Oncolytic viral vectors encoding interleukin-7 (IL-7) polypeptides

Abstract

The present invention provides an oncolytic adenoviral vector comprising a nucleic acid sequence encoding an interleukin 7 (IL-7) polypeptide or a variant thereof as a transgene. The present invention also provides a pharmaceutical composition comprising said oncolytic vector and at least one of the following: a physiologically acceptable carrier, a buffer, an excipient, an adjuvant, an additive, a disinfectant, a preservative, a filler, a stabilizer and / or a thickener. A particular object of the present invention is to provide said oncolytic viral vector or pharmaceutical composition for use in the treatment of cancer or tumor, preferably a solid tumor.

Description

[Technical field]

[0001] The present invention relates to the field of life science and medicine. In particular, the present invention relates to human cancer treatment. More particularly, the present invention relates to an oncolytic viral vector comprising a nucleic acid sequence encoding an interleukin-7 (IL-7 or IL7) polypeptide. [Background technology]

[0002] IL-7 is one of the main cytokines involved in the expansion and proliferation of immune cells. Its main function is to maintain the survival of naive and memory T cells as well as their diversity. IL-7 can improve the effector function of T cells through the suppression of negative regulators of T cell activation and can enhance IFNγ production (Rosenberg et al. 2006). Conversely, IL-7 antagonizes immune suppressive pathways through several mechanisms: it prevents the activation of regulatory T cells and inhibits their ability to suppress effector cells (Pellegrini et al. 2012). Moreover, IL-7 abolishes the inhibition of T cell proliferation and prevents their exhaustion (Heninger at al. 2012).

[0003] Recombinant IL-7 showed promising results in preclinical experiments, but off-target toxicity caused dose-limiting toxicity in phase I clinical trials, with only 1 out of 16 patients experiencing a tumor response (Sportes et al. 2010).

[0004] In the prior art, Huang et al.2021 disclosed an oncolytic adenoviral vector encoding IL-7, the backbone of which is adenovirus serotype 5. This vector was used in combination with adoptive cell therapy (ACT) for the treatment of glioblastoma. This treatment led to the prolongation of survival of tumor-bearing mice. However, the authors state that one major limitation of the study is the design of the IL-7-loaded oncolytic adenoviral vector, which prevents it from infecting mouse-derived glioblastoma cells. Indeed, the adenoviral vector used in this study enters cells through the CXAR receptor, which is not expressed in all tumor cells.

[0005] Nakao et al. 2020 discloses a tumor-specific vaccinia virus vector carrying both IL-7 and IL-12 genes. The vector is used in combination with anti-PD-1 and anti-CTLA4 antibodies to treat contralateral tumors in a mouse model, resulting in tumor regression. However, the authors state that checkpoint inhibitors may interfere with the replication of vaccinia virus.

[0006] After many years of development, oncolytic viruses are now beginning to be used as therapeutic agents for cancer. Although there have been discoveries regarding the mechanisms of action and factors that affect the efficacy of viruses, there is still a need to identify the pathways that determine the overall response to virus therapy. In clinical trials, oncolytic viruses have shown favorable safety profiles and promising efficacy.

[0007] WO2014170389 relates to oncolytic adenoviral vectors and cancer treatment methods, either alone or together with therapeutic compositions for therapeutic use. For example, separate administration of adoptive cell therapy compositions and oncolytic adenoviral vectors is disclosed. Adoptive cell therapy is a powerful approach for treating cancer, as well as other diseases such as infectious diseases and graft-versus-host disease. Adoptive cell transfer is the passive transfer of ex vivo grown cells, most commonly immune derived cells, into a host for the purpose of transferring immunological functionality and transplantation properties. WO2014170389 also discloses the nucleic acid sequences of oncolytic adenoviral vectors.

[0008] WO2016146894 discloses oncolytic adenoviral vectors encoding bispecific monoclonal antibodies.

[0009] EP 3858369 relates to the treatment of cancer by the combination of an oncolytic vaccinia virus with an immune checkpoint inhibitor. The virus encodes two interleukins, IL-7 and IL-12, and is administered together with the immune checkpoint inhibitor to further improve the antitumor effect.

[0010] There is still room for improving oncolytic virus therapy for cancer patients, especially in patients with large metastatic load.Further characterization of the pathways related to the activity of oncolytic virus may reveal potential targets for improving the efficacy of virus therapy.Therefore, the efficacy of oncolytic virus vectors alone or together with other therapies may still be improved.The present invention provides efficient tools and methods for cancer therapeutic agents, for example, adoptive cell therapy and / or immune checkpoint inhibitors, by utilizing specific virus vectors. Summary of the Invention

[0011] The object of the present invention is to overcome the limitations found in the use of oncolytic adenoviruses in cancer treatment. It is therefore an object of the present invention to provide simple methods and tools to overcome the problems of ineffective, dangerous and unpredictable cancer treatment. In embodiments of the present invention, novel approaches and means for cancer treatment are thus provided. The object of the present invention is achieved by specific viral vectors, methods and configurations that are characterized by what is stated in the independent claims. Particular embodiments of the present invention are disclosed in the dependent claims.

[0012] Specifically, the present invention provides an oncolytic adenoviral vector comprising a nucleic acid sequence encoding an interleukin 7 (IL-7) polypeptide as a transgene. The present invention also provides a pharmaceutical composition comprising said oncolytic vector and at least one of the following: a physiologically acceptable carrier, a buffer, an excipient, an adjuvant, an additive, a disinfectant, a preservative, a filler, a stabilizer and / or a thickener. A particular object of the present invention is to provide said oncolytic viral vector or pharmaceutical composition for use in the treatment of cancer or tumor, preferably a solid tumor. [Brief description of the drawings]

[0013] [Figure 1]Functionality of Ad5 / 3-E2F-d24-IL7 in vitro. (A) Schematic representation of chimeric 5 / 3 oncolytic adenovirus containing E2F promoter; 24 base pair deletion in E1A; human IL7 transgene inserted in E3 region; and Ad3 serotype knob in Ad5 fiber. (B) Relative human cancer cell viability after addition of 1, 10, 100 or 1000 viral particles (VP) / cell at day 4 post-infection in epithelial adenocarcinoma (A549) and rhabdomyosarcoma (RD). No significant difference was observed between the tumor cell killing ability of Ad5 / 3-E2F-d24-IL7 and Ad5 / 3-E2F-d24, thus suggesting that the presence of IL7 transgene does not reduce the oncolytic potency of Ad5 / 3-E2F-d24-IL7 in human cancer cells. Statistical significance compared to mock is expressed as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. (C) Relative hamster cancer cell viability after addition of 100, 1000, 5000, or 10000 VP / cell at day 5 post-infection in hamster lung carcinoma (HT100) and hamster leiomyosarcoma (DDT1-MF2) or day 8 post-infection in hamster pancreatic carcinoma (HapT1). There was no significant difference between the cell killing ability of Ad5 / 3-E2F-d24-IL7 and Ad5 / 3-E2F-d24, thus suggesting that the presence of IL7 transgene does not reduce the oncolytic potency of Ad5 / 3-E2F-d24-IL7 in hamster cancer cells. Statistical significance compared to mock is expressed as **p<0.01, ***p<0.001, and ****p<0.0001. (D) IL7 expression was analyzed from transfected cancer cells. IL7 concentration was measured in cell supernatants collected 3 days after infection with 1000VP / cell (A549 and RD) or 10000VP / cell (HT100 and DDT1-MF2). Analysis showed that Ad5 / 3-E2F-d24-hIL7 was able to induce IL7 expression in a number of cancer cell lines. (E) Bioactivity of IL7 measured after infection with 1000VP / cell of A549 cell line. Supernatants were filtered, diluted in growth medium, and applied to the IL7-dependent murine cell line 2E8.Recombinant mouse IL7 (rmIL7) and mouse IL7 (rhIL7) were used as controls at 20 ng / ml. These data suggest that IL-7 produced by cancer cells upon infection with Ad5 / 3-E2F-d24-IL7 is functional. In vitro data sets performed in triplicate and all data are presented as mean ± SEM. [Diagram 2] Efficacy of Ad5 / 3-E2F-d24-IL7 in vivo. (A) Tumor growth up to day 30 from experimental groups treated with PBS, Ad5 / 3-E2F-d24 or Ad5 / 3-E2F-d24-IL7 virus. Tumor volumes were normalized to day 0. Data are presented as median + range. Statistical significance from normalized tumor volumes is expressed as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Treatment with Ad5 / 3-E2F-d24-IL7 caused a significant reduction in tumor volume compared to experimental controls (unarmed virus (Ad5 / 3-D24-E2F) and mock), thus providing the best antitumor effect. (B) Changes in immune-related gene expression in tumor lysates. Total RNA was isolated from tumor tissues, followed by cDNA synthesis and quantitative real-time PCR. PCR was performed in duplicate and data were normalized to mock. Data are presented as mean + SEM. Ad5 / 3-E2F-d24-IL7 treated tumors showed increased transcription of immune-related genes compared to mock treated animals, suggesting that the IL-7 encoding virus can induce superior local immune activation. [Diagram 3]Lytic ability and replication of IL7-armed adenovirus in cancer patients in ex vivo tumor cultures. (A) Viability of tumor lysates from ovarian (HUSOV4 and OvCaS) and head and neck (HUSHN11) cancer patients was evaluated at different days after infection with oncolytic viruses at 100VP / cell. Cell viability data are normalized to uninfected mock. Experiments were performed in triplicate. Statistical significance is expressed as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. No significant differences were observed between the tumor cell killing ability of Ad5 / 3-E2F-d24-IL7 and Ad5 / 3-E2F-d24 in most cancer patient samples, suggesting that the presence of IL7 transgene does not reduce the oncolytic potency of Ad5 / 3-E2F-d24-IL7 in cancer patient tumor cultures. (B) Ad5 / 3 replication assessment through quantitative real-time PCR from ovarian (HUSOV4 and OvCaS) and head and neck (HUSHN11 and HUSHN10) samples. Viral copy numbers were normalized to the amount of genomic DNA in the samples and determined by the expression of human β-actin. PCR was performed in duplicate. The data showed a similar pattern of viral DNA levels between Ad5 / 3-E2F-d24 and Ad5 / 3-E2F-d24-IL7 over time, suggesting that the IL-7 transgene does not interfere with viral replication. (C) IL7 protein concentrations in supernatants from ovarian (HUSOV4 and OvCaS) and head and neck (HUSHN15 and HUSHN17) samples measured by cytokine bead array (CBA) assay. Experiments were performed in triplicate. This suggests that IL-7 is produced in human cancer patient samples infected with Ad5 / 3-E2F-d24-IL7. All data are expressed as mean + SEM. [Figure 4]Evaluation of cytokines and chemokines in the tumor microenvironment. (A) Pro-inflammatory cytokines, (C) anti-inflammatory cytokines and (E) chemokine levels obtained from ovarian cancer samples HUSOV4, HUSOV5 and OvCaS 3 days after infection with oncolytic adenovirus at MOI 100. Pooled (B) pro-inflammatory and (D) anti-inflammatory changes and (F) overall ratio of pro-inflammatory to anti-inflammatory cytokines. Increased pro-inflammatory cytokine and chemokine content indicates the ability of IL-7 virus to favorably polarize the microenvironment of human cancer patient samples towards immunostimulation compared to controls. Furthermore, a general minimal effect or decrease in anti-inflammatory cytokine content was observed in IL-7 virus treated wells. All data were normalized to mock. All experiments were performed in triplicate and the resulting data are expressed as mean + SEM. Statistical significance is expressed as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Diagram 5] Assessment of infiltrating CD4+ and CD8+ T cell activation and cytotoxicity in ovarian cancer ex vivo samples (HUSOV4 and OvCaS). (A) Frequency of CD69+ cells in CD4+ and CD8+ cell populations. (B) Expression of the activating receptor CD69 (specified as MFI, mean fluorescence intensity) in CD4+ and CD8+ cells. (C) Frequency of CD4+ and CD8+ cells expressing perforin and granzyme B in HUSOV4 samples. (D) Frequency of CD4+ and CD8+ cells expressing perforin and granzyme B in OvCaS samples. Overall, the data suggest that treatment of patient-derived ovarian ex vivo tumor cultures with Ad5 / 3-E2F-d24-hIL7 activates and enhances the frequency of T cell subpopulations. All flow cytometry experiments were performed in duplicate and the data presented are expressed as mean + SEM. Statistical significance is expressed as *p<0.05, **p<0.01. [Figure 6]Relative cancer cell viability of patient-derived renal cell carcinoma (RCC) samples (HUSRenca5) treated with Ad5 / 3-E2F-d24-IL7 (TILT-517) and immune checkpoint inhibitors (anti-PD1 and anti-PD-L1). (A) Overall cell viability of HUSRenca5 tumor cells at days 1, 2, 3, 4, and 5. A detailed view of tumor cell killing at (B) day 2 and (C) day 5 as assessed by MTS assay. In (B) and (C), samples are from left to right: mock, TILT-517, anti-PD-1, anti-PD-L1, TILT-517+anti-PD-1, TILT-517+anti-PD-L1; *p<0.05, **p<0.01, and ***p<0.001. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Interleukin 7 (IL-7) and its variants As used herein, "IL-7" or "IL7" refers to a wild-type IL-7 isoform 1 polypeptide, whether natural or recombinant, or a nucleic acid (i.e., gene) encoding said polypeptide. Mature human IL-7 exists as a 152 amino acid sequence (without a signal peptide, consisting of an additional 25 N-terminal amino acids). The amino acid sequence of human IL-7 (SEQ ID NO: 1) can be found in GenBank under the accession number NP_000871.1. In its broadest sense, the term "IL-7" or "IL7" may also refer to any of the IL-7 variants suitable for cancer therapy.

