be presented on the surface of stromal cells (cells located in the stroma) and / or on soluble molecules located in the stromal matrix, particularly molecules that can be targeted.

[0147] 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.

[0148] 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 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], EGFR (such as EGFRvIII), VEGFR (such as VEGFR-1, VEGFR-2), EPHA3, folate receptor α [FRα], GPC3, WT1, CEA, MUC-1, EpCAM, MAGE, mesothelin, PRAME, NYESOAFP, 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 antigenic fragments of any of them], 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 receptor 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 antigenic fragments of any of them.

[0149] In one embodiment, the cancer target is selected from: 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 (such as 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).

[0150] Cancer antigens (also referred to herein as tumor antigens) as used herein include tumor-specific antigens (TSA) and cancer-associated antigens (TAA). Tumor-specific antigens are present only on tumor cells and not on any other cells, and cancer-associated antigens are present on some tumor cells and some normal cells. Tumor antigens include:

[0151] · Products of mutated oncogenes and tumor suppressor genes

[0152] · Products of other mutated genes

[0153] o Overexpressed or abnormally expressed cellular proteins

[0154] o Tumor antigens produced by oncogenic viruses

[0155] o Carcinoembryonic antigens

[0156] o Altered cell surface glycolipids and glycoproteins, and

[0157] o Cell type-specific differentiation antigens.

[0158] Cancer antigens (tumor antigens) are antigens specifically found on cancer cells (i.e., usually not found on healthy cells or highly upregulated on cancer cells), including, for example, CEA, 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, ErbB3, WT1, MUC1, LMP2, idiotype, HPV E6 and E7, EGFRvIII, HER-2 / neu, MAGE A3, NY-ESO-1, wild-type p53, mutant p53, NY-ESO-1, GD2, PSMA, PCSA, PSA, MelanA / MART1, Ras mutant, proteinase 3 (PR1), bcr-abl, tyrosinase, survivin, PSA, hTERT, especially 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 particular target sequence disclosed herein).

[0159] Other cancer antigens include epithelial tumor antigens, CA-125, and alpha-fetoprotein.

[0160] Antibodies targeting cancer include: avelumab, bevacizumab, brentuximab, cemiplimab, cetuximab, daratumumab, dinutuximab, elotuzumab, enfortumab, gemtuzumab, ibritumomab, inotuzumab, ipilimumab, isatuximab, mogamulizumab, moxetumomab, necitumumab, nivolumab, obinutuzumb, ofatumumab, olaratumab, panitumumab, pembrolizumab, pertuzumab, polatuzumab, ramucirumab, rituximab, Sacituzumab, tositumomab, trastuzumab, Fab-G8 and Fab-Hyb3 (targeting EADPTGHSY in MAGE A1); 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 in NY-ESO-1); 7D4, 8A11, 2G12 and 9E6 (targeting FLWGPRALV in MAGE3); RL4B / 3.2G1 and 1B10 (targeting GVLPALPQV in hCGβ); 3F9 (targeting TMTRVLQGV in hCGβ); 1B8 (targeting KIFGSLAFL in Her2 / Neu); CAG10 and CLA12 (targeting EAAGIGILTV in Melan-A / MARR-1); Fab-D2 (targeting FLRNFSLML in TARP); I3.M3-2A6 (targeting LLGRNSFEV in p53); T1-116C, T1-29D, and T1-84C (targeting RMPEAAPPV in p53); T2-108A, T2-2A, T2-116A (targeting GLAPPQHLIRV in p53); T2A (specific for YMDGTMSQV in tyrosinase); RL6A (specific for YLLPAIVHI in p68); RL21A (specific for FLSELTQQL in MIF); 8FA (specific for VLQELNVTV in protease 3); ESK1 F2, F3, and clone 45 (specific for RMFPNAPYL in WT1); #131 (specific for VLHDDLLEA in HA-1H); and Pr20 (specific for ALYVDSLFFL).

[0161] In one embodiment, constructs (such as antibodies or binding fragments thereof) encoded by a virus according to the present disclosure are human or humanized.

[0162] In one embodiment, an "immune checkpoint inhibitor antibody" or fragment is encoded within a virus of the present disclosure, such as anti-PD1, PDL1, CTLA4, LAG3, GITR, TIGIT, CD40.

[0163] In one embodiment, immunosuppression of the microenvironment is inhibited by intrinsic checkpoint blockade, such as inhibiting E3 ubiquitin-protein ligases (such as CBL-B) and / or cytokine-induced SH2-containing proteins (such as CISH).

[0164] Thus, in one embodiment, a virus according to the present disclosure encodes a checkpoint kinase inhibitor, such as selected from checkpoint kinase inhibitor 1 (CHEK1 / CHK1), checkpoint kinase inhibitor 2 (CHEK2 / CHK2), ataxia telangiectasia and Rad3 related (ATR) inhibitor, ataxia telangiectasia mutated (ATM) inhibitor, Wee1 dual-specificity protein kinase (Wee1) inhibitor, poly ADP-ribose polymerase (PARP) inhibitor, and Myt1 inhibitor, and combinations of two or more thereof, such as checkpoint kinase inhibitors, particularly CHK1 inhibitors.

[0165] 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 that is glycosidically linked to the C-terminal amino acid of the mature protein through a carbohydrate-containing linker (glucosamine and mannose glycosidically bound to the inositol residue) and through an ethanolamine phosphate (EtNP) bridge. Two fatty acids within the hydrophobic phosphatidylinositol group anchor the protein to the cell membrane.

[0166] Glycosylphosphatidylinositol (GPI)-anchored proteins contain a signal peptide that directs them into the endoplasmic reticulum (ER). The C-terminus consists of hydrophobic amino acids that insert into the ER membrane. The hydrophobic terminus is then cleaved off and replaced by a GPI anchor. As the protein is processed through the secretory pathway, it is transferred by vesicles to the Golgi apparatus and ultimately to the extracellular space, where it remains attached to the outer leaflet of the cell membrane. Since glycosylphosphatidylinositolation is the only way to attach such proteins to the membrane, cleavage of the group by phospholipase will result in the controlled release of the protein from the membrane. This latter mechanism is used in vitro; i.e., membrane proteins released from the membrane in enzymatic assays are glycosylphosphatidylinositolated proteins.

[0167] Phospholipase C (PLC) is an enzyme known to cleave the phosphoglycerol bond found in GPI-anchored proteins. Treatment with PLC will result in the release of GPI-linked proteins from the outer cell membrane. The T cell marker Thy-1 and acetylcholinesterase, as well as intestinal and placental alkaline phosphatases, are known to be GPI-linked and released by treatment with PLC. GPI-linked proteins are thought to be preferentially located in lipid rafts, indicating a high level of organization within the plasma membrane microdomains.

[0168] A review of GPI anchors by Ferguson, Kinoshita, and Hart can be found in Chapter 11 of the second edition of Essentials of Glycobiology.

[0169] In one embodiment, a combination of a transmembrane domain and a secretory signal sequence is used to express a protein encoded by a virus (such as 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 viral infection (i.e., cancer cells).

[0170] In one embodiment, fragments (such as transmembrane fragments) for expressing a protein on the surface of infected cancer cells are selected from the group consisting of the TM domain sequences (minimal portions) given in the following table.

[0171] SEQ ID NO: Name Sequence 100 PDGFR Receptor A AVLVLLVIVIISLIVLVVIW 101 PDGFR Receptor B VVISAILALVVLTIISLIILI 102 Insulin-like Growth Factor 1 IIIGPPLIFVFLFSVVIGSIYLFL 103 IL6-R SSSVPLPTFLVAGGSLAFGTLLCIAIVL 104 CD28 FWVLVVVGGVLACYSLLVTVAFIIFWV

[0172] The use of non-human sequences may be advantageous as it can increase cancer specificity, thereby increasing the therapeutic window and effectively reducing off-target effects.

[0173] In one embodiment, the cellular immunotherapy is a transgenic cell, particularly a CAR-T, engineered NK cell, and / or engineered NKT cell, more particularly a CAR-T.

[0174] As used herein, a genetically modified cell refers to an engineered cell, e.g., engineered using recombinant techniques to include a non-natural polynucleotide that modifies cell function, i.e., the cell is modified to express a synthetic receptor on its surface.

[0175] As used herein, a 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 generated by linking the variable regions of the heavy and light chains of a monoclonal antibody, but can also be generated with other antibody formats (e.g., camelid single-chain VHH antibodies) or other antigen / ligand binding proteins. The receptor binds to its specific antigen or ligand and stimulates a signaling pathway in the genetically modified cell.

