Oncolytic adenovirus, immune checkpoint inhibitor and chemotherapeutic agent combination therapy for cancer

Abstract

The present invention relates to combination therapy of oncolytic viruses (in particular oncolytic adenoviruses), checkpoint inhibitors and chemotherapeutic agents (such as paclitaxel) for the treatment of cancer, in particular to combinations comprising: (a) an oncolytic adenoviral vector encoding CD40L as a transgene and (b) one or more immune checkpoint inhibitors for use in combination with (c) a chemotherapy treatment in the treatment of cancer.

Description

Technical Field

[0001] This invention generally relates to virology, immunology, and medicine. In some aspects, this invention relates to a combination therapy of oncolytic viruses (particularly oncolytic adenoviruses), checkpoint inhibitors, and chemotherapeutic agents for the treatment of cancer. Background of the Invention Chemotherapy has been used to treat cancer for over half a century. These chemotherapeutic agents have multiple mechanisms of action, are cytotoxic to cancer cells, and are grouped into several classes, including but not limited to antimicrotubule agents, alkylating agents, topoisomerase inhibitors, and antimetabolites. Chemotherapy has become standard clinical practice for several tumor types due to its demonstrated good antitumor responses in patients (Tilsed et al., 2022). However, clinical responses are often short-lived, leading to chemotherapy resistance and, in some cases, even triggering more aggressive relapses. Therefore, new treatment methods are needed to optimize current cancer care.

[0003] Immune checkpoint inhibitors (CPIs) have revolutionized cancer therapy and validated immunotherapy as a viable approach. Unfortunately, only a small percentage of patients respond. Some patients benefit only for a limited time, while most experience no detectable benefit, especially when it involves common types of non-melanoma solid tumors. Therefore, while CPIs have indeed been validated as a method, unmet clinical needs remain due to the limited number of patients who benefit.

[0004] Urothelial carcinoma (UC) is a case in point. It is one of the most common cancers in both men and women, affecting the lower urinary tract (i.e., the bladder) or, less commonly, the upper urinary tract (i.e., the ureters and renal pelvis). Despite advances in surgical resection techniques and the recent approval of several checkpoint inhibitors for UC treatment, the 5-year survival rate for muscle-invasive UC remains largely <50%, often requiring drastic surgical interventions such as radical cystectomy (Knowles and Hurst, 2015). While CPIs and chemotherapy are effective in some patients, many do not respond to or are unable to maintain a response to CPIs and chemotherapy.

[0005] Another example is breast cancer (BC), the most common cancer affecting women worldwide. Based on the expression of hormone receptors (estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 (HER2)), breast cancer can be subtyped. BC subtype classification is particularly useful for selecting appropriate systemic therapy. Among these subtypes, triple-negative breast cancer (TNBC) is negative for ER, PgR, and HER2, and is associated with a more aggressive tumor type, a higher risk of metastasis, and recurrence. Treatment options for TNBC are limited, with standard care including chemotherapy drugs such as anthracyclines, alkylating agents, taxanes, and 5-FU. There is no standard chemotherapy regimen for recurrent TNBC, and treatment responses are typically short-lived, with frequent visceral and brain metastases. While CPI monotherapy has shown some success as first-line treatment for advanced TNBC, several clinical studies are underway to explore the synergistic potential of chemotherapy and CPI.

[0006] Other examples are prostate cancer (PC), one of the most common cancers afflicting men, with cases of advanced prostate cancer currently on the rise. Prostate cancer typically develops slowly and can remain undetected for years due to the abnormal cells dividing within the prostate. Early intervention is often curative, involving surgical removal of the prostate or radiation therapy, either alone or in combination with hormone therapy. Patients with metastatic hormone-sensitive prostate cancer (mHSPC) may initially respond to hormone therapy, but all eventually develop castration-resistant prostate cancer. Early treatment with the cytotoxic chemotherapy drug docetaxel has been shown to improve overall survival in men with mHSPC and remains the most commonly used chemotherapy for advanced PC. Because PC is considered an immune-cold tumor, using oncolytic adenovirus vectors, alone or in combination with other therapeutic compositions, to transform it into an immune-inflammatory tumor has the potential to reduce the tumor burden.

[0007] After years of development, oncolytic viruses are now being used as cancer therapeutics. While some findings have been made regarding the mechanisms and factors influencing viral efficacy, the pathways determining the overall response to viral therapy still need to be identified. In clinical trials, oncolytic viruses have demonstrated good safety and promising efficacy. However, there is still room for improvement in response, particularly in patients with significant metastatic burden. Further characterization of pathways associated with oncolytic viral activity could reveal potential targets for improving the efficacy of viral therapy.