[0015] As used herein, "IL-7 variant", "variant IL-7", "vIL7" or "vIL-7" refers to a polypeptide or a nucleic acid (i.e., gene) encoding said polypeptide, in which certain changes or modifications, such as substitutions, have been made or found to an interleukin-7 polypeptide. The term "polypeptide" as used herein refers to any chain of amino acid residues, regardless of its length or post-translational modification (e.g., glycosylation or phosphorylation). Variant IL-7 polypeptides can also be characterized by insertions, deletions, substitutions and modifications of amino acids at one or more sites or at other residues of the native IL-7 polypeptide chain. Any such insertions, deletions, substitutions and modifications may result in a variant IL-7 that exhibits modified binding to the receptor subunit IL-7R or components thereof, preferably with the intent of improving the properties of the IL-7 variant for cancer therapy. Exemplary variants can include substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids. Variants may also include conservative modifications and substitutions at other positions of IL-7 (i.e., positions that have minimal effect on the activity or secondary or tertiary structure of the variant). IL-7 variants may also include naturally occurring isoforms (Vudattu et al. 2008). Sequences for naturally occurring IL-7 isoform precursors can be found in GenBank under accession numbers NP_001186815.1 (isoform 2), NP_001186816.1 (isoform 3) and NP_001186817.1 (isoform 4), but compared to the sequence of isoform 1, isoform 2 has a 44-amino acid deletion at positions 77-120, isoform 3 has an 18-amino acid deletion at positions 121-138, and isoform 4 has a 62-amino acid deletion at positions 76-138.

[0016] Representative mutant IL-7 polypeptides include an amino acid sequence at least about 80% identical to SEQ ID NO: 1 that binds IL-7R with altered affinity, such as higher or lower than the affinity of the polypeptide represented by SEQ ID NO: 1 to bind IL-7R. Representative mutant IL-7 polypeptides can be at least about 50%, at least about 65%, at least about 70%, at least about 80%, at least about 85%, at least about 87%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% identical to wild-type IL-7. Mutant polypeptides can include changes in the number or content of amino acid residues. For example, mutant IL-7 can have a greater or lesser number of amino acid residues than wild-type IL-7. Alternatively, or in addition, representative mutant polypeptides can contain substitutions of one or more amino acid residues present in wild-type IL-7.

[0017] In another embodiment, IL-7 polypeptide can also be prepared as a fusion polypeptide or chimeric polypeptide comprising IL-7 polypeptide and another heterologous polypeptide. Chimeric polypeptides can be produced that comprise IL-7 and an antibody or an antigen-binding portion thereof. The antibody or antigen-binding component of the chimeric protein can function as a targeting moiety. For example, it can be used to localize the chimeric protein to a specific subset of cells or target molecules.

[0018] The present invention is particularly directed to the design of oncolytic viral vectors that contain, as a transgene, a nucleic acid sequence encoding any of the above-mentioned IL-7 polypeptides.

[0019] Viral Vectors Oncolytic viral vectors are therapeutically useful anti-cancer viruses that can selectively infect, replicate and destroy cancer cells. Most current oncolytic viruses are adapted or designed to be tumor-selective, but there are viruses such as reovirus and mumps virus that have a natural preference for cancer cells. Many genetically engineered oncolytic viral vectors utilize tumor-specific promoter elements to enable autonomous replication only in cancer cells. Surface markers selectively expressed by cancer cells can also be targeted by using them as receptors for viral entry. Numerous viruses, including adenovirus, reovirus, measles, herpes simplex virus, Newcastle disease virus and vaccinia virus, are currently undergoing clinical trials as oncolytic agents.

[0020] Preferably, the oncolytic vector used in the present invention is an adenoviral vector suitable for treating humans or animals.As used herein, "oncolytic adenoviral vector" refers to an adenoviral vector that can infect and kill cancer cells by selectively replicating in tumors versus normal cells.As used herein, the expression "adenovirus serotype 5 (Ad5) nucleic acid backbone" refers to the genome of Ad5.Similarly, "adenovirus serotype 3 (Ad3) nucleic acid backbone" refers to the genome of Ad3."Ad5 / 3 vector" refers to a chimeric vector that includes or has parts of both Ad5 and Ad3 vectors.

[0021] In one embodiment of the present invention, the adenoviral vector is a human virus vector.Specifically, the adenoviral vector is an Ad5 / 3 vector.In one embodiment, the backbone is an Ad5 nucleic acid backbone, further comprising an Ad3 fiber knob.In other words, the construct has a fiber knob from Ad3, but the rest of the genome or most of the rest is from Ad5 (see, for example, WO2014170389).

[0022] Adenoviral vectors may be modified by any method known in the art, for example, by deleting, inserting, mutating or modifying any viral region.Vector is made tumor-specific for replication.For example, adenoviral vectors may include modifications in E1, E3 and / or E4, such as inserting tumor-specific promoters (for example, to drive E1), deleting regions (for example, "Δ24", constant region 2 of E1 used in E3 / gp19k, E3 / 6.7k) and inserting one or more transgenes.

[0023] In certain embodiments, the E1B 19K gene, which is generally known to support the replication of adenoviral vectors, has a disabled deletion dE1B 19K in the vector, as described in WO2020249873.

[0024] One approach for the generation of tumor-specific oncolytic adenoviruses is to engineer a 24 base pair (bp) deletion ("Δ24" or "d24") affecting the constant region 2 (CR2) of E1. In wild-type adenoviruses, CR2 is responsible for binding the cellular Rb tumor suppressor / cell cycle regulator protein for induction of the synthetic (S) phase, i.e., the DNA synthesis or replication phase. The interaction between pRb and E1A requires amino acids 121-127 of the conserved region of the E1A protein. The vector may contain a deletion of nucleotides corresponding to amino acids 122-129 of the vector according to Heise C. et al. (2000, Nature Med 6, 1134-1139) and Fueyo J. et al. (2000, Oncogene 19(1):2-12). Viruses with Δ24 have a reduced ability to overcome the G1-S checkpoint and are known to replicate efficiently only in cells where this interaction is not required, e.g., tumor cells with defects in the Rb-p16 pathway, including most, if not all, human tumors. In one embodiment of the present invention, the vector contains a 24 bp deletion in the Rb-binding constant region 2 of adenovirus E1 ("Δ24" or "d24").

[0025] It is also possible to replace the E1A endogenous viral promoter with, for example, a tumor-specific promoter. For example, the E2F1 (e.g., in an Ad5-based vector) or hTERT (e.g., in an Ad3-based vector) promoter can be used instead of the E1A endogenous viral promoter. The vector may contain the E2F1 promoter for tumor-specific expression of E1A. The E1A promoter can also be deleted.