[0176] In a CAR, signaling is intrinsic or integrated with the receptor. However, alternative technologies are being developed in which signaling is not physically linked to the synthetic receptor. All of these types are technologies suitable for use with the viruses of the present disclosure.

[0177] The CAR-T cell examples given below can be technically appropriately applied to other immunotherapy cells, including NK and NKT cells.

[0178] First-generation CAR-T cells typically have an intracellular signaling unit based on CD3-ζ. However, second-generation CARs typically have a co-stimulatory element, such as CD28, 4-1BB, CD136, CD137, or CD27, and ICOS built into the intracellular signaling domain, while third-generation CARs can contain more than one co-stimulatory element, such as CD28 plus 4-1BB (see, e.g., US7,446,190, 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).

[0179] 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 that is specific for a tumor or other target antigen. In such cases, the TCR recognizes an MHC / antigen peptide complex. These cells can be engineered in a manner similar to the CAR-Ts described herein.

[0180] Immunotherapy cells can also be engineered by techniques such as CRISPR / Cas9 to, for example, remove the expression of inhibitory proteins (such as PD1) and / or remove endogenous receptors such as endogenous TCR (to enhance target specificity).

[0181] Companies such as ProMab Biotechnologies, Inc. make these products commercially available.

[0182] Thus, in one embodiment, the CAR comprises a CD3ζ signaling unit. The following discloses first-generation CARs: Irving and Weiss, Cell, March 8, 1991; 64(5):891-901. Letourmeur, October 15, 1991; 88(20):8905-8909. Romeo, Cell, Vol. 68, No. 5, pp. 889-897, March 6, 1992.*

[0183] In one embodiment, the CAR comprises a CD28 signaling unit, see for example Maher et al., Nat Biotechnol, January 2002; 20(1); 70-75 and Carpenito et al., PNAS, March 3, 2009, 106(9):3360-3365.* In one embodiment, the CAR comprises a CD27 signaling unit. The latter makes an important contribution to 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 co-stimulator. In one embodiment, the CAR comprises 4 1BBs, see for example Imai 2004, Leukemia 18, 676-684.* In one embodiment, the CAR therapy comprises a co-stimulatory factor. In one embodiment, the CAR therapy comprises a combination of co-stimulatory factors, for example 2, 3 or 4, 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, which is incorporated herein by reference, discloses ICOS-based chimeric antigen receptors.* Duong, PLoS, May 7, 2013; 8(5) discloses engineering T cell function using chimeric antigen receptors.* A signaling unit as used herein is an element that contributes to the cellular signaling of the CAR. Chimeric T cell receptors are disclosed in US2004043401.*

[0184] *The structural features (in terms of signaling rather than specificity) of the CARs disclosed herein are incorporated by reference and can be used as a basis for amending the claims.

[0185] The binding domain of the CAR is antibody-like and can, for example, comprise a scFv, see, e.g., Kuwana et al., Biochem Biophys Res Commun., Dec. 31, 1987, 149(3); and Eshhar et al., Proc Natl Acad Sci USA, Jan. 15, 1993; 90(2):270-724. *Second-generation CARs.

[0186] In one embodiment, the binding domain of the CAR is specific for a hematopoietic cell antigen, such as CD19, CD30, CD123, FLT (including combinations such as CD19 and CD20 or CD22), which hematopoietic cell antigens are 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, disclose CAR-modified T cells in CLL. Grupp et al., N Engl J Med, Apr. 18, 2013; 368(16)1509-1518, disclose CAR-modified T cells for ALL. Maude et al., N Engl J Med 2014, 371:1507-1517, disclose CAR-T cells for durable remission in leukemia. Garfall et al., N Engl J Med 2015; 373:1040-1047, disclose CAR T cells directed against CD19 for multiple myeloma.

[0187] In one embodiment, the recombinant receptor (such as a CAR) is specific for a solid tumor and / or stromal tissue.

[0188] In one embodiment, the recombinant receptor (such as a CAR) is specific for a cancer antigen. Cancer antigens are as described above.

[0189] In one embodiment, the binding domain of the recombinant receptor (such as a CAR) targets abnormal sugars on the surface of cancer cells.

[0190] In one embodiment, the recombinant receptor (such as a CAR) is specific for a stromal antigen, as defined herein, for example.

[0191] In one embodiment, the recombinant receptor (such as a CAR) binding domain is specific for an antigen 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-3CD20, 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 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], 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 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.

[0192] In one embodiment, the engineered cell encodes at least two entities, such as a CD19 CAR and a PD-1 siRNA, a CD19 TIGIT siRNA, a BCMA-CS1, or a BCMA-CD33.

[0193] In one embodiment, the recombinant receptor (such as a CAR) is specific for:

[0194] CD19 (such as CD19 - CD28, CD19scFv - CD28 - CD3ζ, CD19scFv - 4 - 1BB - CD3ζ, CD19scfv - CD28 - 4 - 1BB, CD19scFv - CD28 - 4 - 1BB - CD3ζ or iCas9 - T2A - anti - CD19scFv - CD28 - CD3ζ, CD19FLAG - CD28 - - CD3ζ or iCas9 HA - T2A - anti - CD19scFv - CD28 - CD3ζ - GGGS - FLAG, humanized CD19 scFv - TM28 - CD28 - CD3ζ, CD19scFv - Beam - TM28 - CD28 - CD3ζ or humanized CD19 scFv - Beam - TM28 - CD28 - CD3ζ, CD19scFv - CD22 scFv - 4 - 1BB - CD3 - T2A - tEGFR or CD19 scFv - TM28 - GITR - CD3ζ or CD19scFv - TM8 - GITR - CD3ζ); Mesothelin (such as 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 (such as VGFR2 scFv - CD28 CD3ζ); GPC3 (such as GPC3 scFv - CD28 - CD3ζ); CD133 (CD133 scFv - CD28 - CD3ζ); EpCAM (such as EpCAMscFv - CD28 - CD3ζ, such as the version in which an Nhel restriction site is introduced, the N - terminus of the scFv amino acids); EGFR (such as EGFR scFv - CD28 - CD3ζ, EGFR scFv - 4 - 1BB - CD3ζ, EGFR scFv - TM28 - GITR - CD3ζ, scFv - TM28 - CD3ζ - GITR; CD33 (such as CD33 scFv - TM28 - CD28 - CD3ζ or CD33 scFv - Beam 2 - TM28 - CD28 - CD3ζ); CD38 (such as CD38 scFv - TM28 - CD28 - CD3ζ); CD138 (such as CD138 scFv - Beam - TM28 - CD28 - CD3ζ);CD22 (such as CD22 scFv-TM28-CD28 CD3ζ, -CD22 scFv-TM28-4-1BB CD3ζ or CD22 scFV-Beam-TM28-CD28-CD3ζ); BCMA (such as BCMA-4-CD28 CD3ζ or humanized BCMA-4scFv-TM8-4-1BB-CD3ζ or BCMA-2scFv-Tm-CD28-CD3ζ); HER2 (such as HER2scFv-CD28-CD3ζ, HER2 scFv-4-1-BB-CD3Z-EGFRt or HER scFV-4-1BB-CD3ζ-GFP); CD4 (such as CD4 scFv-Beam-TM28-CD28-CD3ζ); ROR-1 (such as ROR-1scFv TM28-CD28-CD3ζ, ROR-1scFv TM28-4-1BB-CD3ζ or humanized ROR-1scFvTM28-4-1BB-CD3ζ); CD19 and CD22 (such as CD19 scFv CD22 scFv-4-1BB-CD3ζ or CD19 scFv-CD22scFv-4-1BB-CD3-T2A-RQR8); CEA (such as CEA scFv-TM28-CD28 CD3ζ or humanized CEA scFv-TM28-CD28 CD3ζ; NGFR (such as NGFR scFv-TM28-CD28-CD3ζ); MCAM (such as MCAM scFv-TM28-CD28-CD3ζ); CD47 (such as CD47 scFv-TM28-Cd28-CD3ζ or humanized CD47 scFv-TM28-CD28-CD3ζ); PDL-1 (such as PDL-1scFV-TM28-CD28-CD3ζ); CD123 (such as CD123 scFv-TM28-CD28-CD3ζ); CD37 (such as CD37 scFv-TM28-CD28-CD3ζ, CD37scFv-TM28-4-1-BB-CD3ζ; CS1 (such as CS1 scFv-TM28-CD28-CD3ζ); B7H4 (such as B7H4scFv-TM28-CD28-CD3ζ); CD24 (such as CD24 scFv-TM28-CD28-CD3ζ) and CD20 (such as CD20 scFv-TM28-CD28-CD3ζ); NKG2D such as CYAD-01; FLT3, such as AMG 553; DLL3.;

[0195] 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.