[0008] WO 2014170389 relates to the therapeutic use and treatment methods of oncolytic adenovirus vectors, alone or in combination with therapeutic compositions, for cancer. Summary of the Invention

[0009] This disclosure provides a method for improving cancer therapy response by enhancing immune activation, wherein an immunogenic agent (particularly oncolytic adenovirus and checkpoint inhibitor) is administered co-administered with a chemotherapeutic agent. Oncolytic viruses are capable of selectively replicating and lysing cancer cells, and can carry genetic material to enhance therapeutic efficacy.

[0010] CD40L is such a transgene that is transiently expressed by T cells and binds to its receptor CD40 on antigen-presenting cells. The CD40-CD40L interaction can trigger a potent immune response involving dendritic cells and B cells. In addition to adenoviruses carrying CD40L (preferably TILT-234) and checkpoint inhibitors (preferably anti-PD1), incorporation of chemotherapeutic agents, preferably paclitaxel (also known as PTX), can provide unexpected synergistic effects for combination therapy.

[0011] PTX is an antimicrotubule agent used to treat a variety of cancers and has been further demonstrated to induce immunogenic cell death. This disclosure shows that adenovirus therapy carrying CD40L enhances the efficacy of anti-PD-1 and chemotherapy in an unexpected way by transforming the tumor microenvironment (TME) into an immunogenic environment, thereby reducing tumor burden.

[0012] By administering TILT-234 to enhance tumor immunogenicity, it is hoped that immune-cold tumors can be transformed into inflammatory tumors, making them responsive to CPIs. TILT-234-induced lysis releases viruses and tumor-associated antigens, which can activate the innate and adaptive immune systems, leading to immune cell infiltration of tumors (Kyo et al., 2008). Furthermore, PTX can also induce the release of immunogenic cell death-related damage-associated molecular patterns and enhance the infiltration of tumor-infiltrating T cells. The combination of the immunogenic agents TILT-234 and PTX may contribute to the generation of an inflammatory tumor microenvironment, thereby leading to enhanced immune cell infiltration and enabling CPIs to function effectively.

[0013] This invention is based on the finding that, for clinically relevant cancer models, co-administration of an oncolytic adenovirus encoding CD40L and an immune checkpoint inhibitor anti-PD-L1 or PD-1 with paclitaxel chemotherapy significantly enhances cytotoxicity against the treated cancer and significantly reduces tumor growth, compared to administration of the adenovirus vector and immune checkpoint inhibitor alone or administration of paclitaxel chemotherapy alone. Therefore, in several embodiments, this application provides combination therapies for treating and / or preventing cancer and / or establishing metastasis in mammals and / or for initiating, enhancing, or prolonging antitumor responses in mammals, comprising administering to a mammal (a) an oncolytic adenovirus vector encoding CD40L as a transgenic vector, (b) preferably selectively binding one or more immune checkpoint inhibitors of PD-L1 or PD-1, and (c) a chemotherapeutic agent. In a particular embodiment, the chemotherapeutic agent may be an antimicrotubule agent (such as paclitaxel), a topoisomerase inhibitor (such as epirubicin), an alkylating agent (such as cyclophosphamide or cisplatin), or an antimetabolite (such as gemcitabine).

[0014] In some respects, administering oncolytic viruses and immune checkpoint inhibitors to subjects with cancer during chemotherapy provides enhanced and even synergistic antitumor effects compared to either treatment alone.

[0015] In other related aspects, methods are provided for optimizing, enhancing, or prolonging the effects of checkpoint inhibitors and chemotherapy, or for reducing the toxicity, dosage, or number of treatments of checkpoint inhibitors and chemotherapy, including administering to mammals in need of this (a) a transgenic oncolytic adenovirus vector encoding CD40L, (b) preferably selectively binding to one or more immune checkpoint inhibitors of PD-L1 or PD-1, and (c) a chemotherapeutic agent.