[0026] Although the E3 region is not essential for ex vivo viral replication, the E3 protein plays an important role in regulating the host immune response, i.e., in inhibiting both innate and adaptive immune responses. In one embodiment of the present invention, the deletion of nucleic acid sequence in the E3 region of the oncolytic adenoviral vector is the deletion of viral gp19k and 6.7k reading frames. The gp19k / 6.7k deletion in E3 refers to the deletion of 965 base pairs from the adenoviral E3A region. In the resulting adenoviral construct, both gp19k and 6.7k genes are deleted (Kanerva A et al. 2005, Gene Therapy 12, 87-94). The gp19k gene product is known to bind and sequester major tissue-binding complex I (MHC1, known as HLA1 in humans) molecules in the endoplasmic reticulum and prevent the recognition of infected cells by cytotoxic T lymphocytes. Since many tumors lack HLA1 / MHC1, the deletion of gp19k enhances the tumor selectivity of the virus (the virus is cleared faster than wild-type virus from normal cells, but not from tumor cells). The 6.7k proteins are expressed on the cell surface and are involved in the downregulation of TNF-associated apoptosis involving ligand (TRAIL) receptor 2.

[0027] In one embodiment of the present invention, the transgene, i.e., the gene encoding interleukin 7 (IL7), is placed in the gp19k / 6.7k deleted E3 region under the E3 promoter. This restricts the expression of the transgene to tumor cells and allows viral replication and subsequent activation of the E3 promoter. In a particular embodiment, the nucleic acid sequence encoding interleukin 7 is inserted into the location of the deleted nucleic acid sequence of the viral gp19k and 6.7k reading frame. In another embodiment of the present invention, E3 gp19k / 6.7k is maintained in the vector, but one or many other E3 regions are deleted (e.g., E3 9kDa, E3 10.2kDa, E3 15.2kDa and / or E3 15.3kDa).

[0028] The E3 promoter can be any exogenous (e.g., CMV or E2F promoter) or endogenous promoter known in the art, specifically, the endogenous E3 promoter. The E3 promoter is mainly activated by replication, but some expression occurs when E1 is expressed. Because the selectivity of Δ24 type viruses occurs after E1 expression (when E1 cannot bind Rb), these viruses express E1 even in transduced normal cells. Therefore, it is crucial to regulate E1 expression as well in order to limit the transgene expression mediated by the E3 promoter to tumor cells.

[0029] Certain embodiments of the invention include oncolytic adenoviral vectors (e.g., Ad5 / Ad3 vectors) whose replication is restricted to the retinoblastoma (Rb) / p16 pathway by a dual selectivity device: an E2F (e.g., E2F1) tumor-specific promoter placed in front of the adenoviral E1A gene that is mutated in constant region 2, such that the resulting E1A protein is unable to bind Rb in cells. Additionally, the fiber is modified with 5 / 3 chimerism to allow efficient entry into tumor cells.

[0030] In a particular embodiment of the invention, the oncolytic adenoviral vector comprises: 1) a 24-bp deletion in the Rb-binding constant region 2 of adenovirus E1 (Δ24); 2) deletion of nucleic acid sequences in the viral gp19k and 6.7k reading frames; and 3) a nucleic acid sequence encoding an interleukin 7 (IL7) transgene in place of the deleted nucleic acid sequence defined in point 2); Includes.

[0031] In the following experimental section, we constructed and characterized an oncolytic adenovirus based on the Ad5 / 3-E2F-d24 backbone and armed it with IL7. The virus has a 24 base pair deletion ("D24") in the E2F promoter and E1A constant region 2, allowing its replication only in rb / p16 pathway defective cells, which is one of the common features of all cancer cells. The E1B region is deleted to induce cancer cell apoptosis (dE1B 19K). Furthermore, to improve its ability to transduce cancer cells and enhance its antitumor effect, the virus features a fiber knob from serotype 3, while the rest of the genome is from serotype 5. Most importantly, the Ad5 / 3 virus has a good safety profile in humans. Preferably, the oncolytic virus armed with IL-7 can be used as a potential platform with T cell combination therapy or checkpoint inhibitors to safely and effectively treat currently incurable solid tumors. In particular, tumor types in which T cells are dysfunctional are preferably treated.

[0032] In one embodiment, the present invention is directed to an oncolytic viral vector, preferably an oncolytic adenoviral vector, comprising a nucleic acid sequence encoding an interleukin 7 (IL7) transgene.

[0033] In a preferred embodiment, the backbone of the oncolytic adenoviral vector is an adenovirus serotype 5 (Ad5) or serotype 3 (Ad3) nucleic acid backbone.

[0034] In a more preferred embodiment, the nucleic acid sequence encoding an interleukin 7 (IL7) transgene is in place of a nucleic acid sequence deleted in the E3 region of the oncolytic adenoviral vector. Most preferably, the deletion of the nucleic acid sequence in the E3 region is a deletion of the viral gp19k and 6.7k reading frames.

[0035] In another preferred embodiment, the vector also comprises a 24 bp deletion (Δ24) in the adenoviral E1 sequence of said oncolytic adenoviral vector.

[0036] In another preferred embodiment, the vector also contains a disabling deletion in E1B (dE1B 19K).

[0037] In another preferred embodiment, the vector also contains an Ad5 / 3 fiber knob.

[0038] In another preferred embodiment, the vector comprises a nucleic acid sequence encoding a further transgene. More preferably, the further transgene encodes a cytokine. In one embodiment, the cytokine is: TNF alpha, interferon alpha, interferon beta, interferon gamma, complement C5a, CD40L, IL-2, IL-12, IL-23, IL-21, IL-15, IL-17, IL-18, CCL1, CCL11, CCL12, CCL13, CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23-1, CCL23-2, CCL24, CCL25-1, CCL25-2, CCL26, CCL27, CCL28, CCL29, CCL30, CCL31, CCL32, CCL33-1, CCL33-2, CCL34, CCL35-1, CCL35-2, CCL36, CCL37, CCL38, CCL39, CCL40, CCL41, CCL42, CCL43-2, CCL44, CCL45-1, CCL45-2, CCL46, CCL47, CCL48, CCL49, CCL50, CCL51, CCL52, CCL53, CCL54, CCL55, CCL56, CCL57, CCL58, CCL59, CCL60, CCL61, CCL62, CCL63, CCL64, CCL65, CCL66, CCL67, CCL68, CCL69, CCL70, CCL71, CCL72, CCL73, CCL74 L28, CCL3, CCL3L1, CCL4, CCL4L1, CCL5(=RANTES), CCL6, CCL7, CCL8, CCL9, CCR10, CCR2, CCR5, CCR6, CCR7, CCR8, CCRL1, CCRL2, CX3CL1, CX3CR, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCR1, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7 and XCL2.

[0039] In a more preferred embodiment, the cytokine is TNF alpha or IL-15.

[0040] The viral vectors used in the present invention may also contain other modifications than those described above. Any additional components or modifications may optionally be used, but are not essential to the present invention.

[0041] The insertion of exogenous elements may enhance the effect of the vector in the target cell. The use of exogenous tissue- or tumor-specific promoters is common in recombinant vectors and can also be used in the present invention.

[0042] Adoptive Cell Therapy One approach of the present invention is the development of a treatment for patients with cancer using the transfer of immune effector cells that can react and destroy the cancer. Isolated immune effector cells, such as tumor infiltrating lymphocytes (TILs), are expanded in large quantities in culture and infused into the patient. In the present invention, an oncolytic vector encoding an interleukin 7 (IL7) transgene can be utilized to enhance the influence of immune effector cells. As used herein, "enhancing the efficacy of adoptive cell therapy" refers to a situation in which an oncolytic vector of the present invention, when used together with an adoptive cell therapy composition, can cause a stronger therapeutic effect in a subject compared to the therapeutic effect of the adoptive cell therapy composition alone. A particular embodiment of the present invention is a method of treating cancer in a subject, the method comprising administering to the subject an oncolytic vector of the present invention, the method further comprising administering to the subject an adoptive cell therapy composition. The adoptive cell therapy composition and the vector of the present invention are administered separately. The separate administration of the adoptive cell therapy composition and the adenoviral vector may be preceded by myeloablative or non-myeloablative preconditioning chemotherapy and / or radiation. The adoptive cell therapy treatment aims to reduce or eliminate the cancer in the patient.

[0043] A particular embodiment of the present invention relates to a therapy using an adenoviral vector and an adoptive cell therapy composition, such as tumor infiltrating lymphocytes (TIL), T cell receptor (TCR) modified lymphocytes or chimeric antigen receptor (CAR) modified lymphocytes. Any other adoptive cell therapy, such as T cell therapy, particularly natural killer (NK) or CAR-NK cell therapy, may also be used in the present invention. Indeed, according to the present invention, the adoptive cell therapy composition may comprise unmodified cells, for example in TIL therapy, or genetically modified cells. There are two general methods to achieve gene targeting of T cells to tumor-specific targets. One is the transfer of human leukocyte antigen (HLA, known in rodents as major tissue-associated complex) type-matched T cell receptors (TCRs) with known antigen specificity. The other is the modification of cells with artificial receptors, such as chimeric antigen receptors (CARs). This approach is not dependent on HLA and is more flexible in terms of target molecules. For example, single chain antibodies can be used and CARs can also incorporate costimulatory domains. However, the target of the CAR cell must be on the membrane of the target cell, whereas TCR modification can use intracellular targets.

[0044] As used herein, "adoptive cell therapy composition" refers to any composition comprising cells suitable for adoptive cell transfer. In one embodiment of the present invention, the adoptive cell therapy composition comprises a cell type selected from the group consisting of TILs, TCR (i.e., heterologous T cell receptor) modified lymphocytes, and CAR (i.e., chimeric antigen receptor) modified lymphocytes. In another embodiment of the present invention, the adoptive cell therapy composition comprises a cell type selected from the group consisting of T cells, CD8+ cells, CD4+ cells, NK-cells, dendritic cells, gamma-delta T cells, regulatory T cells, and peripheral blood mononuclear cells. In another embodiment, TILs, T cells, CD8+ cells, CD4+ cells, NK-cells, gamma-delta T cells, regulatory T cells, or peripheral blood mononuclear cells form the adoptive cell therapy composition. In one particular embodiment of the present invention, the adoptive cell therapy composition comprises T cells. As used herein, "tumor infiltrating lymphocytes" or TILs refer to white blood cells that have left the bloodstream and migrated to a tumor. Lymphocytes can be divided into three groups, including B cells, T cells and NK cells. In another specific embodiment of the present invention, the adoptive cell therapy composition comprises T cells that are modified with target-specific CAR or specially selected TCR. As used herein, "T cells" refers to CD3+ cells, including CD4+ helper cells, CD4+ cytotoxic cells, CD8+ cytotoxic T cells, gamma-delta T cells and NK T cells.