[0196] In one embodiment, the CAR is provided in T cells (such as autologous T cells or allogeneic T cells, more specifically HLA-matched T cells). In one embodiment, the CAR-T cells are selected from: tisagenlecleucel, axicabtagene ciloleucel, lisocabtagene maraleucel, idecabtagene vicleucel, brexucabtagene autoleucel, JCAR015 (CD19 CAR T from Juno), Descartes-08 (BCMA), and AMG119.

[0197] In one embodiment, the immune cell is a phagocyte, for example encoding a recombinant receptor (such as a CAR) listed herein, such as a CD19 scFv-CAR or a mesothelin scFv CAR. In one embodiment, the phagocyte is a macrophage.

[0198] In one embodiment, the recombinant receptor, such as a CAR, is provided in NKT cells. The advantage of NKT cell vehicles is that they do not require HLA matching with the patient. Thus, they can be used to provide "off-the-shelf products", for example having 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.

[0199] In one embodiment, the immune cell is an NK cell, see for example Tran et al., J Immunol July 1995, 155(2); 1000-1009, which is incorporated herein by reference.

[0200] In one embodiment, the engineered cells are derived from pluripotent stem cells, such as iPSCs.

[0201] Thus, in one embodiment, the immune cell therapy further comprises a transgene (i.e., an engineered gene) encoding a cytokine, for example selected from IL-2, IL-5, IL-7, IL-12, and IL-15.

[0202] In one embodiment, the engineered cells do not contain a transgene encoding a cytokine.

[0203] In one embodiment, the engineered cells contain only a transgene that expresses a recombinant receptor, such as a CAR, although this does not exclude deleting certain wild-type genes if this is beneficial (e.g., to increase specificity).

[0204] In one embodiment, the immunotherapy does not contain a transgene (i.e., an engineered gene) encoding a cytokine, such as selected from IL-2, IL-5, IL-7, IL-12, and IL-15

[0205] Immune cells, such as T cells, NKT cells, can be activated by IL-15 expressed by the virus of the present invention or have an activity maintained by IL-15 expressed by the virus of the present invention. This may help counteract the non-responsive / hypoxic microenvironment of the tumor. The hypoxic microenvironment can have the ability to neutralize natural cells and even the killing power of the engineered cell therapy used in combination with the present invention. Thus, the use of the virus of the present disclosure can trigger several cancer-killing mechanisms, especially when used in combination with cell therapy.

[0206] As used herein, a therapeutic dose refers to the amount of a virus (such as an oncolytic adenovirus) that is suitable to achieve the desired therapeutic effect (e.g., to improve the symptoms or conditions of a disease, especially without dose-limiting toxicity) when used in a suitable treatment regimen. A dose can be considered a therapeutic dose for treating cancer or metastasis when the number of virus particles is sufficient to cause the growth of the tumor or metastasis to slow down or stop, or the size of the tumor or metastasis is found to shrink, and / or the lifespan of the patient is extended. Infection of cancer cells after systemic delivery of the virus of the present disclosure is an indication of a therapeutic dose, i.e., it has been delivered to the target cells. A suitable therapeutic dose is generally a balance between the therapeutic effect and tolerable toxicity, e.g., considering the benefits obtained from the therapy, the side effects and toxicity are tolerable.

[0207] In one embodiment, the virus or therapeutic construct according to the present disclosure (including the formulation containing it) is administered once every 3 to 4 weeks, e.g., a dose is administered on days 1, 3, and 5 of the first week, and then further multiple doses are administered 3 to 4 weeks later.

[0208] In one embodiment, the virus or therapeutic construct according to the present disclosure (including the formulation containing it) is administered every two weeks or every three weeks, e.g., on days 1, 3, and 5 of the first week, and also on days 1, 3, and 5 of the third or fourth week. This dosing regimen can be repeated appropriately multiple times.

[0209] In one embodiment, the first dose is lower than subsequent doses, e.g., the first dose is in the range of 1x10 11 to 1x10 12 virus particles, and subsequent doses are in the range of 1x10 12 to 1x10 13 virus particles.

[0210] In one embodiment, six doses are administered over a two-week period, such as on days 1, 3, 5, 8, 10, and 12, such that each dose can be administered within + / - 1 day, including where the dose on day 1 is lower than the other doses.

[0211] In one embodiment, the virus or therapeutic construct according to the present disclosure (including formulations comprising the same) is administered monthly.

[0212] In one embodiment, a first dose of the virus is administered prior to treatment with immunocyte therapy, such as 7 to 28 days prior to the cell therapy.

[0213] In one embodiment, the viruses and constructs of the present disclosure are prepared by recombinant techniques. Those skilled in the art will understand that an armed adenovirus genome can be manufactured by other technical means, including a fully synthetic genome or a plasmid containing a portion of all genomes. Those skilled in the art will understand that in the case of a synthetic genome, the inserted region may not contain restriction site nucleotides, as the latter are an artifact after inserting genes using cloning methods.

[0214] The disclosure herein further extends to adenoviruses of formula (I) or sub-formulae thereof, which are obtained or obtainable by inserting a transgene or transgene cassette.

[0215] As used herein, "comprises" means includes.

[0216] In the context of the present specification, "comprising" shall be construed as "including".

[0217] Embodiments of the invention comprising certain features / elements are also intended to extend to alternative embodiments "consisting of" or "consisting essentially of" the relevant elements / features.

[0218] Where technically appropriate, embodiments of the invention may be combined.

[0219] Technical references such as patents and applications are incorporated herein by reference.

[0220] Any embodiment specifically and expressly recited herein may form the basis of a disclaimer, either alone or in combination with one or more additional embodiments.

[0221] The background section contains technically relevant details that can be used as a basis for amendment. BRIEF DESCRIPTION OF THE DRAWINGS

[0222] Figure 1 Schematic diagram of a transgene cassette encoding a series of bispecific proteins and membrane-anchored antigens (with or without additional transgenes)

[0223] Figure 2The figure shows the design of bispecific proteins and how they recognize tumor cells through a cell therapy agent (taking CAR-T cells as an example) and enhance the activity of cell therapy through a transporter

[0224] Figure 3 Screen the ability of NG-1100 - 1126 designs to bind to A549 tumor cells and present cell therapy ligands in vitro. A. Expression of transmembrane CD19 on A549 cells after transfection of NG-1108 with pUC57 plasmid; B. Supernatants from A549 cells transfected with pUC57 plasmid of NG-1100, NG-1101, NG-1102, NG-1106, NG-1107 or control pUC57 vector were incubated with fresh A549 cells, and the binding of bispecific proteins was measured by flow cytometry to detect the C-terminal 2A peptide tag; C to E. Supernatants from A549 cells transfected with pUC57 plasmid of NG-1109 to NG-1118 or control plasmid were incubated with fresh A549 cells, and the binding of bispecific proteins to EpCAM, i.e., the reduction in binding of AF647-labeled anti-EpCAM antibody, was measured using flow cytometry; F. Supernatants from A549 cells transfected with pUC57 plasmid of NG-1106, NG-1107, NG-1109, NG-1110, NG-1113 to NG-1121 or NG-1123 to NG-1126 or control plasmid were incubated with fresh A549 cells, and the binding of bispecific proteins was measured by detecting CD19 by flow cytometry

[0225] Figure 4 A and B. Binding of anti-HER2ScFv-CD19 fusion protein to A549 and SKOV3 tumor cells after incubation with the infection supernatant. A549 tumor cells were infected with EnAd or NG-1124, and the infection supernatant was collected and incubated with uninfected A549 (A) or SKOV3 (B), and then the CD19 expression of the cells was analyzed by flow cytometry. C. Transmembrane CD19 expression 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, and then the CD19 expression of the two groups of cells was analyzed by flow cytometry

[0226] Figure 5In the co-culture system, binding of virus-encoded bispecific proteins secreted by infected A549 tumor cells to uninfected SKOV3 cells. A and B. A549 cell cultures, SKOV3 cell cultures, and A549+SKOV3 cell co-cultures were infected with EnAd or NG-1100, and binding of the anti-EpCAM–RAE1TG3 fusion protein to the surface of SKOV3 (A) or A549 (B) cells was assessed by flow cytometry for the C-terminal 2A peptide tag peptide. C and D. A549 cell cultures, SKOV3 cell cultures, and A549+SKOV3 cell co-cultures were infected with EnAd or NG-1124, and binding of the anti-HER2-CD19 fusion protein to the surface of SKOV3 (C) or A549 (D) cells was assessed by flow cytometry for the CD19 antigen.