[0016] Furthermore, the present invention relates to the use of a serotype 3 (Ad3) oncolytic adenovirus vector comprising: a deletion in the E3 region and a tumor-specific promoter for expressing the CD40L transgene in the E3 deletion region. Attached Figure Description

[0017] Figure 1. The combination of paclitaxel and TILT-234 exhibited the highest cytotoxic activity in both UM-UC-3 cells and ex vivo tumor tissue cultures. A statistically significant enhancement in cytotoxicity was observed in the paclitaxel plus TILT-234 combination therapy group compared to monotherapy alone (p<0.05) (Figure 1a). Patient sample 1 showed a trend toward enhanced cytotoxicity in the combination therapy group (Figure 1b). Patient sample 2 showed decreased susceptibility to TILT-234 treatment alone; however, the combination with paclitaxel resulted in the lowest cell viability among all treatment groups (Figure 1c). Statistical significance of the datasets was analyzed using an unpaired t-test. Values ​​are expressed as mean ± SEM. p-value < 0.05 p-value < 0.001 p-value < 0.0001.

[0018] Figure 2 Treatment regimen overview: Adenovirus will be administered every 3 days, followed by aPD-1 and PTX every 6 days.

[0019] Figure 3 Triple therapy with anti-PD1, paclitaxel, and TILT-234 improved tumor control in a humanized mouse model. The in vivo tumor response to the combination of TILT-234 with paclitaxel and aPD-1 was characterized. Statistical significance was analyzed using a linear mixed model. Results are expressed as mean ± SEM. p-value < 0.001.

[0020] Figure 4 To evaluate the cytotoxic effects of TILT-123, paclitaxel, and anti-PD-1 in surgically resected urothelial tumors. Single-cell suspensions of resected tumor cells were plated and treated with different combinations of TILT-234 (250 VP), paclitaxel (0.01 μM), and anti-PD-1 (20 μg / ml). After 6 days of incubation, cell proliferation was assessed by MTS assay. Data represent mean ± SEM. p-value < 0.001 p-value < 0.0001, evaluated using one-way ANOVA and Tukey post-hoc test.

[0021] Figure 5. Flow cytometry analysis of T cell activation in mouse tumors. a) Median fluorescence intensity of granzyme B and IFNG in CD4+ T cells and b) CD8+ T cells. Data are expressed as mean ± SEM for each treatment group n = 6–7. p-value < 0.05 p-value < 0.001 p-values ​​< 0.0001 and < 0.00001 were evaluated using one-way ANOVA and Tukey post-hoc tests. Detailed Implementation

[0022] In several embodiments, a combination therapy is provided for treating cancer and / or establishing metastases in mammals, the combination therapy comprising administering to the mammal: (i) an oncolytic adenovirus vector encoding CD40L in combination with (ii) an immune checkpoint inhibitor and (iii) a first chemotherapeutic agent (such as paclitaxel) and / or with (iv) a second chemotherapeutic agent (such as cisplatin, epirubicin, gemcitabine, or cyclophosphamide). In a preferred embodiment, the immune checkpoint inhibitor selectively binds to PD-L1 or PD-1. In a preferred embodiment, the oncolytic adenovirus vector is administered simultaneously or sequentially with the immune checkpoint inhibitor and the first chemotherapeutic agent (such as paclitaxel). In embodiments, the oncolytic adenovirus vector is administered sequentially with the immune checkpoint inhibitor and the first chemotherapeutic agent over 24 or 48 hours, or over 3-21 days.

[0023] Oncolytic virus In a preferred embodiment, the oncolytic virus in the combination therapy is an oncolytic adenovirus.

[0024] As used herein, "oncolytic adenovirus vector" refers to an adenovirus vector capable of infecting and killing cancer cells through selective replication in tumor cells relative to normal cells. WO 2014170389 discloses examples of oncolytic adenovirus vectors encoding the cytokine CD40L that can be used as one or more transgenic vectors in this invention.

[0025] The vector can be modified in any manner known in the art, such as by deletion, insertion, mutation, or modification of any viral region. The vector exhibits tumor specificity in its replication.

[0026] As used in this article, the expression “adenovirus serotype 3 (Ad3) nucleotide backbone” refers to the genome of Ad3.

[0027] One approach to generating tumor-specific oncolytic adenoviruses is to engineer a 24-base deletion (D24) affecting the constant region 2 (CR2) of E1. In wild-type adenoviruses, CR2 is responsible for binding to cellular Rb tumor suppressor / cell cycle regulator proteins to induce the synthetic (S) phase, i.e., DNA synthesis or replication. The interaction between Rb and E1A requires eight amino acids (121 to 127) of the conserved region of the E1A protein, which can be deleted. Vectors can include the deletion of nucleotides corresponding to amino acids 122–129 of the vector according to Heise C. et al. (2000, Nature Med 6, 1134–1139). Viruses with D24 are known to have reduced ability to overcome the G1-S checkpoint and replicate efficiently only in cells where this interaction is not essential, such as in Rb-p16 pathway-deficient tumor cells, which include most (if not all) human tumors.