[0045] In addition to suitable cells, the adoptive cell therapy compositions used in the present invention may contain any other agents, such as pharma- ceutically acceptable carriers, buffers, excipients, adjuvants, additives, disinfectants, fillers, stabilizers and / or thickeners, and / or any components normally found in the corresponding products. The selection of suitable components and suitable manufacturing methods for formulating the compositions is within the general knowledge of one of ordinary skill in the art.

[0046] The adoptive cell therapy composition may be in any form, such as a solid, semi-solid or liquid form suitable for administration. The formulation may be selected from the group consisting of, but not limited to, a liquid, an emulsion, a suspension, a tablet, a pellet and a capsule. The composition is not limited to a particular formulation; instead, the composition may be formulated into any known pharma- ceutically acceptable formulation. The pharmaceutical composition may be produced by any of the conventional processes known in the art.

[0047] The combination of the oncolytic adenoviral vector and the adoptive cell therapy composition of the present invention refers to the oncolytic adenoviral vector and the adoptive cell therapy composition being used together, not as separate compositions.It is clear to those skilled in the art that the oncolytic adenoviral vector and the adoptive cell therapy composition of the present invention are not used as one composition.Indeed, the adenoviral vector is not used to directly modify the adoptive cell, but to modify the target tumor, so that the tumor is more susceptible to the desired effect of cell transplantation.In particular, the present invention promotes the recruitment of the adoptive cell to the tumor and enhances its activity there.In a particular embodiment of the present invention, the combined oncolytic adenoviral vector and the adoptive cell therapy composition are for administration to the subject simultaneously or sequentially, in any order.

[0048] Immune checkpoint inhibitors Immune checkpoint proteins interact with specific ligands to send signals to T cells and inhibit T-cell function. Cancer cells exploit this by promoting high levels of expression of checkpoint proteins on their surface, thereby suppressing anti-cancer immune responses.

[0049] The immune checkpoint inhibitor (also called CPI or ICI) described herein is any compound that can inhibit the function of immune checkpoint protein. Inhibition includes reducing and completely blocking the function. In particular, the immune checkpoint protein is a human checkpoint protein. Therefore, the immune checkpoint inhibitor is preferably an inhibitor of human immune checkpoint.

[0050] Immune checkpoint proteins include, but are not limited to, CTLA-4, PD-1 (and its ligands PD-L1 and PD-L2), B7-H3, B7-H4, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, BTLA, TIGIT and / or IDO. It is recognized in the art that the pathway involving LAG3, BTLA, B7-H3, B7-H4, TIM3 and KIR constitutes an immune checkpoint pathway similar to the CTLA-4 and PD-1 dependent pathway. The immune checkpoint inhibitor can be an inhibitor of CTLA-4, PD-1 (and its ligands PD-L1 and PD-L2), B7-H3, B7-H4, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, BTLA, TIGIT and / or IDO. In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-L1 or PD-1. Preferably, the immune checkpoint inhibitor is a monoclonal antibody which selectively binds to PD-L1, more preferably selected from the group consisting of: BMS-936559, LY3300054, atezolizumab, durvalumab, embafolimab, cosibelimab and avelumab, or a monoclonal antibody which selectively binds to PD-1, more preferably selected from the group consisting of: pembrolizumab, nivolumab, cemiplimab, sintilimab, tislelizumab, spartalizumab, toripalimab, dostallimab, INCMGA00012, AMP-514.

[0051] In some embodiments, the combined immune checkpoint inhibitor is an antibody. The term "antibody" as used herein includes naturally occurring and engineered antibodies as well as full-length antibodies or their functional fragments or analogs thereof, e.g., capable of binding a target immune checkpoint or epitope (e.g., capable of retaining an antigen-binding portion). Antibodies for use with the methods described herein may be from any source, including but not limited to human, humanized, animal, or chimeric, may be of any isotype, although IgG1 or IgG4 isotypes are preferred, and may be glycosylated or non-glycosylated. The term antibody also includes bispecific or multispecific antibodies, so long as the antibody exhibits the binding specificity described herein.

[0052] cancer The recombinant vector of the present invention is capable of autonomous replication in tumor cells. In one embodiment of the present invention, the vector is capable of autonomous replication in cells that are defective in the Rb-pathway, specifically the Rb-p16 pathway. These defective cells include all tumor cells in animals and humans. As used herein, "defects in the Rb-pathway" refers to mutations and / or epigenetic changes in any gene or protein in the pathway. Due to these defects, tumor cells overexpress E2F and therefore do not require the binding of Rb by E1A CR2, which is normally required for efficient replication. Further selectivity is mediated by the E2F promoter, which is only active in the presence of free E2F, as seen in Rb / p16 pathway-deficient cells. Without free E2F, transcription of E1A does not occur and the virus does not replicate. The inclusion of the E2F promoter is important to prevent E1A from being expressed in normal tissues, which may cause toxicity, either directly or indirectly, by allowing expression of the transgene from the E3 promoter.

[0053] The present invention relates to an approach for treating cancer in a subject. In one embodiment of the present invention, the subject is a human or mammal, particularly a mammal or a human patient, more particularly a human or mammal suffering from cancer.

[0054] This approach can be used to treat any cancer or tumor, including both malignant and benign tumors, and both primary and metastatic tumors can be targets of the approach.In one embodiment of the present invention, cancer is characterized by tumor-infiltrating lymphocytes.The tool of the present invention is particularly attractive for the treatment of metastatic solid tumors characterized by tumor-infiltrating lymphocytes.In another embodiment, the T cell graft is modified with tumor or tissue-specific T cell receptor or chimeric antigen receptor.

[0055] As used herein, the term "treatment" or "treating" refers to the administration of at least an oncolytic adenoviral vector to a subject, preferably a mammalian or human subject, for purposes including not only complete cure, but also prevention, amelioration, or alleviation of cancer or tumor-related disorders or symptoms. The efficacy of treatment may be assessed by monitoring the patient's symptoms, tumor markers in the blood, or, for example, tumor size or patient survival.

[0056] In another embodiment of the invention, the cancer or tumor is selected from the group consisting of nasopharyngeal cancer, synovial cancer, hepatocellular carcinoma, renal cancer, cancer of connective tissue, melanoma, lung cancer, intestinal cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, throat cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, adrenal gland cancer, anal cancer, bile duct cancer, bladder cancer, ureter cancer, oligodendroglioma, neuroblastoma, meningioma, spinal tumor, bone cancer, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, carcinoid of the digestive tract, fibrosarcoma, breast cancer, Paget's disease, cervical cancer, esophageal cancer, gallbladder cancer, head and neck cancer, eye cancer, kidney cancer, cancer), Wilms' tumor, Kaposi's sarcoma, prostate cancer, testicular cancer, Hodgkin's disease, non-Hodgkin's lymphoma, skin cancer, mesothelioma, multiple myeloma, ovarian cancer, endocrine pancreatic cancer, glucagonoma, parathyroid cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus cancer, thyroid cancer, trophoblastic cancer, hydatidiform mole, uterine cancer, endometrial cancer, vaginal cancer, vulvar cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gingival cancer, heart cancer, lip cancer, meningeal cancer, mouth cancer, nerve cancer, palate cancer, parotid gland cancer, peritoneal cancer, pharyngeal cancer, pleural cancer, salivary gland cancer, tongue cancer, and tonsil cancer. Preferably, the cancer or tumor to be treated is selected from the group consisting of renal cancer, ovarian cancer, bladder cancer, prostate cancer, breast cancer, lung cancer (such as small cell lung cancer, non-small cell lung cancer and squamous non-small cell lung cancer), gastric cancer, soft tissue sarcoma, classical Hodgkin's lymphoma, mesothelioma, and liver cancer. In a preferred embodiment of the invention, the cancer or tumor to be treated is a mesothelin-negative cancer or tumor.

[0057] Before classifying a human or animal patient as suitable for the treatment of the present invention, a clinician may examine the patient. Based on results that deviate from normal and reveal a tumor or cancer, the clinician may suggest the treatment of the present invention for the patient.

[0058] Pharmaceutical Compositions The pharmaceutical composition of the present invention comprises at least one viral vector of the present invention. Preferably, the present invention provides a pharmaceutical composition containing (a) an oncolytic virus alone or in combination with (b) an adoptive cell composition and / or (c) an immune checkpoint inhibitor. The present invention also provides said pharmaceutical combination for use in the treatment of cancer. Furthermore, the composition may comprise at least two, three or four different vectors. In addition to the vector and the adoptive cell composition or the immune checkpoint inhibitor, the pharmaceutical composition may also comprise other therapeutically active agents and any other agents such as pharma- ceutical acceptable carriers, buffers, excipients, adjuvants, additives, preservatives, disinfectants, fillers, stabilizers and / or thickeners, and / or any ingredients that are usually found in the corresponding products. The selection of suitable ingredients and suitable manufacturing methods for formulating the composition belongs to the general knowledge of the skilled person.

[0059] The pharmaceutical composition may be in any form, such as solid, semi-solid or liquid form suitable for administration.The formulation may be selected from the group consisting of, but not limited to, liquid, emulsion, suspension, tablet, pellet and capsule.The composition of the present invention is not limited to a particular formulation, instead, the composition may be formulated into any known pharma-ceutically acceptable formulation.The pharmaceutical composition may be produced by any conventional process known in the art.

[0060] The pharmaceutical kit of the present invention comprises an oncolytic adenoviral vector encoding IL-7 as a transgene and one or more immune checkpoint inhibitors. The oncolytic adenoviral vector encoding IL-7 as a transgene is formulated in a first formulation, and the one or more immune checkpoint inhibitors are formulated in a second formulation. Alternatively, the pharmaceutical kit of the present invention comprises an oncolytic adenoviral vector encoding IL-7 as a transgene in a first formulation, and an adoptive cell composition in a second formulation. In another embodiment of the present invention, the first and second formulations are for administration to a subject simultaneously or sequentially, in any order. In another embodiment, the kit is for use in the treatment of cancer or tumor.