[0227] Figure 6 Anti-CD19 CAR-T cell-mediated cytotoxicity against SKOV3 tumor cells in the presence of cell culture supernatants (SN) from NG-1124-infected A549 cells. SKOV3 cells were incubated in the presence of cell culture supernatants from A549 cells infected with EnAd (A) or NG-1124 (B), and then anti-CD19 CAR or control (Ctrl) T cells were added, and tumor cell death was monitored in real time.

[0228] Figure 7 Anti-CD19 CAR-T-mediated cytotoxicity against A549 tumor cells in the presence of cell culture supernatants from NG-1124-infected cells. A549 cells were infected with NG-1124 or EnAd, and virus particles were removed from the culture supernatants by exclusion size column filtration before testing. A. Levels of the anti-HER2-CD19 bispecific protein were shown to be similar before and after removal of virus particles. B to D. A549 cells were incubated in the presence of these virus-depleted culture supernatants (VR SN), and then anti-CD19 CAR or control (Ctrl) T cells were added, and tumor cell death was monitored in real time.

[0229] Figure 8 A. Effects of encoding multiple transgenes on bispecific protein expression levels and binding to tumor cells. B. A549 tumor cells infected with NG-1101, NG-1104, and NG-1125 expressed CXCL9, CCL21, IFNα, and IL-15 transgene proteins.

[0230] Figure 9In vivo expression and tumor cell binding of NG-1125-encoded anti-HER2-CD19 fusion protein. A. After viral intracellular staining, cells were gated by flow cytometry and separated into negative (uninfected) or cells with low (low VP) or high (high VP) infection levels. B. Frequencies of tumor cells remaining uninfected or having low VP or high VP after administration of EnAd or NG-1125IV. C. Levels of cell surface-bound anti-HER2-CD19 on subsets of A549 tumor cells that were uninfected, low VP, or high VP in tumor masses after administration of EnAd or NG-1125IV.

[0231] Figure 10 In vivo demonstration of enhanced CAR-T cell activity in A549 xenograft tumors after administration of NG-1125IV expressing an anti-HER2-CD19 bispecific protein plus two additional transgenes, compared to that observed with NG-1124 or EnAd expressing only the anti-HER2-CD19 protein. Excised tumors were processed into single-cell suspensions and total human CD45 was analyzed by flow cytometry + cells (A), activated cytotoxic human CD8 + CD107a + cells (B), or activated cytotoxic human CD4 + CD107a + cells (C) for their presence.

[0232] Figure 11 Shows the general structure of a chimeric antigen receptor

[0233] Figure 12 In vivo demonstration of enhanced CAR-T cell recruitment and activation in A549 xenograft tumors after administration of NG-1125 (expressing an anti-HER2-CD19 bispecific protein plus CXCL9 and IFNα), NG-641 (expressing CXCL9, CXCL10, IFNα, and an irrelevant bispecific protein), or EnAd IV. Excised tumors were processed into single-cell suspensions and total human CD45 was analyzed by flow cytometry + cells (corresponding to transferred T cells) (A), activated cytotoxic human CD45 + CD107a + cells (B), or activated human CD45 + CD25 + cells (C) for their presence. Blood was also processed and assayed for the presence of circulating human CD45 + cells (D).

[0234] Sequence Listing

[0235] SEQ ID NO:1 Short Splice Acceptor (SSA) sequence (CAGG)

[0236] SEQ ID NO:2 Splice Acceptor sequence

[0237] SEQ ID NO:3 Splice Acceptor sequence

[0238] SEQ ID NO:4 Polyadenylation (PA) sequence (SV40 late polyA sequence)

[0239] SEQ ID NO:5 Highly efficient self-cleaving T2A peptide sequence

[0240] SEQ ID NO:6 Highly efficient self-cleaving E2A peptide sequence

[0241] SEQ ID NO:7 Highly efficient self-cleaving F2A peptide sequence

[0242] SEQ ID NO:8 Highly efficient self-cleaving P2A peptide sequence

[0243] SEQ ID NO:9 Flexible linker (FL)

[0244] SEQ ID NO:10 Rigid linker (RL)

[0245] SEQ ID NO:11 Myc peptide tag

[0246] SEQ ID NO:12 His tag

[0247] SEQ ID NO:13 Human CXCL9

[0248] SEQ ID NO:14 Human CXCL9-T2A

[0249] SEQ ID NO:15 Human CXCL9-P2A

[0250] SEQ ID NO:16 Human CXCL9-E2A

[0251] SEQ ID NO:17 Human CCL21 with N-terminal signal peptide

[0252] SEQ ID NO:18 Human CCL21 with N-terminal signal peptide and C-terminal E2A peptide

[0253] SEQ ID NO:19 Human IL-15Ra sushi domain with signal sequence

[0254] SEQ ID NO:20 Human IL-15Ra sushi domain with signal sequence and C-terminal P2A peptide

[0255] Human IL-15 with immunoglobulin leader sequence, SEQ ID NO:21

[0256] (Gly4Ser)3 linker (G4S), SEQ ID NO:22

[0257] Short Gly4Ser spacer (sG4S), SEQ ID NO:23

[0258] Long (Gly4Ser)4 linker (G4S4), SEQ ID NO:24

[0259] Human CD19 (extracellular domain), SEQ ID NO:25

[0260] Human CD19m1 (extracellular domain, mutated), SEQ ID NO:26

[0261] Human CD19m2 (extracellular domain, mutated), SEQ ID NO:27

[0262] Protein sequence encoding the anti-EpCAM-ScFv-OKT3-ScFv fusion protein (without C-terminal tag) in NG-611, including the leader sequence, SEQ ID NO:28

[0263] Protein sequence encoding the anti-EpCAM-ScFv-RAET1G3 fusion protein with F2A C-terminal tag in NG-1100, NG-1101, including the leader sequence, SEQ ID NO:29

[0264] Protein sequence encoding the RAET1G3-anti-EpCAM-ScFv fusion protein with F2A C-terminal tag in NG-1102, including the leader sequence, SEQ ID NO:30

[0265] Protein sequence encoding the anti-EpCAM-ScFv-OKT3-ScFv fusion protein with F2A C-terminal tag in NG-1104, including the leader sequence, SEQ ID NO:31

[0266] Protein sequence encoding the anti-EpCAM-ScFv-human CD19 EC domain fusion protein with P2A C-terminal tag in NG-1106, NG-1119, including the leader sequence, SEQ ID NO:32

[0267] Protein sequence encoding the human CD19 EC domain-anti-EpCAM-ScFv fusion protein with P2A C-terminal tag in NG-1107, including the leader sequence, SEQ ID NO:33

[0268] The protein sequence of transmembrane human CD19 with a short cytoplasmic tail and a P2A C-terminal tag encoded by SEQ ID NO:34 in NG-1108, including the leading sequence

[0269] The protein sequence of anti-EpCAM-ScFv-human CD19 EC domain fusion protein (without C-terminal tag) encoded by SEQ ID NO:35 in NG-1109, including the leading sequence

[0270] The protein sequence of human CD19 EC domain-anti-EpCAM-ScFv fusion protein (without C-terminal tag) encoded by SEQ ID NO:36 in NG-1110, including the leading sequence

[0271] The protein sequence of anti-EpCAM-ScFv-RAET1G3 fusion protein (without C-terminal tag) encoded by SEQ ID NO:37 in NG-1111, including the leading sequence

[0272] The protein sequence of RAET1G3-anti-EpCAM-ScFv fusion protein (without C-terminal tag) encoded by SEQ ID NO:38 in NG-1112, including the leading sequence

[0273] The protein sequence of anti-EpCAM-ScFv-flexible linker-human CD19 EC domain fusion protein (without C-terminal tag) encoded by SEQ ID NO:39 in NG-1113, NG-1115, including the leading sequence