[0028] The endogenous viral promoter of E1A can also be replaced, for example, by a tumor-specific promoter. hTERT (human telomerase reverse transcriptase) is regulated by the gene products of various tumor repressor genes, which at least partially lead to cancer-specific upregulation of the hTERT promoter. This property can be used to develop oncolytic viruses that specifically replicate in cancer cells. The vector of the present invention includes a tumor-specific hTERT promoter for expressing E1A.

[0029] In one embodiment of the invention, the oncolytic adenovirus vector is based on an adenovirus serotype 3 (Ad3) nucleic acid backbone and includes: a deletion in the E3 region, and a tumor-specific promoter (e.g., CMV or E2F) for expressing a transgene (e.g., CD40L) at the location of the E3 deletion region. In one embodiment of the invention, the adenovirus vector is based on human adenovirus. (The construction of the TILT-234 viral vectors Ad3-hTERT-E3del-CMV-CD40L and Ad3-hTERT-E3del-E2F-CD40L is disclosed in WO 2014170389.) In other embodiments of the invention, E3 gp19kis is retained in the carrier, but one or more other E3 regions (e.g., E3 9-kDa, E3 10.2 kDa, E3 15.2 kDa and / or E3 15.3 kDa) are missing.

[0030] In a specific embodiment of the invention, the oncolytic adenovirus vector is based on an adenovirus serotype 3 (Ad3) nucleic acid backbone and includes: a promoter (e.g., hTERT) for tumor-specific expression of E1A, deletions in the E3 region (e.g., deletions affecting E3 9-kDa, E3 10.2 kDa, E3 15.2 kDa, and E3 15.3 kDa), and a tumor-specific promoter (e.g., CMV or E2F) for expressing a transgene (e.g., CD40L) at the location of the E3 deletion region. In one embodiment of the invention, the nucleic acid backbone of the vector is entirely adenovirus serotype 3. In one embodiment of the invention, the Ad3 delE3 virus is missing the following features: E3 9 kDa, E3 10.2 kDa, E3 15.2 kDa, and E3 15.3 kDa, and CD40L having the promoter (CMV or E2F) is inserted at their locations. These viruses induce tumor cell apoptosis and trigger several immune mechanisms, including a type 1 T helper cell (TH1) response, which in turn activates cytotoxic T cells and reduces immunosuppression.

[0031] The exact function of the early region (E3) proteins in adenovirus 3 remains unclear. Normally in adenoviruses, their deletion does not appear to impair replication, and they appear to affect the antiviral host response to adenovirus. Of the six adenoviruses (AF) found in humans, the human adenovirus genome contains the highest level of genetic diversity in its E3 region. This diversity in genetic content is primarily located between the highly conserved E3-gp19K and E3-RIDα open reading frames (ORFs), where species-specific gene arrays are encoded.

[0032] The killing of virus-infected cells is mediated by cytotoxic T cells via E3-gp19K. This is achieved by blocking the transport of MHC class I cells to the plasma membrane and inhibiting the formation of the TAP-MHC class I complex.

[0033] Therefore, in one aspect of the invention, the important molecule E3-gp19K is included in the adenovirus vector to make viral replication more covert and allow more time for oncolytic activity and its beneficial effects. Furthermore, retaining E3-gp19K can reduce the induction of anti-adenovirus cytotoxic T cells, thereby generating more anti-tumor T cells.

[0034] Cytokines participate in immune responses through various mechanisms, including recruiting T cells to tumors. The nucleotide sequences encoding cytokine transgenes can originate from any animal, such as humans, apes, rats, mice, hamsters, dogs, or cats, but specifically, they are encoded by human sequences. The nucleotide sequences encoding transgenes can be modified to improve their function, or remain unmodified, i.e., wild-type.

[0035] Specific embodiments of the present invention include a viral vector encoding at least one cytokine. The cytokine used in the present invention may be selected from any cytokine known in the art. In one embodiment of the present invention, the cytokine is selected from the group consisting of: interferon α, interferon β, interferon γ, complement C5a, IL-2, TNFα, CD40L, IL12, IL-23, IL15, IL17, CCL1, CCL11, CCL12, CCL13, CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL17, CCL18, CCL19, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23-1, CCL23-2, CCL24, CCL25-1, CCL25-2, CCL26, CCL27, and CCL28. The cytokines are CCL3, CCL3L1, CCL4, CCL4L1, CCL5, 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, CXCL9, CXCR1, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7, and XCL2. In a specific embodiment of the invention, the cytokine is CD40L.