[0061] Administration The vector or pharmaceutical composition of the present invention may be administered to any mammalian subject. In a particular embodiment of the present invention, the subject is a human. The mammal may be selected from the group consisting of pets, livestock and production animals.

[0062] Any conventional method may be used for administering vector or composition to a subject.The route of administration is determined according to the composition formulation or form, disease, tumor location, patient, comorbidities and other factors.Therefore, the dosage and frequency of administration of each therapeutic agent in the combination is determined in part according to the specific therapeutic agent, the severity of the cancer being treated, and the characteristics of the patient.Preferably, the dosing schedule maximizes the amount of each therapeutic agent delivered to the patient, consistent with an acceptable level of side effects.

[0063] The effective amount of the vector depends at least on the subject requiring treatment, the tumor type, and the location and stage of the tumor. The dose is, for example, about 1×10 8 from about 1 × 10 14 VP, specifically, about 5 × 10 9 About 1×10 from VP 13 VP, more specifically, approximately 3 × 10 9 About 2 x 10 from VP12 In one embodiment, the oncolytic adenoviral vector encoding IL-7 is 1×10 10 ~1×10 14 In another embodiment of the invention, the dose is about 5×10 10 ~5×10 11 It is within the VP range.

[0064] In one embodiment of the present invention, administration of the oncolytic virus is carried out through intratumoral, intraarterial, intravenous, intrapleural, intravesicular, intracavitary, intranodal or intraperitoneal injection, or through oral administration. Any combination of administrations is possible. The approach can provide a systemic effect despite local injection.

[0065] In one embodiment of the present invention, the separate administration of (a) an oncolytic adenoviral vector encoding IL-7 as a transgene and (b) one or more immune checkpoint inhibitors to a subject is carried out simultaneously or consecutively, in any order. This means that (a) and (b) may be provided in a single dosage form for taking together or in separate entities (e.g. in separate containers) that are administered simultaneously or with a certain time difference. This time difference may be within 1 hour to 2 weeks, preferably 12 hours to 3 days, more preferably within 24 hours or 48 hours. In a preferred embodiment, the first administration of the adenoviral vector is carried out before the first administration of the immune checkpoint inhibitor. Furthermore, it is possible to administer the virus by a different administration method from the immune checkpoint inhibitor. In this regard, it may be advantageous to administer either the virus or the immune checkpoint inhibitor intratumorally and the other systemically or orally. In a particular preferred embodiment, the virus is administered intratumorally and the immune checkpoint inhibitor is administered intravenously. In another particular preferred embodiment, both the virus and the checkpoint inhibitor are administered intravenously. Preferably, the virus and the checkpoint inhibitor are administered as separate compounds. Combination treatment with the two agents is also possible.

[0066] In a preferred embodiment, the immune checkpoint inhibitor is administered in an amount of about 0.2 mg / kg to 50 mg / kg, more preferably about 0.2 mg / kg to 25 mg / kg.

[0067] As used herein, "separate administration" or "separate" refers to a situation in which (a) an oncolytic adenoviral vector encoding IL-7 as a transgene and (b) one or more immune checkpoint inhibitors are two different products or compositions that are distinct from one another.

[0068] Any other treatment or combination of treatments may be used in addition to the treatment of the present invention. In certain embodiments, the method or use of the present invention further comprises simultaneous or sequential administration to the subject of a targeted therapy such as radiation therapy, chemotherapy, angiogenesis inhibitors or alkylating agents, nucleoside analogues, cytoskeleton modifying agents, cytostatic agents, monoclonal antibodies, kinase inhibitors or other anti-cancer agents or intervention (including surgery).

[0069] The terms "treat" or "enhance," as well as their variants, as used herein, do not necessarily imply 100% or complete treatment or improvement, but rather the degree to which one of skill in the art would recognize a potential benefit or therapeutic effect.

[0070] It is obvious to a person skilled in the art that with the advancement of technology, the inventive concept can be implemented in various ways. The present invention and its embodiments are not limited to the above examples but can vary within the scope of the claims.

[0071] Experimental Section material and method cell line Human cancer cell lines A549 (epithelial adenocarcinoma) and RD (rhabdomyosarcoma) were purchased from the American Type Culture Collection (ATCC) (Manassas, USA). Golden hamster cancer cell line DDT1-MF2 (leiomyosarcoma) was kindly provided by Dr. William Wold, hamster HapT1 (pancreatic ductal adenocarcinoma cell line) was obtained from the Leibniz Institute (DSMZ, Braunschweig, Germany), and hamster HT100 (lung adenocarcinoma) was obtained from the Japan Collection of Research Bioresource Cell Bank (Osaka, Japan). All cell lines were cultured under recommended conditions.

[0072] Virus construction Ad5 / 3-E2F-d24-hIL7 virus was constructed by previously described techniques (Havunen et al. 2017). Tumor-specific replication was achieved by two modifications that determine tumor selectivity for viral replication: a 24-base pair deletion in the E2F promoter and the constant region of E1A. Through a bacterial artificial chromosome (BAC) recombineering strategy, the human IL7 coding sequence was introduced into the E3 region in place of the gp19k and 6.7k genes. The resulting viral vector sequences were confirmed by next-generation sequencing.

[0073] Viral particles were generated by transfection of the A549 cell line at a multiplicity of infection (MOI) of 1 and subsequently purified by cesium chloride gradient. The infectivity and concentration of the resulting viruses were determined by TCID50 assay following the protocol described in Lock et al. 2019.

[0074] Cell viability assay Human cell lines A549 and RD were cultured in 96-well plates (flat bottom) at 1 × 10 4Cells / well were seeded in triplicate for 24 hours and infected with either Ad5 / 3-E2F-d24 virus (also referred to herein as backbone or unarmed backbone) or Ad5 / 3-E2F-d24-IL7 (also referred to herein as IL7 virus, IL7 armed virus, IL7 encoding virus or IL7 oncolytic adenovirus) at 1, 10, 100 or 1000VP / cell. Similarly, hamster cell lines HT100, DDT1-MF2 and HapT1 were infected with either Ad5 / 3-E2F-d24 virus (also referred to herein as backbone or unarmed backbone) or Ad5 / 3-E2F-d24-IL7 (also referred to herein as IL7 virus, IL7 armed virus, IL7 encoding virus or IL7 oncolytic adenovirus) at 1, 10, 100 or 1000VP / cell. 4 Cells / well were seeded in triplicate for 24 hours and infected with either the backbone virus or Ad5 / 3-E2F-d24-hIL7 at 100, 1000, 5000 or 10000VP / cell.

[0075] After 4 (A549 and RD), 5 (HT100 and DDT1-MF2) or 8 days (HapT1), cell viability was measured by incubating wells for 2 h with 20% CellTiter 96 AQueous One Solution Proliferation Assay reagent (Promega, Wisconsin, USA). Absorbance was read at 490 nM using a Fluostar OPTIMA analyzer (BMG Labtech, Offenburg, Germany). Data were normalized to uninfected mock controls.

[0076] Cytokine expression and bioactivity assays The aforementioned human and hamster cell lines were infected with either 1000VP / cell (A549 and RD) or 10000VP / cell (HT100 and DDT1-MF2) of Ad5 / 3-E2F-d24-hIL7 for 3 days. Human IL7 was measured from cell supernatants using the BD Cytometric Bead Array Human Soluble Protein Master Buffer Kit (BD Biosciences, New Jersey, USA) together with the Human IL7 Flex Set (BD Biosciences, New Jersey, USA) according to the manufacturer's instructions. Beads were detected using a BD Accuri flow cytometer and results were analyzed using FCAP Array software (version 3.0.1; BD Biosciences, New Jersey, USA).

[0077] To confirm that the virus-produced human IL7 was biologically active, the murine IL7-dependent cell line 2E8 (ATCC, Virginia, USA) was cultured at 2.5 × 10 cells in McCoy's 5A medium (Thermo Fisher, Massachusetts, USA) supplemented with 10% FBS, 1% L-glutamine, and 1% Pen / strep. 5 Cells were cultured in triplicate at 1000 cells / ml for 24 hours. Filtered supernatants from A549 cells infected with Ad5 / 3-E2F-d24-hIL7 were added at dilutions of 1:1, 1:4, 1:16, 1:64 and 1:256 and incubated for 5 days before viability assays using 20% ​​CellTiter 96 AQueous One Solution Proliferation Assay reagent as described above. As positive controls, recombinant mouse and human IL7 were used at a concentration of 20ng / ml.

[0078] Animal testing The efficacy of Ad5 / 3-E2F-d24-hIL7 was tested in vivo using immunocompetent golden hamsters, a model semi-permissive for adenovirus replication. Five-week-old male Syrian golden hamsters (Mesocricetus auratus) (Envigo, Indiana, USA) were inoculated with 2 × 10 6 After tumors reached a diameter of 5–6 mm, animals were randomized and treated with either Ad5 / 3-E2F-d24 or Ad5 / 3-E2F-d24-hIL7 at 1 × 10 9 Control animals received an intratumoral injection of PBS. Tumors were measured with digital calipers and tumor volumes were calculated (length × width). 2 The tumor-to-body ratio was calculated as 1 / 2. Hamsters received all 12 rounds of virus treatment before being euthanized on day 64, and their tumors and organs were harvested for subsequent analysis. Alternatively, animals were euthanized when they reached the maximum allowable tumor volume (22.0 mm), tumors became ulcerated, or as soon as their welfare was compromised.