[0274] The protein sequence of anti-EpCAM-ScFv-rigid linker-human CD19 EC domain fusion protein (without C-terminal tag) encoded by SEQ ID NO:40 in NG-1114, NG-1116, including the leading sequence

[0275] The protein sequence of anti-EpCAM-ScFv-flexible linker-RAET1G3 fusion protein (without C-terminal tag) encoded by SEQ ID NO:41 in NG-1117, including the leading sequence

[0276] The protein sequence of anti-EpCAM-ScFv-rigid linker-RAET1G3 fusion protein (without C-terminal tag) encoded by SEQ ID NO:42 in NG-1118, including the leading sequence

[0277] The protein sequence of anti-EpCAM-ScFv-human CD19m1EC domain fusion protein with a P2A C-terminal tag encoded by SEQ ID NO:43 in NG-1120, including the leading sequence

[0278] The protein sequence of the human CD19 EC domain - anti - HER2 - ScFv fusion protein with a C - terminal P2A tag encoded by SEQ ID NO:44 in NG - 1121, including the leading sequence

[0279] The protein sequence of the human CD19 EC domain - anti - HER2 - ScFv fusion protein with a C - terminal Myc tag encoded by SEQ ID NO:45 in NG - 1122, including the leading sequence

[0280] The protein sequence of the human CD19m2 EC domain - anti - HER2 - ScFv fusion protein with a C - terminal P2A tag encoded by SEQ ID NO:46 in NG - 1123, including the leading sequence

[0281] The protein sequence of the anti - HER2 - ScFv - CD19m1 fusion protein with a C - terminal P2A tag encoded by SEQ ID NO:47 in NG - 1124, NG - 1125, including the leading sequence

[0282] The protein sequence of the anti - HER2 - ScFv - CD19 fusion protein with a C - terminal P2A tag encoded by SEQ ID NO:48 in NG - 1126, including the leading sequence

[0283] The protein sequence encoded by the NG - 611 transgenic cassette

[0284] The protein sequence encoded by the NG - 1100 transgenic cassette

[0285] The protein sequence encoded by the NG - 1101 transgenic cassette

[0286] The protein sequence encoded by the NG - 1102 transgenic cassette

[0287] The protein sequence encoded by the NG - 1104 transgenic cassette

[0288] The protein sequence encoded by the NG - 1106 transgenic cassette

[0289] The protein sequence encoded by the NG - 1107 transgenic cassette

[0290] The protein sequence encoded by the NG - 1108 transgenic cassette

[0291] Protein sequence encoded by the NG-1109 transgene cassette, SEQ ID NO:57

[0292] Protein sequence encoded by the NG-1110 transgene cassette, SEQ ID NO:58

[0293] Protein sequence encoded by the NG-1111 transgene cassette, SEQ ID NO:59

[0294] Protein sequence encoded by the NG-1112 transgene cassette, SEQ ID NO:60

[0295] Protein sequence encoded by the NG-1113 transgene cassette, SEQ ID NO:61

[0296] Protein sequence encoded by the NG-1114 transgene cassette, SEQ ID NO:62

[0297] Protein sequence encoded by the NG-1115 transgene cassette, SEQ ID NO:63

[0298] Protein sequence encoded by the NG-1116 transgene cassette, SEQ ID NO:64

[0299] Protein sequence encoded by the NG-1117 transgene cassette, SEQ ID NO:65

[0300] Protein sequence encoded by the NG-1118 transgene cassette, SEQ ID NO:66

[0301] Protein sequence encoded by the NG-1119 transgene cassette, SEQ ID NO:67

[0302] Protein sequence encoded by the NG-1120 transgene cassette, SEQ ID NO:68

[0303] Protein sequence encoded by the NG-1121 transgene cassette, SEQ ID NO:69

[0304] Protein sequence encoded by the NG-1122 transgene cassette, SEQ ID NO:70

[0305] Protein sequence encoded by the NG-1123 transgene cassette, SEQ ID NO:71

[0306] Protein sequence encoded by the NG-1124 transgene cassette, SEQ ID NO:72

[0307] Protein sequence encoded by the NG-1125 transgene cassette, SEQ ID NO:73

[0308] Protein sequence encoded by the NG-1126 transgene cassette, SEQ ID NO:74

[0309] Genomic sequence of the NG-1100 virus, SEQ ID NO:75

[0310] Genomic sequence of the NG-1101 virus, SEQ ID NO:76

[0311] Genomic sequence of the NG-1102 virus, SEQ ID NO:77

[0312] Genomic sequence of the NG-1104 virus, SEQ ID NO:78

[0313] Genomic sequence of the NG-1106 virus, SEQ ID NO:79

[0314] Genomic sequence of the NG-1107 virus, SEQ ID NO:80

[0315] Genomic sequence of the NG-1108 virus, SEQ ID NO:81

[0316] DNA sequence of the pUC57 CMV expression plasmid containing the transgene cassette insert of NG-1109, SEQ ID NO:82

[0317] DNA sequence of the pUC57 CMV expression plasmid containing the transgene cassette insert of NG-1110, SEQ ID NO:83

[0318] DNA sequence of the pUC57 CMV expression plasmid containing the transgene cassette insert of NG-1111, SEQ ID NO:84

[0319] DNA sequence of the pUC57 CMV expression plasmid containing the transgene cassette insert of NG-1112, SEQ ID NO:85

[0320] DNA sequence of the pUC57 CMV expression plasmid containing the transgene cassette insert of NG-1113, SEQ ID NO:86

[0321] DNA sequence of the pUC57 CMV expression plasmid containing the transgene cassette insert of NG-1114, SEQ ID NO:87

[0322] DNA sequence of the pUC57 CMV expression plasmid containing the transgene cassette insert of NG-1115, SEQ ID NO:88

[0323] DNA sequence of the pUC57 CMV expression plasmid containing the transgenic cassette insert of NG-1116, SEQ ID NO:89

[0324] DNA sequence of the pUC57 CMV expression plasmid containing the transgenic cassette insert of NG-1117, SEQ ID NO:90

[0325] DNA sequence of the pUC57 CMV expression plasmid containing the transgenic cassette insert of NG-1118, SEQ ID NO:91

[0326] DNA sequence of the pUC57 CMV expression plasmid containing the transgenic cassette insert of NG-1119, SEQ ID NO:92

[0327] DNA sequence of the pUC57 CMV expression plasmid containing the transgenic cassette insert of NG-1120, SEQ ID NO:93

[0328] DNA sequence of the pUC57 CMV expression plasmid containing the transgenic cassette insert of NG-1121, SEQ ID NO:94

[0329] DNA sequence of the pUC57 CMV expression plasmid containing the transgenic cassette insert of NG-1122, SEQ ID NO:95

[0330] DNA sequence of the pUC57 CMV expression plasmid containing the transgenic cassette insert of NG-1123, SEQ ID NO:96

[0331] Genomic sequence of the NG-1124 virus, SEQ ID NO:97

[0332] Genomic sequence of the NG-1125 virus, SEQ ID NO:98

[0333] DNA sequence of the pUC57 CMV expression plasmid containing the transgenic cassette insert of NG-1126, SEQ ID NO:99

[0334] Transmembrane protein sequence from human PDGFR receptor A, SEQ ID NO:100

[0335] Transmembrane protein sequence from human PDGFR receptor B, SEQ ID NO:101

[0336] Transmembrane protein sequence from human insulin-like growth factor 1, SEQ ID NO:102

[0337] Transmembrane protein sequence from human IL-6R, SEQ ID NO:103

[0338] SEQ ID NO: 104 Transmembrane protein sequence from human CD28

[0339] Examples

[0340] Example 1: Generation of expression plasmids and viruses encoding bispecific proteins comprising a tumor cell binding antibody fused to a target ligand for CAR / TCR or other cell therapies

[0341] A series of transgenic cassette designs were initially synthesized as pUC57 plasmids, in which expression of the transgenic protein was under the control of the CMV promoter, enabling preliminary studies of different bispecific proteins and transgenic combinations to be evaluated by transient transfection experiments. In each transgenic cassette, the cDNA encoding the protein sequence is flanked by a short splice acceptor sequence (SSA, SEQUENCE ID NO: 1) at the 5' end. At the 3' end of the protein sequence, the SV40 late poly (A) sequence (PA, SEQUENCE ID NO: 4) is encoded. In viruses with more than one encoding transgene, a single sequence is linked to a 2A ribosomal skipping sequence (T2A, E2A, F2A or P2A) (SEQ ID NO: 5 to 8) to enable each individual protein to be translated and produced as a separate chain. In some designs, the 2A sequence is also added to the final transgene in the cassette as an epitope tag for analytical purposes. Schematic diagrams of different transgenic cassettes are shown in Figure 1 are listed in Table 1.