[0036] The viral vector of the present invention can encode one, two, three, four, five, or more cytokines. In one embodiment of the present invention, the oncolytic adenovirus vector encodes two or more cytokines. These two cytokines can be any known cytokines, such as, but not limited to, those listed above. These two cytokines can be different cytokines. In one embodiment of the present invention, the oncolytic adenovirus vector encodes any two or more cytokines selected from the group of cytokines comprising: interferon α, interferon β, interferon γ, complement C5a, GMCSF, IL-2, TNFα, CD40L, IL12, IL-23, IL15, IL17, CCL1, CCL11, CCL12, CCL13, CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL17, CCL18, CCL19, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23-1, CCL23-2, CCL24, CCL25-1, CCL25-2, CCL... 26, CCL27, CCL28, CCL3, CCL3L1, CCL4, CCL4L1, CCL5, 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, CXCL9, CXCR1, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7, and XCL2.Alternatively, an oncolytic adenovirus vector encoding CD40L and one or more cytokines selected from the following group of cytokines: interferon α, interferon β, interferon γ, complement C5a, GMCSF, TNFα, IL-2, IL-12, IL-23, IL-15, IL-17, CCL1, CCL11, CCL12, CCL13, CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL17, CCL18, CCL19, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23-1, CCL23-2, CCL24, CCL25-1, CCL25-2, CCL26, CCL... 27, CCL28, CCL3, CCL3L1, CCL4, CCL4L1, CCL5, 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, CXCL9, CXCR1, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7, and XCL2. In a specific embodiment of the invention, the cytokine is CD40L. In an embodiment, the oncolytic adenovirus vector encodes only one transgene, which is CD40L. Preferably, the CD40L transgene expresses the CD40L cytokine secreted by infected tumor cells. More preferably, the CD40L transgene does not express trimer CD40L (TMZ-CD40L). Danger signaling provided by oncolytic virus replication and activation of pathogen-associated molecular pattern recognition receptors by viral DNA, along with the action of one or more transgenes, can reduce tumor immunosuppression.

[0037] In one embodiment of the invention, the viral vector includes an internal ribosome entry site (IRES) or optionally a ribosome shunt site 2A between the two transgenes. Thus, the IRES or ribosome shunt site 2A can be located between any cytokines, such as CD40L and any other cytokines, preferably selected from the group of cytokines listed above. As used herein, “IRES” refers to a nucleotide sequence capable of initiating translation in the middle of a messenger RNA sequence during protein synthesis. The IRES can be derived from any virus, but in one embodiment of the invention, the IRES is derived from encephalocarditis virus (EMCV). As used herein, “ribosome shunt site 2A” refers to a translation initiation site in which a ribosome physically bypasses a portion of the untranslated region to reach the start codon. Both IRES and A2 enable the virus to produce two transgenes from a single promoter (E3 promoter).

[0038] In summary, the main advantages of the present invention using a viral vector comprising at least one cytokine transgene are: i) the cytokines and the virus themselves induce a danger signal that recruits T cells and other immune cells to the tumor; ii) the cytokines induce T cell proliferation in both the tumor and local lymphoid organs; iii) the cytokines and the virus themselves can induce T cell (natural anti-tumor T cells and innate anti-tumor T cells, and even adopted T cell grafts) to expand at the tumor site; and vi) cytokine and viral replication advantageously alter the tumor microenvironment by reducing immunosuppression and cell dysfunction.

[0039] The viral vector used in this invention may also include other modifications besides those described above. Any additional components or modifications may be used optionally, but these additional components or modifications are not essential to this invention.

[0040] The insertion of exogenous elements can enhance the effect of the vector in target cells. The use of exogenous tissue or tumor-specific promoters is common in recombinant vectors, and they can also be used in this invention.

[0041] In summary, this invention reveals that oncolytic virus replication can recruit T cells and induce danger signals at tumor sites, thereby reducing immunosuppression and cell dysfunction. These effects are mediated through pathogen-associated molecular pattern recognition receptors, an evolutionarily conserved mechanism for inducing immunity that is unaffected by tolerance. This invention also reveals the additional benefits of oncolytic platforms capable of replicating in tumors but not in normal cells, exhibiting self-amplification at the tumor site. Furthermore, oncolysis itself can enhance overall antitumor activity in humans.

[0042] Checkpoint inhibitors Immune checkpoint proteins interact with specific ligands to send signals to T cells that inhibit T cell function. Cancer cells exploit this by driving high levels of expression of checkpoint proteins on their surface, thereby suppressing the anti-cancer immune response.