[0079] Gene expression assays Fragments of animal tumor samples were preserved in RNAlater (Sigma-Aldrich, MO, USA) and stored at -20°C until further use. RNA from samples was isolated using the RNeasy extraction kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and RNA concentration was measured using a Qubit4 fluorometer (Thermo Fisher, MA, USA). 250 ng of purified total RNA was used to synthesize cDNA using the High capacity cDNA Reverse Transcription kit (Thermo Fisher, MA, USA) according to the manufacturer's instructions. The resulting cDNA was used for quantitative real-time PCR. The following primers and probes were used: granzyme B (forward primer 5'-CACTGTTCAGGGAGCTCAATAA-3' (SEQ ID NO: 2), reverse primer 5'-TGGAGTAGTCCTTGGGATTATAGTC-3' (SEQ ID NO: 3), probe 5'-Fam-CCTCCTTTTCTTTGATGTTGTGGGC-BBQ-3' (SEQ ID NO: 4), perforin (forward primer 5'-TGAGTGCCCTTCTGAAATCG-3' (SEQ ID NO: 5), reverse primer 5'-TGTCGCCTGTACAGTTTTCG-3' (SEQ ID NO: 6), probe 5'-Fam-CTGGTACAGAGACCCCCACTGCAC-BBQ-3' (SEQ ID NO: 7), CD25 (forward primer 5'-TC ATCAGTTTCCAGCCAGTG-3' (SEQ ID NO: 8), reverse primer 5'-GTATAAATGTCTCCATAGTTGTAGCTGC-3' (SEQ ID NO: 9), probe 5'-Fam-TCCTGAGAGTGAGACTTCCTGTCCCATC-BBQ-3' (SEQ ID NO: 10), CD137 (forward primer 5'-AGTGCATCGAGGGACTCC-3' (SEQ ID NO: 11), reverse primer 5'-CAGTTTTTACAACCCTGCTCTG-3' (SEQ ID NO: 12), probe 5'-Fam-CTCTTGACCCGGCTTGCAATCC-BBQ-3' (SEQ ID NO: 13), interferon gamma (forward primer 5'-GAACTGGCAAAAGGCTGG-3' (SEQ ID NO: 14),Gene expression levels were measured using reverse primer 5'-CCTTCAAGGCTTCAAAGAGTTT-3' (SEQ ID NO:15), probe 5'-Fam-CATTGAGAGCCAGATCGTCTCCTTCTACTT-BBQ-3' (SEQ ID NO:16), Ki67 (forward primer 5'-GACCGATCTTTTAGGTATGAAAACG-3' (SEQ ID NO:17), reverse primer 5'-GTGGTTTTTGAAGCTTCAGCAT-3' (SEQ ID NO:18), probe 5'-Fam-ACGGCGAGCCTCGAGAGCTAG-BBQ-3' (SEQ ID NO:19), PD1 (forward primer 5'-GTGTCCTAGTGGGTGTCCC-3' (SEQ ID NO:20), reverse primer 5'-GCCCTCCTTCAGAGGCT-3' (SEQ ID NO:21), probe 5'-GCTGCTGGCCTGGGTCCTA-BBQ-3') (SEQ ID NO:22). Results were normalized to the content of hamster gamma actin housekeeping gene cDNA and to mock (ΔΔCt). All PCR reactions were performed in duplicate.

[0080] Patient sample processing and establishment of ex vivo tumor cultures Fresh single-cell tumor lysates were prepared from tumors using a protocol previously validated by our group. Briefly, tumors were minced into small fragments and placed in 50 mL Falcon tubes containing RPMI 1640 supplemented with 1% L-glutamine, 1% Pen / strep, collagenase type I (170 mg / L), collagenase type IV (170 mg / L), DNase I (25 mg / mL) and elastase (25 mg / mL) (all enzymes from Worthington Biochemical) with shaking at +37 °C for overnight enzymatic digestion. After digestion, the cell suspension was filtered through a 70 μm filter and treated with ACK lysis buffer (Sigma-Aldrich, MO, USA) for removal of undigested fragments and red blood cells. The resulting single-cell suspension was used to lyse 3 × 10 5Ex vivo tumor cultures were established by seeding cells in triplicate into 96-well plates (U-bottom) and treating them with 100VP / cell of either Ad5 / 3-E2F-d24 or Ad5 / 3-E2F-d24-hIL7; non-infected cells were used as mock controls. Cells used for further flow cytometry were harvested on day 3 into cryopreservation medium containing 90% fetal bovine serum (FBS) and 10% dimethyl sulfoxide and stored at -140°C or below until further use.

[0081] Cytotoxicity and viral replication assays Cell viability of ex vivo tumor cultures was measured as described above. Briefly, cell viability was measured on days 3, 5, and 7 by incubation for 2 hours with CellTiter 96 AQueous One Solution Proliferation Assay reagent. Data were normalized to uninfected controls.

[0082] To measure viral replication in ex vivo tumor cultures, cells were harvested in phosphate-buffered saline on days 1, 2 and 3 after viral infection and DNA was extracted using the QIAmp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The presence of virus was confirmed by quantitative real-time PCR targeting the E4 region using forward primer (5'-GGAGTGCGCCGAGACAAC-3') (SEQ ID NO: 23), reverse primer (5'-ACTACGTCCGGCGTTCCAT-3') (SEQ ID NO: 24) and probe (Fam-TGGCATGACACTACGACCAACACGATCT-Tam) (SEQ ID NO: 25). Quantification of E1A was calculated according to a standard curve generated using a plasmid encoding a known concentration of virus. Results were normalized to the content of human beta-actin housekeeping gene DNA. All PCR reactions were performed in duplicate.

[0083] Chemokine and cytokine analysis Supernatants from infected ex vivo tumor cultures were collected on day 3 and the presence of CC motif ligand 2 (Ccl2), CC motif ligand 5 (Ccl5), C-X-C motif chemokine 10 (Cxcl10), interleukin 2 (IL2), tumor necrosis factor alpha (TNFa), IFNg, interferon 1 beta (IFN1b), interleukin 4 (IL4), interleukin 6 (IL6), interleukin 10 (IL10) and transforming growth factor beta-1 (TGF-b1) was measured using the Essential Immune Response LEGENDplex panel (Biolegend, CA, USA) according to the manufacturer's instructions.

[0084] Levels of C-X-C motif chemokine 9 (Cxcl9; also known as MIG) and IL7 in the supernatants were measured using human MIG and IL7 flex sets (BD Biosciences, New Jersey, USA), respectively. Samples were measured in triplicate using an Accuri C6 flow cytometer and analyzed either through the LEGENDplex data analysis software suite (Biolegend, California, USA) or FCAP Array software. The concentration of each specimen was normalized to the total protein content measured by a Qubit4 fluorometer (Thermo Fisher, Massachusetts, USA). The data were then normalized to the uninfected control group.

[0085] Flow cytometry The following antibodies were purchased: human CD3 (clone SK7, fluorescent dye Alexa Fluor 700, Biolegend, California, USA), CD4 (clone RPA-T4, fluorescent dye V500, BD Biosciences, New Jersey, USA), CD8 (clone RPA-T8, fluorescent dye FITC, BD Biosciences, New Jersey, USA), CD69 (clone FN50, fluorescent dye PE / Cyanine7, Biolegend, California, USA), and CD127 (clone HIL-7R-M21, fluorescent dye PE-CF594, BD Biosciences, New Jersey, USA). Analysis of immune cell populations from ex vivo tumor cultures was performed using antibodies specific for granzyme B (clone GB11, fluorochrome BV421, BD Biosciences, New Jersey, USA), perforin (clone B-D48, fluorochrome PerCP / Cyanine5.5, Biolegend, California, USA), and CD107a (clone H4A3, fluorochrome APC / Cyanine7, Biolegend, California, USA). Intracellular staining was performed using the BD Cytofix / Cytoperm Plus kit (with BD GolgiPlug) (BD Biosciences, New Jersey, USA) according to the manufacturer's instructions. All samples were stained using human TruStain FcX receptor blocking solution (Biolegend, California, USA) after Fc blocking. Samples were acquired in duplicate using a FACS Aria II cell sorter (BD Biosciences, New Jersey, USA) and data analysis was performed using FlowJo software v10 (FlowJo LLC, BD Biosciences, New Jersey, USA).

[0086] statistical analysis GraphPad Prism v.8.4.2 (GraphPad Software) was used for statistical analysis and graphical presentation of data. Normality of tumor progression data was performed using the Shapiro-Wilk test, and for homogeneity of variance, Levene's test was used. According to these tests, data were non-normal and variances between groups were different, so multigroup comparisons were performed using the non-parametric Friedman test, and pairwise comparisons were performed using Dunn's test. Groups in in vitro experiments were compared using unpaired t-tests. Results were considered statistically significant when p<0.05.

[0087] Example 1. Oncolytic adenovirus armed with human IL7 can deliver transgenes to cancer cells and reduce the viability of multiple cancer cell lines Previously, we demonstrated the ability of Ad5 / 3-E2F-d24 virus to penetrate and replicate in several cancer cell lines (Havunen et al. 2017). The oncolytic adenovirus encoding human IL7 utilizes the same backbone as adenovirus serotype 5 carrying the fiber knob from serotype 3, with a 24-bp deletion in the constant region 2 of the E1A gene (d24) and an insertion of a tumor-specific E2F promoter upstream of the E1A region. The human IL7 gene was introduced into the partially deleted E3 gene region, linking transgene expression to viral replication (Figure 1A).

[0088] The ability of Ad5 / 3-E2F-d24-hIL7 to infect and lyse cancer cells was evaluated in vitro using several human and hamster cell lines. Human lung and rhabdomyosarcoma cancer cell lines (A549 and RD, respectively), as well as hamster leiomyosarcoma, lung and pancreatic cancer cell lines (DDT1-MF2, HT100 and HapT1, respectively) were infected with various concentrations of Ad5 / 3-E2F-d24-hIL7; uninfected cells and cells infected with the previously described (Havunen et al. 2017) unarmed Ad5 / 3-E2F-d24 virus were used as controls. Thus, the lytic effect of Ad5 / 3-E2F-d24-hIL7 virus was comparable to the unarmed virus (Figure 1B and Figure 1C). These results indicate that the presence of the IL7 transgene does not affect the viral oncolytic characteristics.

[0089] To evaluate the ability of the virus to induce transgene expression levels in cancer cells, the latter were infected with Ad5 / 3-E2F-d24-hIL7 and the supernatants were analyzed for the presence of IL7 protein levels. Interestingly, human cell lines showed higher concentrations of IL7, with the highest amount of 17.5 pg / ml produced by A549 (Figure 1D). The hamster cell line HT100 had the lowest protein amount (0.4 pg / ml), whereas the DDT1-MF2 and RD cell lines were equally productive (Figure 1D).

[0090] We next assessed the bioactivity of the virally produced human IL7. To do this, we incubated the IL7-dependent murine cell line 2E8 with several dilutions of A549 supernatants, followed by MTS cell proliferation assays. A dose-dependent cell proliferation was observed, with a better result at the 1:4 dilution compared to the control group (Figure 1E).

[0091] Overall, it was confirmed that Ad5 / 3-E2F-d24-hIL7 was able to kill cancer cells and induce the expression of bioactive IL7 in the latter.