[0342] Table 1

[0343]

[0344]

[0345] 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; 22 SEQ ID NO.43; 23 SEQ ID NO.44; 24 SEQ ID NO.46; 25 SEQ ID NO.47; 26 SEQ ID NO.48;

[0346] Virus production

[0347] Plasmid pColoAd2.4 was used to generate different viral vectors by restriction enzyme digestion through direct insertion of the transgene cassette sequences taken from the pU57 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.

[0348] To generate the virus, the plasmid was linearized by restriction digestion with AscI enzyme to generate the viral genome. The virus was amplified and purified according to the method given below.

[0349] The digested DNA was purified by phenol / chloroform extraction and precipitated at -20 °C for 16 ± 2 hours in 600 μl of 95% molecular biology grade ethanol and 15 μl of 3M sodium acetate. The precipitated DNA was pelleted by centrifugation at 13000 rpm for 5 minutes and washed twice in 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. Then the transfection mixture was added dropwise to a T-25 flask containing HEK-293 cells grown to 70% confluence. After incubating the cells with the transfection mixture at 37 °C for about 2 hours, 4 ml of cell medium (DMEM high glucose with glutamine supplemented with 2% FBS) was added to the cells, and the flask was incubated at 37 °C, 5% CO2.

[0350] Transfected HEK-293 cells were monitored every 24 hours and additional medium was supplemented as needed. Viral production was monitored by observing significant cytopathic effect (CPE) in the cell monolayer. Once extensive CPE was observed, the virus was harvested from HEK-293 cells by three freeze-thaw cycles. The harvested virus was used to reinfect HEK-293 cells to amplify the virus stock. Live virus production during the amplification process was confirmed by observing significant CPE in the cell monolayer. Once CPE was observed, the virus was harvested from HEK-293 cells by three freeze-thaw cycles. The amplified virus stock was used for further amplification and then the virus was purified by density gradient centrifugation to produce a purified virus stock.

[0351] Example 2: Screening the ability of bispecific protein designs to bind to tumor cells in vitro.

[0352] To verify the correct folding of bispecific proteins prior to generating viral vectors encoding the bispecific proteins, transgenic cassette designs NG-1100 to NG-1126 were created as pUC57 plasmid DNA vectors. These plasmids were transfected into A549 human lung tumor cells and the cells were cultured for 72 hours. Cell culture supernatant (SN) containing the secreted bispecific protein was collected and added to freshly suspended untransfected A549 cells and incubated at room temperature for 1.5 hours. During this period, the bispecific protein present in the SN could bind to A549 cells via its anti-EpCAM ScFv or anti-HER2 ScFV moiety. Binding to A549 was detected by flow cytometry by: a) decreasing the mean fluorescence intensity (MFI) of EpCAM staining (due to competitive binding between anti-EpCAM ScFv and anti-EpCAM antibody clone 9C4); b) for designs that have a 2A peptide tag, detecting the 2A peptide tag using an anti-P2A antibody. For designs that use CD19 as the CAR-T target, the extracellular domain portion of the protein's CD19 was detected by flow cytometry using an anti-CD19 antibody. For all of these readouts, a background level of signal intensity was established using SN from cells transfected with a control plasmid encoding an unrelated protein.

[0353] We first tested the design encoding the transmembrane CD19 target protein NG-1108. To verify transmembrane CD19 expression, A549 cells were transfected with a pUC57 plasmid encoding the NG-1108 transgenic cassette (SEQ ID NO:56) or a negative control plasmid and the transfected cells were stained with an anti-CD19 antibody by flow cytometry. High levels of transmembrane CD19 expression were detected on pUC-1108 transfected A549 compared to the control ( Figure 3 A)

[0354] Data from a series of experiments performed with different sets of plasmids (Figure 3 (B to 3F) indicate that different secreted bispecific protein designs can bind to the surface of tumor cells. The data indicate that a bispecific protein design with an anti-EpCAM ScFv as the tumor cell-binding component functions only when the ScFv antibody portion is placed at the N-terminus of the antigen (RAET1G3 or CD19), but not when placed at the C-terminus, while the anti-Her2 ScFv antibody can function at the N-terminal or C-terminal positions.

[0355] Example 3: Virally encoded bispecific proteins spread from infected tumor cells to uninfected tumor cells.

[0356] We hypothesized that when a secreted bispecific protein is encoded by a viral vector, the secreted protein will be able to bind to both infected and nearby uninfected tumor cells, thereby spreading the target antigen in the microenvironment. To demonstrate this in vitro, two different cell culture systems were used. In the first model, A549 cells were infected with 0.1 or 1 ppc of the EnAd or NG-1124 (SEQ ID NO:97) viral vector for 7 days or with 10 or 50 ppc for 3 days, and the infected supernatant was collected and incubated with uninfected A549 or SKOV3 cells for 1.5 hours, and then the CD19 expression of the cells 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 on SKOV3 and A549 under all tested conditions ( Figure 4 A and 4B). In contrast, when CD19 was encoded in the virus as a transmembrane protein rather than as part of a bispecific soluble molecule (such as NG-1108), CD19 expression was expected to be detected only in infected cells and not transferred to uninfected cells. Consistently, we found that CD19 was expressed on the membrane of A549 cells infected with 1 ppc of NG-1108 (SEQ ID NO:81) after 3 days of infection. However, when the infected supernatant from these cells was transferred and incubated with uninfected A549 cells, no CD19 was detected on the latter ( Figure 4 C).

[0357] In the second model, cell types that readily permit viral replication and transgene expression during a short culture period (notably A549 lung tumor cells), and cell lines that hardly permit viral replication and transgene expression within this time frame (notably SKOV3 ovarian cancer cells), were cultured and infected either alone or in co-cultures containing both cell types. Prior to the assay, both cell lines were shown to express the EpCAM and HER2 cell surface target antigens. SKOV3 cells were labeled with Cell Trace Violet dye to enable their differentiation from A549 cells using flow cytometry. Cells were infected with 10 ppc of the EnAd, NG-1100 (SEQ ID NO:75) or NG-1124 (SEQ ID NO:97) viral vectors, and harvested after 3 days and analyzed by flow cytometry to quantify the bispecific protein bound to each cell type based on P2A tag or CD19 detection. After NG-1100 infection, when cultured and infected alone, SKOV3 did not show any P2A signal above background (EnAd infection) because the virus was unable to produce sufficient transgene protein in this cell type at the time of the assay ( Figure 5 A). However, when co-cultured with A549, approximately 13% of SKOV3 cells were positive for 2A staining, indicating transfer and binding of the bispecific protein produced by A549 cells present in the culture ( Figure 5 A). As a control, due to the strong viral replication ability of A549, A549 could produce the bispecific protein and show positive P2A staining even when cultured without SKOV3 ( Figure 5 B). In a similar experiment, it was shown that SKOV3 cells did not produce or secrete any bispecific protein after NG-1124 (SEQ ID NO:97) infection. However, when co-cultured with A549 cells, SKOV3 cells could bind to the bispecific construct (SEQ ID NO:47) secreted by A549 cells, resulting in approximately 100% CD19 positivity of SKOV3 cells ( Figure 5 C). As a control, A549 could produce and bind the bispecific protein and show a positive CD19 signal both when cultured alone and in co-culture with SKOV3 cells ( Figure 5 D).

[0358] Example 4: Anti-CD19 CAR-T mediated cytotoxicity against SKOV3 and A549 tumor cells in the presence of cell culture supernatants from NG-1124 infected cells.

[0359] The ability of the anti-HER2 ScFv-CD19m1 fusion protein (SEQ ID NO: 47) encoded by NG-1124 (SEQ ID NO: 97) to engage anti-CD19 CAR-T cells and direct their cytotoxic activity against HER2-expressing tumor cells was assessed by real-time cytotoxicity assay (RTCA) using the SKOV3 or A549 cell lines as target cells. In the assay using the SKOV3 cell line, cells were incubated for 3 days in the presence of cell culture supernatant (diluted 1:10 in medium) from A549 cells infected with 10 ppc of NG-1124 or EnAd. 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 the xCELLigence instrument. Control conditions using medium containing 4% Tween20 (100% lysis control) were set for complete tumor cell death. In the presence of SN from NG-1124 infection (but not from EnAd infection), addition of anti-CD19 CAR-T cells to SKOV3 target cells resulted in complete killing of the target cells within less than 24 hours after addition of CAR-T ( Figure 6 A and 6B). As a control, control T cells pre-activated non-specifically with anti-CD3 / anti-CD28 antibodies in the presence of NG-1124 infection SN did not show any detectable cytotoxicity ( Figure 6 A and 6B).