[0043] Immune checkpoint inhibitors (also known as CPIs) as described herein are any compounds capable of inhibiting the function of immune checkpoint proteins. Inhibition includes both reduction and complete blockade. In particular, immune checkpoint proteins are human checkpoint proteins. Therefore, immune checkpoint inhibitors are preferably inhibitors of human immune checkpoints.

[0044] 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. Pathways involving LAG3, BTLA, B7-H3, B7-H4, TIM3, and KIR are recognized in the art as constituting immune checkpoint pathways similar to CTLA-4 and PD-1 dependent pathways. Immune checkpoint inhibitors can be inhibitors 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.

[0045] In some embodiments, the combined checkpoint inhibitor is an antibody. As used herein, the term "antibody" includes naturally occurring and engineered antibodies, as well as full-length antibodies or functional fragments thereof or the like capable of binding to, for example, target immune checkpoints or epitopes (e.g., retaining antigen-binding portions). Antibodies used according to the methods described herein can be from any source, including but not limited to human, humanized, animal, or chimeric, and can be of any isotype, preferably IgG1 or IgG4 isotypes, and additionally can be glycosylated or non-glycosylated. The term "antibody" also includes bispecific or multispecific antibodies, provided that one or more antibodies exhibit the binding specificity described herein. Preferably, the immune checkpoint inhibitor is a monoclonal antibody that selectively binds to PD-L1, more preferably selected from the group consisting of: BMS-936559, LY3300054, atezolizumab, durvalumab, and avelumab. Examples of monoclonal antibodies binding to human PD-1 are described in US 7521051, US 8008449, and US 8354509. Specific anti-human PD-1 mAbs that can be used as PD-1 antagonists in therapeutic applications include, but are not limited to: pembrolizumab (MK-3475), nivolumab (BMS-936558), cimiprimab, dotalimab, toripalimab, retifanlimab, and humanized antibodies h409A11, h409A16, and h409A17, described in WO2008156712.

[0046] Humanized antibodies are non-human (e.g., mouse, rat, etc.) antibodies whose protein sequences have been modified to increase their similarity to human antibodies. Chimeric antibodies are antibodies that include one or more elements from one species and one or more elements from other species, such as non-human antibodies that include at least a portion of the constant region (Fc) of human immunoglobulins.

[0047] Various forms of antibodies can be engineered for use in the combinations of the present invention, representative examples of which include Fab fragments (monovalent fragments consisting of VL, VH, CL, and CHI domains), F(a'')2 fragments (bivalent fragments comprising two Fab fragments linked by at least one disulfide bond in the hinge region), Fd fragments (consisting of VH and CHI domains), Fv fragments (consisting of VL and VH domains of an antibody single arm), dAb fragments (consisting of a single variable domain fragment (VH or VL domain)), and single-chain Fv (scFv) which comprises two domains, VL and VH, of an Fv fragment fused together, ultimately forming a single protein chain with a linker.

[0048] In some embodiments, the checkpoint inhibitor (also known as a CPI) of the combination therapy is an antibody or fragment thereof that specifically binds to the immune checkpoint protein PD-L1 or PD-1. In a particularly preferred embodiment, the immune checkpoint inhibitor is a monoclonal antibody, fully human antibody, chimeric antibody, humanized antibody, or fragment thereof capable of at least partially antagonizing PD-L1 or PD-1.

[0049] Chemotherapy drugs As used herein, the term "chemotherapy" refers to treatment with cell inhibitors or cytotoxic agents (i.e., chemotherapeutic agents) to reduce or eliminate the growth or proliferation of unwanted cells, such as cancer cells. Therefore, the use of chemotherapeutic agents or combinations thereof aims to stop the growth of cancer cells by killing them or by halting their division. When chemotherapeutic drugs are administered orally or injected intravenously or intramuscularly, the drugs enter the bloodstream and can reach cancer cells throughout the body (systemic chemotherapy). When chemotherapeutic drugs are placed directly into the cerebrospinal fluid, organs, or body cavities (such as the abdomen), the drugs primarily affect cancer cells in these areas (regional chemotherapy).

[0050] As used in this article, the term "chemotherapeutic agent" refers to any compound or agent that directly or indirectly kills tumor cells as part of its antitumor action. This category of therapeutic agents is broad, encompassing many chemotherapeutic agents with different mechanisms of action. Generally, chemotherapeutic agents can be classified according to their mechanism of action.