[0092] Example 2. IL7 oncolytic adenovirus promotes tumor regression in a hamster model of pancreatic cancer The efficacy of Ad5 / 3-E2F-d24-hIL7 in vivo was tested in immunocompetent golden hamsters, a preclinical model semi-permissive to adenovirus replication. The pancreatic cancer cell line HapT1 was implanted subcutaneously to allow ectopic tumor formation and subsequent intratumoral virus injection. Thirty days after treatment initiation, mock control group hamsters had significantly larger tumors when compared to virus-treated animals. Both backbone and IL7-coding virus-treated groups showed a trend toward reduced tumor volume, but IL7-coding virus-treated animals had significantly smaller tumors than mock and Ad5 / 3-E2F-d24-treated animals (p values ​​of 0.0013 and 0.0155, respectively) (Figure 2A).

[0093] To gain a better understanding of the immunological mechanism of action of IL7-armed adenovirus in more detail, we measured the expression levels of several immune-related genes in tumors harvested from animals at day 64. In both included virus-treated groups, we observed a considerable upregulation of the canonical T cell activation markers CD25, CD137, Ki67 and PD1 (Figure 2B). The expression levels of IFNg and perforin were also substantially higher in the virus group, potentially suggesting enhanced cytotoxicity of immune cells in the tumor microenvironment. The Ad5 / 3-E2F-d24-treated group showed higher amounts of most analyzed cytokines. No significant differences were observed in the amount of granzyme B across all groups (Figure 2B).

[0094] In conclusion, we show that Ad5 / 3-E2F-d24-hIL7 viral treatment results in effective in vivo tumor regression and allows the activation of key immune stimulators.

[0095] Example 3. IL7-armed oncolytic adenovirus infects and replicates in patient-derived ex vivo tumor models. To further explore the mechanism of action of IL7-armed oncolytic adenoviruses, we moved to more clinically relevant ex vivo tumor models. As expected, cell viability studies showed comparable lytic capacities for both backbone- and IL-7-encoding viruses in the case of ovarian cancer (HUSOV4 and OvCaS): after 3 days of incubation, viability dropped to 70-80% and continued to drop until day 7 (Figure 3A). A drop in viability was also observed in head and neck samples (HUSHN11) at day 7 after viral infection with backbone- and IL7-encoding viruses (Figure 3A).

[0096] We next assessed viral copy number in ex vivo tumor samples through quantitative real-time PCR targeting the adenovirus E4 gene. Overall, viral load was similar for backbone and IL-7-encoding viruses across all samples tested, with an overall trend toward increased viral genome copies over time (Figure 3B). However, there were clear differences in total viral copy number between sample cancer types. OvCaS and HUSHN10 samples had the highest viral DNA load, whereas HUSHN11 had 100-fold lower viral copy number. HUSOV4 showed intermediate viral copy number (Figure 3B).

[0097] Finally, human IL7 concentrations were measured in the supernatants. An increase in IL7 concentrations was observed from day 1 to day 3 in all samples tested, but as expected, the amount of protein differed between samples. HUSOV4 and HHUSHN15 samples had higher amounts of IL7, reaching 0.65 pg / ug and 0.57 pg / ug of total IL7 protein, respectively, whereas IL7 concentrations in OvCaS and HUSHN17 samples were 3-fold lower (Figure 3C).

[0098] Overall, the data demonstrate the ability of IL7 oncolytic adenovirus to infect patient-derived tumor lysates, replicate, and lyse cancer cells, despite variability between samples.

[0099] Example 4. IL7-armed oncolytic adenovirus transforms the local tumor microenvironment into an immunopromoting one To assess the immune status of ex vivo ovarian cancer samples from infected patients, we measured the concentrations of key immune signaling molecules - chemokines and cytokines. First, we compared the amounts of inflammatory cytokines: IL2, TNFa, IFNg, and IL1b. All samples tested showed a significant increase in IFNg when infected with the IL7-encoding virus group compared to uninfected cells and the backbone virus group. OvCaS and HUSOV5 samples also showed higher expression of IL2 when infected with the IL7-encoding virus, whereas HUSOV4 showed no significant cytokine changes between the different experimental groups. Furthermore, OvCaS samples showed a significantly higher concentration of TNFa in the IL7-encoding virus group than in any other treatment group. Interestingly, no change was observed in the amount of IL1b in all samples tested (Figure 4A). Overall, the amount of inflammatory cytokines pooled in OvCaS and HUSOV5 was significantly higher in the IL7 virus treatment group compared to the other groups (Figure 4B).

[0100] Next, the amounts of anti-inflammatory cytokines: IL4, IL6, IL10 and TGFb were measured. Overall, no significant increase in cytokine amounts was observed in virus-treated groups compared to non-infected cells (Figure 4C). HUSOV4 showed a marked decrease in IL4, IL6 and IL10 concentrations, whereas OvCaS and HUSOV5 showed reduced amounts of TGFb upon infection with IL7-encoding virus. Pooled amounts of anti-inflammatory cytokines were decreased in two out of three samples (OvCaS and HUSOV5) in cultures infected with IL7-encoding virus. The decrease was statistically significant between the backbone virus and the IL7-encoding virus in OvCaS samples (Figure 4D).

[0101] Finally, the amounts of chemokines: Ccl2, Ccl5, Cxcl9, Cxcl10 in ex vivo infected samples were measured. Notably, virus-treated samples showed a marked increase in Ccl5 and Cxcl10 production, with the virus encoding IL7 resulting in significantly higher production than the backbone (Figure 4E). HUSOV4 samples did not show any other changes in chemokine concentrations between groups, whereas OvCaS samples showed upregulation of Ccl2 and Cxcl9 when treated with the virus encoding IL7. HUSOV5 samples showed a marked upregulation of Ccl5, Cxcl9, and Cxcl10 upon infection with the virus encoding IL7.

[0102] Importantly, IL-7-encoding viruses induced the highest prevalence of proinflammatory cytokines over anti-inflammatory ones in most ovarian cancer patient samples tested (Figure 4F). Overall, these data demonstrate the ability of Ad5 / 3-E2F-d24-hIL7 to induce the production of signaling molecules to convert the tumor microenvironment to a proinflammatory one, which is particularly useful for recruiting T cells to tumors.

[0103] Example 5. IL7-armed oncolytic adenovirus activates infiltrating CD4+ and CD8+ T cells To evaluate the impact of Ad5 / 3-E2F-d24-hIL7 on tumor-infiltrating lymphocytes, we first examined the expression of activation and cytotoxicity markers on CD4+ and CD8+ cells from HUSOV4 and OvCaS ex vivo tumor cultures treated with oncolytic adenoviruses separately through flow cytometry. Upon infection with IL7-encoding virus, HUSOV4 cultures showed a significant increase in the amount of CD69+CD4+T cells and CD69+CD8+T cells compared to the other groups (Figure 5A), whereas CD8+ cells also had a higher expression of the CD69+ receptor on their surface (Figure 5B). A similar scenario was observed in OvCaS cultures infected with IL7-encoding virus, where an increase in CD69+CD4+T cells was observed compared to uninfected cultures, whereas no significant changes were detected in CD8+T cells (Figure 5A).

[0104] Furthermore, changes in the number of cytotoxic T cells expressing perforin (Perf) and granzyme B (GrzmB), two potent cytolytic agents, were evaluated. A significant increase in both CD4+ and CD8+ GrzmB+ T cells was observed in HUSOV4 cultures treated with IL7-encoding virus (Figure 5C), whereas no changes were detected in GrzmB+ cells in OvCaS samples (Figure 5D). However, in the same samples, an increase in CD8+Perf+ cells was detected in the IL7 group (Figure 5D), and an increase in the frequency of both CD4+Perf+ and CD8+Perf+ populations was observed in HUSOV4 treated with IL7-encoding virus.

[0105] Overall, our data show that Ad5 / 3-E2F-d24-hIL7 is able to activate infiltrating lymphocytes and increase the proportion of cytotoxic CD4+ and CD8+ cells.

[0106] Example 6. Oncolytic adenovirus armed with IL7 in combination with human immune checkpoint inhibitors (PD-1 and / or PDL-1) Viral constructs and immune checkpoint inhibitors (ICIs) The virus used in this proof-of-concept study is TILT-517 (Ad5 / 3-E2F-d24-hIL7). The virus was prepared by bacterial artificial chromosome recombineering technology, incorporating modifications on the E2F promoter and a 24-base pair deletion in the constant region of E1A and human interleukin-7 protein as a transgene. ICIs such as human anti-PD-L1 Tecentriq® (atezolizumab, Roche; referred to in the text as anti-PD-L1) and human anti-PD-1 KEYTRUDA® (pembrolizumab, Merck; referred to in the text as anti-PD-1) were used in the study either alone and / or in combination with TILT-517.

[0107] Patient sample processing and ex vivo tumor culture Tumor samples from patients with renal cell carcinoma (RCC) were obtained from Helsinki University Hospital (HUS). Briefly, tumors were minced into small fragments and placed in 50 mL Falcon tubes containing RPMI 1640 supplemented with 1% L-glutamine, 1% Pen / strep, collagenase type I (170 mg / L), collagenase type IV (170 mg / L), DNase I (25 mg / mL) and elastase (25 mg / mL) (all enzymes from Worthington Biochemical) for overnight enzymatic digestion with shaking at +37 °C. After digestion, the cell suspension was filtered through a 70 μm filter and treated with ACK lysis buffer (Sigma-Aldrich, MO, USA) for removal of undigested fragments and red blood cells. All this process resulted in a heterogeneous single-cell suspension, the latter of which was used to establish ex vivo tumor cultures, in this case HUSRenca5, for use in cell viability assays.

[0108] Cell viability assay The MTS assay was used to assess cell viability of infected tumor cells. 20,000 viable patient tumor cells were seeded in triplicate in 96-well plates (U-bottom). The following day, tumors were treated with either TILT-517, anti-PD-1, anti-PDL-1, TILT-517 + anti-PD-1 or TILT-517 + anti-PD-L1. Uninfected cells were used as mock controls. Viruses were infected at a concentration of 100 VP / cell. The concentrations of both ICIs used were 0.1 mg / ml. Cell viability was assessed 1, 2, 3, 4, and 5 days after treatment by 2-h incubation with CellTiter 96 AQueous One Solution Proliferation Assay reagent (Promega, Wisconsin, USA). Absorbance was read at 490 nm using a Hidex Sense plate reader (Hidex, Turku, Finland). Data were normalized to the uninfected mock control group.