[0360] Since A549 tumor cells are highly sensitive to adenovirus replication and adenovirus-induced oncolysis, to assess the specific cytotoxicity of CAR-T against these cells without any confounding virus-related cell death, virus-reduced cell culture supernatant (VR SN) was generated for testing in the CAR-T cell-A549 RTCA assay. To generate VR SN, we infected A549 cells with 0.1 ppc of NG-1124 or EnAd for 7 days, collected cell culture SN and filtered them through a 300 KDa size exclusion column to separate virus particles from the remaining SN containing the bispecific protein. The effect of the filtration process on the anti-HER2-CD19 protein content in the SN was minimal, as assessed by incubating SN with A549 cells before and after virus removal and detecting CD19 expression ( Figure 7A). To assess the ability of the secreted anti-HER2-CD19 bispecific protein to engage anti-CD19 CAR-T cells against tumor cells, A549 cells were incubated with VR SN for 1.5 hours and then CAR-T cells or control T cells were added at a T cell:tumor cell ratio of 3:1, and tumor cell cytotoxicity was monitored by RTCA. Complete tumor cell death was defined as 100% lysis of the control in the presence of 4% Tween20. Complete death of A549 cells was observed within less than 24 hours after addition of CAR-T when the cells were incubated in the presence of NG-1124 VR SN containing the anti-Her2 ScFv-CD19 protein, but not in the presence of EnAd VR SN or normal cell culture medium ( Figure 7 B to 7D). Control-activated T cells (generated as Figure 5 described) showed low levels of non-specific killing activity, independent of the presence or type of added viral SN.

[0361] These RTCA-based assays demonstrated that the anti-Her2 ScFv-CD19 m1 bispecific construct (SEQ ID NO:47) encoded by the NG-1124 adenovirus (SEQ ID NO:97) was functional and able to redirect the cytotoxic activity of anti-CD19 CAR-T cells against HER2+ tumor cells that do not endogenously express CD19.

[0362] Example 5: Effects of encoding multiple transgenes on bispecific protein activity. To maximize the ability of adenovirus to synergistically enhance the efficacy of cell therapy, a series of viruses encoding CAR-T cell-targeting bispecific proteins and additional transgenes (such as chemokines, cytokines, and other immunomodulators) were generated, and the transgenes could provide additional signals to promote the activity of cell therapy (such as CAR or TCR T cell therapy). To verify that these more complex designs could still effectively express bispecific proteins, A549 cells were infected with various transgene-encoding NG viruses or EnAds at 0.1 ppc for 7 days. SNs were collected 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 from patent application WO2019 / 043020), NG-1104 (SEQ ID NO:78), or EnAd and incubated with uninfected A549 cells for 1.5 hours, and then the presence of cell-bound bispecific proteins was determined by flow cytometry for the P2A tag, CD19 antigen, or OKT3 ScFv according to the virus design. For each staining, an anti-2A peptide antibody, an anti-CD19 antibody, or an anti-OKT3 ScFV antibody was used, and when fluorescence signals were measured above background fluorescence in cells incubated with SNs from EnAd-infected cells, the fluorescence signals were considered positive. Surprisingly, we found that adding two or more transgenes to the same transgene cassette encoding a bispecific protein led to a significant increase in bispecific protein expression compared to the corresponding virus designs encoding only the bispecific protein. Specifically: I) Compared with approximately 15% of the cells incubated with NG-1100 SN, approximately 45% of the A549 cells incubated with SNs from NG-1101 (SEQ ID NO:76) infection were positive for the 2A tag peptide (indicating the binding of the anti-EpCAM ScFv-RAET1G3 protein); II) Compared with approximately 7.5% of the cells incubated with NG-611 SN, approximately 12% of the A549 cells incubated with SNs from NG-1104 (SEQ ID NO:78) infection showed a positive signal for anti-OKT3 ScFv (representing the binding of the anti-EpCAM-OKT3 ScFv protein); III) Compared with approximately 20% of the cells incubated with NG-1124 SN, approximately 55% of the A549 cells incubated with SNs from NG-1125 (SEQ ID NO:98) infection were positive for CD19 staining (indicating the binding of the anti-HER2-CD19m1 protein)( Figure 8A). Production of other encoded transgenes (CXCL9, CCL21, IFNa, and IL-15) was demonstrated by specific ELISA assays after treatment with NG-1101, NG-1104, or NG-1125 Figure 8 B).

[0363] Example 6: In vivo binding of the NG-1125-encoded anti-HER2 ScFv-CD19 bispecific protein in A549 tumors. To study the efficiency of in vivo production and tumor cell binding of the anti-HER2 ScFv-CD19m1 bispecific protein, 5x10 9 A549 lung tumor cells were subcutaneously inoculated into NSG mice to generate xenografts, and when the tumors reached approximately 100 to 200 mm 3 , 5x10 9 virus particles (VPs) of EnAd or NG-1125 (SEQ ID NO:98) were administered intravenously to the mice (administered on days 0 and 3). Mice were euthanized 9 days after the first virus administration, and the tumors were processed into single-cell suspensions and analyzed by flow cytometry. Tumor cells were gated as live cells (i.e., negative for LIVE / DEAD TM fixable Aqua dead cell viability dye staining), CD45 - EpCAM + cells. Based on nuclear staining of the adenovirus capsid protein, uninfected tumor cells (no positive adenovirus staining above the negative isotype antibody control) could be distinguished from infected tumor cells with low VP load (dim fluorescence intensity of adenovirus staining) and infected tumor cells with high VP load (bright fluorescence intensity of adenovirus staining) Figure 9 A). Infected tumor cells with low and high VP load accounted for 20% and 5% of the total tumor cells on average, respectively Figure 9 B). In each tumor cell subset, we used surface CD19 staining to quantify the percentage of tumor cells that bound to the NG-1125-encoded anti-HER2-ScFv-CD19m1 protein (SEQ ID NO:47). In both the uninfected and infected tumor cell populations, we found subsets of tumor cells that were CD19 + Figure 9 C). These data indicate that the anti-HER2 ScFv-CD19 bispecific protein is efficiently secreted in vivo by tumor cells infected with NG-1125 virus and can diffuse in the TME and bind to both infected and uninfected tumor cells, potentially allowing anti-CD19 CAR-T cells to recognize and destroy these cells.

[0364] In further in vivo studies, 5x10 were subcutaneously inoculated into NSG mice6 A549 lung tumor cells were inoculated to generate xenografts, and when the tumors reached approximately 100 to 200 mm 3 , 5x10 6 viral particles (VPs) of EnAd or NG-1125 (SEQ ID NO:98) were administered intravenously to the mice (administered on day 0 and day 3). On day 6, 1.7x10 7 CD19-specific human CAR-T cells (produced and provided by ProMab Biotechnologies Inc, Richmond, California, USA) were administered intravenously to the mice. Then, two additional intravenous reinjections of the same virus were given to the mice on day 30 and day 34, and then the accumulation of adoptively transferred human T cells in the tumors was evaluated on day 50. The tumors were removed and gently disrupted to form a cell suspension, and flow cytometry was used to determine the total human CD45+ cells and the activated human CD8 + and CD4 + T cell numbers. Figure 10 showed that compared with NG-1124 (SEQ ID NO:97) expressing only the CD19 bispecific protein (SEQ ID NO:47) or the empty vector (EnAd), NG-1125 (SEQ ID NO:98) expressing the anti-HER2 ScFv-CD19m1 bispecific protein (SEQ ID NO:47) as well as human CXCL9 and human IFNα led to higher total human T cells (A) and cytotoxicity-activated CD8 + (B) and CD4 + (C) T cell densities in human tumor xenografts.