[0051] As used herein, the term "anti-microtubule agent" includes, but is not limited to, taxanes such as paclitaxel and docetaxel; and vinblastine alkaloids such as vincristine sulfate, vinblastine sulfate, and vinorelbine.

[0052] As used herein, the term "alkylating agent" includes, but is not limited to, chemotherapeutic agents such as cyclophosphamide, ifosfamide, melphalan, nitrosourea, cisplatin, carboplatin, oxaliplatin, heptaplatin, denaplatin, and enloplatin.

[0053] As used herein, the term "antometabolite" includes, but is not limited to, chemotherapeutic agents such as gemcitabine, 5-FU, capecitabine, and methotrexate.

[0054] As used herein, the term "topoisomerase inhibitor" includes, but is not limited to, chemotherapeutic agents such as epirubicin and doxorubicin.

[0055] In one embodiment of the invention, the chemotherapeutic agent is selected from the group consisting of antimicrotubule agents. Some examples of antimicrotubule agents are paclitaxel, docetaxel, and cabazitaxel.

[0056] In one embodiment of the invention, the chemotherapeutic agent is selected from the group consisting of: topoisomerase inhibitors (such as epirubicin or doxorubicin), alkylating agents (such as cyclophosphamide or cisplatin), and antimetabolites (such as gemcitabine).

[0057] In some implementations, the combination therapy includes the use of an antimicrotubule agent (such as paclitaxel) to enhance the efficacy of the combination therapy. The combination therapy may further include the use of a first and second chemotherapeutic agent (such as cisplatin) together with the antimicrotubule agent to further enhance the efficacy of the combination therapy.

[0058] In embodiments of the present invention, paclitaxel (PTX) may be nab-paclitaxel, wherein paclitaxel is bound to albumin nanoparticles. In a preferred embodiment, in this combination therapy, the patient receives only one type of chemotherapy agent, which is paclitaxel (PTX).

[0059] cancer The recombinant vector of the present invention replicates in tumor cells. In one embodiment of the invention, the vector is capable of replicating in cells defective in the Rb pathway, particularly the Rb-p16 pathway. These defective cells include all tumor cells in animals and humans. As used herein, “defective in the Rb pathway” refers to mutations and / or epigenetic changes in any gene or protein of this pathway. Due to these defects, tumor cells overexpress E2F, and therefore, the binding of E1ACR2 to Rb, which is normally required for efficient replication, is unnecessary. Further selectivity is mediated by the E2F promoter, which is activated only in the presence of free E2F, as seen in Rb / p16 pathway defective cells. In the absence of free E2F, E1A transcription does not occur, and the virus does not replicate. The inclusion of the E2F promoter is important for preventing E1A expression in normal tissues, which can cause toxicity directly and indirectly by allowing transgenic expression of the E3 promoter.

[0060] In other embodiments, the presence of a human telomerase (hTERT) promoter in the viral construct enables tumor-selective viruses to replicate in cancer cells. Telomerase, responsible for maintaining telomere length, is inactive in adult cells but highly active in cancer cells. Telomerase activity in cancer cells allows cells to replicate and proliferate uncontrollably. Therefore, the hTERT promoter is active only in telomerase-activated cancer cells and drives the expression of genes required for viral replication, thereby conferring selective replication capabilities to the colony.

[0061] This invention relates to a method of treating cancer in a subject. In one embodiment of the invention, the subject is a human or mammal, particularly a mammal or human patient, and more particularly a human or mammal suffering from cancer.

[0062] This method can be used to treat any cancer or tumor, including both malignant and benign tumors, and both primary and metastatic tumors can be targets of this method. In one embodiment of the invention, the cancer is characterized by tumor-infiltrating lymphocytes. The tool of the present invention is particularly attractive for the treatment of metastatic solid tumors characterized by tumor-infiltrating lymphocytes.

[0063] As used herein, the terms "treatment" or "treating" refer to the administration of at least an oncolytic adenovirus vector and a selectively binding PD-L1 or PD-1 checkpoint inhibitor to a subject, preferably a mammal or human subject, for purposes including not only complete cure but also prevention, improvement, or reduction of ailments or symptoms associated with cancer or tumors. In one embodiment, the treatment may include administration of a chemotherapeutic agent, such as paclitaxel, to provide a synergistic effect of treatment. In another embodiment, the treatment may include administration of a second chemotherapeutic agent to further provide a synergistic effect of treatment. The therapeutic effect can be assessed by monitoring the patient's symptoms, tumor markers in the blood, such as tumor size, patient survival time, or progression-free survival time of the patient.