[0109] statistical analysis GraphPad Prism v9.2.0 (GraphPad Software) was used for statistical analysis and graphical display of data. Statistical testing was performed by using unpaired t-test between groups, and p<0.05 was considered significant.

[0110] result The results in Figure 6A show that the use of TILT-517 and its combination with ICI works more effectively compared to the use of either anti-PD-1 or anti-PD-L1 alone in RCC samples. It is important to acknowledge that ICI is a common immunotherapy used as a standard treatment for RCC. However, as can be observed in Figure 6A, anti-PD-1 and anti-PD-L1 treatment reduces the relative cell viability of tumor cultures to only about 90%, while the TILT-517-involved group continues to reduce the relative cell viability of tumors with each passing day, even reaching less than 40%.

[0111] Of note, on day 2, tumor killing mediated by the combination groups of TILT-517 + anti-PD-1 and TILT-517 + anti-PD-L1 is statistically significant compared to mock (Figure 6B). Importantly, this combination group showed to be a promising strategy as it showed more than 20% improvement in reducing sample cell viability in culture compared to TILT-517, anti-PD-1 or anti-PD-L1 monotherapy (Figure 6B). By day 5, this combination group showed statistically significant higher tumor cell killing compared to either anti-PD-1 or anti-PD-L1 alone (Figure 6C). One possible mechanism behind the better tumor killing by the combination treatment group is due to higher immune cell activation.

[0112] Overall, the rapid and effective responses of TILT-517 plus anti-PD-L1 or anti-PD-1 compared with either therapy alone suggest a promising approach for clinical implementation.

[0113] References Cited non-patent literature: Havunen R, Siurala M, Sorsa S, Gronberg-Vaha-Koskela S, Behr M, Tahtinen S, Santos JM, Karell P, Rusanen J, Nettelbeck DM, et al.: Oncolytic Adenoviruses Armed with Tumor Necrosis Factor Alpha and Interleukin-2 Enable Successful Adoptive Cell Therapy. Mol Ther Oncolytics 2017, 4:77-86. Heninger AK, Theil A, Wilhelm C, Petzold C, Huebel N, Kretschmer K, Bonifacio E, Monti P: IL-7 abrogates suppressive activity of human CD4+CD25+FOXP3+ regulatory T cells and allows expansion of alloreactive and autoreactive T cells. J Immunol 2012, 189:5649-5658. Huang J, Zheng M, Zhang Z, Tang X, Chen Y, Peng A, Peng X, Tong A, Zhou L: Interleukin-7-loaded oncolytic adenovirus improves CAR-T cell therapy for glioblastoma. Cancer Immunology, Immunotherapy 2021, 70:2453-2465. Lock, M., et al., Measuring the Infectious Titer of Recombinant Adenovirus Using Tissue Culture Infection Dose 50% (TCID(50)) End-Point Dilution and Quantitative Polymerase Chain Reaction (qPCR). Cold Spring Harb Protoc, 2019. 2019(8). Nakao S, Arai Y, Tasaki M, Yamashita M, Murakami R, Kawase T, Amino N, Nakatake M, Kurosaki H, Mori M, et al.: Intratumoral expression of IL-7 and IL-12 using an oncolytic virus increases systemic sensitivity to immune checkpoint blockade. Science Translational Medicine 2020, 12:eaax7992. Pellegrini M, Calzascia T, Elford AR, Shahinian A, Lin AE, Dissanayake D, Dhanji S, Nguyen LT, Gronski MA, Morre M, et al.: Adjuvant IL-7 antagonizes multiple cellular and molecular inhibitory networks to enhance immunotherapies. Nat Med 2009, 15:528-536. Rosenberg SA, Sportes C, Ahmadzadeh M, Fry TJ, Ngo LT, Schwarz SL, Stetler-Stevenson M, Morton KE, Mavroukakis SA, Morre M, et al.: IL-7 administration to humans leads to expansion of CD8+ and CD4+ cells but a relative decrease of CD4+ T-regulatory cells. Journal of immunotherapy (Hagerstown, Md. : 1997) 2006, 29:313-319. Sportes C, Babb RR, Krumlauf MC, Hakim FT, Steinberg SM, Chow CK, Brown MR, Fleisher TA, Noel P, Maric I, et al.: Phase I study of recombinant human interleukin-7 administration in subjects with refractory malignancy. Clin Cancer Res 2010, 16:727-735. Vudattu, N., Magalhaes, I., Hoehn, H., Pan D., and Maeurer MJ (2009). Expression analysis and functional activity of interleukin-7 splice variants. Genes Immun 10, 132-140. White, E., Sabbatini, P., Debbas, M., Wold, WSM, Kusher, DI, and Gooding, L. (1992). The 19-kilodalton adenovirus E1B transforming protein inhibits programmed cell death and prevents cytolysis by tumor necrosis factor alpha. Mol. Cell. Biol. 1992; 12: 2570-2580.

[0114] Cited Patent Publications: International Publication No. 2014170389 International Publication No. 2016146894 International Publication No. 2020249873 European Patent No. 3858369

Claims

1. 1. An oncolytic adenoviral vector comprising a nucleic acid sequence encoding an interleukin-7 (IL-7) polypeptide or a variant thereof as a transgene, wherein the backbone of the oncolytic adenoviral vector is an adenovirus serotype 5 (Ad5) backbone having an adenovirus serotype 3 (Ad3) fiber knob.

2. 2. The oncolytic adenoviral vector of claim 1, wherein the nucleic acid sequence encoding an interleukin-7 (IL-7) polypeptide or a variant thereof is located in place of a deleted nucleic acid sequence in the E3 region of the oncolytic adenoviral vector.

3. 3. The oncolytic adenoviral vector of claim 2, wherein the deletion of nucleic acid sequences in the E3 region is a deletion of the viral gp19k and 6.7k reading frames.

4. 2. The oncolytic adenoviral vector of claim 1, comprising a 24 bp deletion (Δ24) in the adenoviral E1 sequence of said oncolytic adenoviral vector.

5. 2. The oncolytic adenoviral vector of claim 1, comprising an Ad5 / 3-E2F-d24 backbone.

6. An oncolytic adenoviral vector as described in claim 5, having the structure Ad5 / 3-E2F-d24-IL-7.

7. The oncolytic adenoviral vector of claim 1 , which comprises a nucleic acid sequence encoding an additional transgene.

8. The oncolytic adenoviral vector of claim 7 , wherein the additional transgene encodes a cytokine.

9. The cytokines include TNF alpha, interferon alpha, interferon beta, interferon gamma, complement C5a, CD40L, IL-12, IL-23, IL-21, IL-15, IL-17, IL-18, IL-2, CCL1, CCL11, CCL12, CCL13, CCL14-1, CCL1 4-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CC L22, CCL23-1, CCL23-2, CCL24, CCL25-1, CCL25-2, CCL26, CCL27, CCL28, CCL3, CCL3L1, CCL4, CCL4L1, CCL5 (=RANTES), CCL6, CCL7, CCL8, CCL9, CCR10, CCR2, CCR5, CCR6, CCR7 , CCR8, CCRL1, CCRL2, CX3CL1, CX3CR, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, 9. The oncolytic adenoviral vector of claim 8, selected from the list consisting of CXCL15, CXCL16, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCR1, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7 and XCL2.

10. The oncolytic adenoviral vector of claim 9, wherein the cytokine is TNF alpha or IL-15.

11. A pharmaceutical composition comprising an oncolytic adenoviral vector according to any one of claims 1 to 10 and at least one of a physiologically acceptable carrier, buffer, excipient, adjuvant, additive, disinfectant, preservative, filler, stabilizer and / or viscosity enhancer.

12. 12. The pharmaceutical composition according to claim 11 for use in the treatment of cancer or tumors, preferably solid tumors.

13. The cancer or tumor is selected from the group consisting of nasopharyngeal cancer, synovial cancer, hepatocellular carcinoma, renal cancer, cancer of connective tissue, melanoma, lung cancer, intestinal cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, throat cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, adrenal gland cancer, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, bone cancer, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, carcinoid of the digestive tract, fibrosarcoma, breast cancer, Paget's disease, cervical cancer, esophageal cancer, gallbladder cancer, head cancer, eye cancer, neck cancer, kidney cancer 13. The pharmaceutical composition of claim 12, wherein the cancer is selected from the group consisting of: uterine cancer, Wilms' tumor, Kaposi's sarcoma, prostate cancer, testicular cancer, Hodgkin's disease, non-Hodgkin's lymphoma, skin cancer, mesothelioma, multiple myeloma, ovarian cancer, endocrine pancreatic cancer, glucagonoma, parathyroid cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus cancer, thyroid cancer, trophoblastic carcinoma, hydatidiform mole, uterine cancer, endometrial cancer, vaginal cancer, vulvar cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gum cancer, heart cancer, lip cancer, meningeal cancer, mouth cancer, nerve cancer, palate cancer, parotid gland cancer, peritoneal cancer, pharyngeal cancer, pleural cancer, salivary gland cancer, tongue cancer, and tonsil cancer.

14. The pharmaceutical composition of claim 12 for use in conjunction with an adoptive cell therapy composition.

15. The pharmaceutical composition of claim 12 for use in combination with an immune checkpoint inhibitor.

16. The pharmaceutical composition of claim 15, wherein the immune checkpoint inhibitor selectively binds to PD-L1 or PD1.

17. 17. The pharmaceutical composition of claim 16, wherein the immune checkpoint inhibitor that selectively binds to PD-L1 or PD-1 is selected from the group consisting of BMS-936559, LY3300054, atezolizumab, durvalumab, avelumab, emvafolimab, cosibelimab, pembrolizumab, nivolumab, cemiplimab, sintilimab, tislelizumab, spartalizumab, toripalimab, dostallimab, INCMGA00012, and AMP-514.

18. 13. The pharmaceutical composition of claim 12 for use in conjunction with radiation therapy, monoclonal antibodies, chemotherapy, small molecular inhibitors, hormone therapy or other anti-cancer agents or interventions in a subject.

19. 13. The pharmaceutical composition of claim 12 for use in combination with an immune checkpoint inhibitor, wherein the adenoviral vector has the structure Ad5 / 3-E2F-d24-IL-7, and the immune checkpoint inhibitor selectively binds to PD-L1 or PD-1.

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