[0365] Example 7: NG-641 and NG-1125 enhance T cell recruitment to tumors in vivo. In a further in vivo study, A549 tumor cells were subcutaneously inoculated into NSG immunodeficient mice as described in Example 6, and when the tumors reached approximately 100 to 200 mm 3 , 5x10 9Virus particles (VPs) of NG-641 (SEQ ID NO: 84 in WO2019 / 043020), NG-1125 (SEQ ID NO: 98), or empty vector (EnAd) were all administered on days 0, 3, and 5, or received no viral treatment. NG-641 expresses CXCL9 and CXCL10 chemokines, IFNα, and FAP-TAC (a bispecific molecule consisting of an antibody fragment targeting human fibroblast activation protein (FAP), which does not bind to murine FAP linked to an anti-CD3 agonistic antibody fragment (and human FAP is not expressed in the tumor microenvironment of A549 xenografts)). In this model, the anti-FAP-anti-CD3 construct is expected to be inactive, and thus the NG-641 virus was used as a "non-antigen" control to explain that the effects of CXCL9 and IFNα on CAR-T cell recruitment and activation are independent of the presence of the CD19 antigen. On day 12, 2.5x10 7 CD19-specific human CAR-T cells (produced and provided by ProMab Biotechnologies Inc, Richmond, California, USA) were administered intravenously to the mice. Ten days after CAR-T cell injection, tumor-derived single-cell suspensions and blood samples were analyzed by flow cytometry to determine the frequency of human CD45 + cells (corresponding to all transferred T cells) and the expression of the CD25 activation marker and the CD107a degranulation marker (the latter is used to identify active cytotoxic T cells). Figure 12 showed that compared with EnAd and the condition without viral administration, administration of NG-641 and NG-1125 (SEQ ID NO: 98) both led to an increase in the intratumoral density of total human CD45 + cells ( Figure 12 A) and activated human CD45 + CD25 + ( Figure 12 B) and CD45 + CD107 + ( Figure 12 C) cells, indicating the effects of CXCL9 and IFNα (expressed by these two viruses) on CAR-T cell recruitment and activation in the tumor. Together with CXCL9 and IFNα, NG-1125 also expresses the anti-HER2 ScFv-CD19m1 bispecific protein (SEQ ID NO: 47) that induces a higher overall frequency of total and activated human CD45 + cells in the tumor, indicating that tumor-specific CD19 antigen expression has an additive effect on CXCL9- and IFNα-mediated CAR-T cell enhancement, most likely as a result of the antigen specificity of the anti-CD19 CAR-T receptor participating. ( Figure 12A, 12B, and 12C). The frequency of human CD45 + T cells in the blood of mice not receiving viral vectors was higher than that in mice receiving NG-641, NG-1125, and EnAd administration ( Figure 12 D), indicating that viral infection alone can promote the recruitment of circulating CAR-T cells into solid tumors, and (as Figure 12 shown in A to 12C) this can then be further enhanced by the expression of selected transgenes.

Claims

1. An oncolytic adenovirus serotype B suitable for treating solid tumors (such as sarcomas, carcinomas, and / or lymphomas), comprising the sequence of formula (I): 5’ITR - B1 - BA - B2 - BX - BB - BY - B3 - 3’ITR (I) wherein: B1 is a linker or comprises: E1A, E1B, or E1A - E1B; BA comprises - E2B - L1 - L2 - L3 - E2A - L4; B2 is a linker or comprises: E3; BX is a linker or a DNA sequence that comprises: a restriction site, one or more transgenes, or both; BB comprises L5; BY is a DNA sequence encoding at least two transgenes, namely a first transgene and a second transgene, for example under the control of a major late promoter; and B3 is a linker or comprises: E4, wherein: · The first transgene encodes a polypeptide that comprises a target sequence specific for a binding domain on the cells of a cell - based immunotherapy, such as a (heterologous) recombinantly surface - expressed protein, such as a chimeric antigen receptor or an NKG2D receptor, particularly wherein the target sequence specifically binds to the surface - expressed protein (more particularly the chimeric antigen receptor) on the immunotherapy cells, and · The second transgene encodes a polypeptide that comprises a molecule that promotes the entry of the cell - based immunotherapy into the tumor and its trafficking within the tumor.

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

3. The oncolytic adenovirus serotype B according to claim 1 or 2, wherein the target sequence is specific for a recombinant receptor on the immunotherapy cells 4. The oncolytic adenovirus group B according to any one of claims 1 to 3, wherein the target sequence is 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, claudins [such as 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], EGFRvIII, EPHA3, folate receptor α [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 of them], for example, a tumor antigen, 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 receptor 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 antigenic fragment of any one of them.

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

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

7. The oncolytic adenovirus of group B according to any one of claims 1 to 6, wherein the first transgene encoding the target sequence is suitable for expression on the surface of the infected cancer cell, particularly a membrane-anchored form that allows cell-based therapy to directly bind to the cancer cell, for example, wherein the membrane-anchored form comprises a transmembrane domain or a GPI anchor.

8. The oncolytic adenovirus of group B according to any one of claims 1 to 7, wherein the target sequence is non-human, such as a murine or yeast antigen, such as GCN4 or a fully synthetic molecule (particularly of non-human origin) that contains an amino acid sequence selected for recognition by a specific receptor embodied by the cell-based immunotherapy.

9. The oncolytic adenovirus of group B according to any one of claims 1 to 8, wherein the target sequence is a marker, such as an HA tag (amino acids 98 to 106 of human influenza hemagglutinin), a His tag (e.g., containing at least 6 histidine residues), a FLAG tag, or a 2A peptide tag (such as P2A, T2A, E2A, and / or F2A).

10. The oncolytic adenovirus of group B according to any one of claims 1 to 9, wherein the first transgene encodes a fusion protein that comprises: a. a target sequence that binds to and activates the cell-based immunotherapy, and b. a first binding protein that is specific for a protein expressed on a cancer cell, a stromal cell, or a stromal tissue, particularly allowing the cell-based immunotherapy to indirectly bind to the cancer cell and / or stromal cell or stromal tissue through the fusion protein 11. The oncolytic adenovirus group B according to 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. For example, one fusion protein is encoded by the first transgene, and the second fusion protein is encoded by the second transgene.

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

13. The oncolytic adenovirus group B according to any one of claims 11 or 12, wherein the target sequence of part a) is 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 adenovirus group B 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 cancer and / or stromal cells.

15. The oncolytic adenovirus group B according to any one of claims 1 to 14, wherein encoding the second transgene increases the effectiveness of the cell-based immunotherapy.

16. The oncolytic adenovirus group B according to claim 15, wherein an additional transgene (such as the second transgene or the third transgene) increases the effectiveness of the cell-based immunotherapy by modulating the tumor microenvironment (such as blocking the inhibitory characteristics of the tumor microenvironment). For example, the microenvironment is modulated to be more permissive for the cell-based therapy and / or to make the microenvironment more inflammatory.

17. The oncolytic adenovirus group B according to any one of claims 1 to 36, wherein the second transgene or the additional transgene encodes a chemokine to assist in the recruitment of immunotherapy cells to the tumor.

18. The oncolytic according to any one of claims 1 to 17, wherein the additional transgene encodes a synthetic protein that is designed to engage an additional recombinant receptor expressed by the cells of the therapy to enhance the ability of the additional recombinant receptor to enter and function within the tumor and / or its survival in the patient, such as an orthogonal IL-2 (synthetic receptor) that is a synthetic ligand for an orthogonal IL-2Rβ - for example, Zhang et al., Sci. Transl. Med. 13(625) eabg6986, 2021, which is incorporated herein by reference; the TIM3 / CD28 switch receptor in CAR-T that interacts with secreted TIM3 ligands such as galectin-9 and HMGB1, which can be engineered to be fused to an antibody fragment so that the antibody fragment binds to tumor cells (Zhao et al., J. Immunotherapy Cancer 9, e003176, 2021, which is incorporated herein by reference); the synNotch fusion receptor on CAR-T (for example, the CAR is an anti-CD19 scFv that is coupled to the IC domain of Notch to signal a response in the engineered CAR when they recognize CD19 (including where the CD19 is in a fusion protein such as an anti-HER2 ScFv-CD19 fusion protein) - Roybal et al., Cell 167(2), 419 - 432, 2016, which is incorporated herein by reference).

19. The oncolytic group B adenovirus according to any one of claims 1 to 18, wherein the cells expressing the (exogenous) recombinant antigen receptor are selected from the group consisting of or comprising: 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. The oncolytic group B adenovirus according to any one of claims 1 to 42, which is for use in therapy, particularly for the treatment of cancer such as solid tumors, for example where the cancer is transformed epithelial cancer cells.

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

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

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

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

26. A virus obtainable from claim 25.

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