[0064] In other embodiments of the invention, the cancer or tumor is selected from the group consisting of: nasopharyngeal carcinoma, synovial carcinoma, hepatocellular carcinoma, kidney cancer, connective tissue cancer, melanoma, lung cancer, intestinal cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, laryngeal cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, von Hippel-Lindaudisease, and Zollinger-Ellison syndrome. (syndrome), adrenal cancer, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, Ewing sarcoma, cancer of unknown primary site, carcinoid, gastrointestinal carcinoid, fibrosarcoma, breast cancer, Paget's disease, cervical cancer, esophageal cancer, gallbladder cancer, head and neck cancer, eye cancer, kidney 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, thymic cancer, thyroid cancer, trophoblastic cancer. Cancer, vesicular fetal mass, uterine cancer, endometrial cancer, vaginal cancer, vulvar cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gingival cancer, heart cancer, lip cancer, meningeal cancer, oral cancer, nerve cancer, palate cancer, parotid gland cancer, peritoneal cancer, pharyngeal cancer, pleural cancer, salivary gland cancer, tongue cancer, and tonsil cancer.

[0065] Preferably, the cancer or tumor being treated is selected from the group consisting of: urothelial carcinoma, bladder cancer, prostate cancer, and breast cancer.

[0066] Clinicians may examine patients before classifying them for treatments applicable to this invention, whether human or animal. Based on deviations from normal and findings revealing tumors or cancer, clinicians may recommend the treatment of this invention to the patient.

[0067] In one embodiment, this treatment is the subject's or patient's first cancer treatment. In other embodiments of the invention, the subject or patient to be treated according to the invention has failed at least one prior chemotherapy treatment. In other embodiments of the invention, the subject or patient to be treated according to the invention has failed at least one prior immunotherapy treatment (such as CPI treatment), i.e., the patient's cancer is a checkpoint inhibitor (CPI) refractory tumor. In a preferred embodiment, the invention relates to the treatment of CPI-refractory tumors.

[0068] Pharmaceutical Composition The pharmaceutical compositions of the present invention comprise at least one type of the viral vector of the present invention. Preferably, the present invention provides a pharmaceutical composition comprising: (a) an oncolytic virus combined with (b) a checkpoint inhibitor and (c) a chemotherapeutic agent (such as paclitaxel, cyclophosphamide, epirubicin, cisplatin, and gemcitabine). The present invention also provides said pharmaceutical compositions for the treatment of cancer. Furthermore, the composition may comprise at least two, three, or four different vectors. In addition to the vector, checkpoint inhibitor, and said chemotherapeutic agent, the pharmaceutical composition may also comprise other therapeutically effective agents, any other agents such as pharmaceutically acceptable loads, buffers, excipients, adjuvants, additives, preservatives, antiseptics, fillers, stabilizers, and / or thickeners and / or any components common in the corresponding products. The selection of suitable ingredients and appropriate manufacturing methods for formulating the composition is common knowledge to those skilled in the art.

[0069] The pharmaceutical composition may be in any form suitable for administration, such as solid, semi-solid, or liquid. The formulation may be selected from the group consisting of, but is not limited to, solutions, emulsions, suspensions, tablets, pills, and capsules. The compositions of the present invention are not limited to a particular formulation; alternatively, the compositions may be formulated into any known pharmaceutically acceptable formulation. The pharmaceutical composition may be produced by any conventional method known in the art.

[0070] The pharmaceutical kit of the present invention comprises an oncolytic adenovirus vector encoding CD40L as a transgene, one or more immune checkpoint inhibitors selectively binding to PD-L1 or PD-1, and a chemotherapeutic agent (preferably paclitaxel). The oncolytic adenovirus vector encoding CD40L as a transgene is formulated in a first formulation, and the one or more immune checkpoint inhibitors selectively binding to PD-L1 or PD-1 are formulated in a second formulation. A third formulation comprises a chemotherapeutic agent, preferably paclitaxel, cyclophosphamide, epirubicin, cisplatin, or gemcitabine. In other embodiments of the invention, the first, second, and third formulations are used to administer the kit to a subject simultaneously or sequentially in any order. In other embodiments, the kit is used to treat cancer or tumors.

[0071] Dosage Adenovirus vectors, checkpoint inhibitors, chemotherapeutic agents, or pharmaceutical compositions comprising any of these can be administered to any mammalian subject. In a particular embodiment of the invention, the subject is a human. Mammals may be selected from the group consisting of pets, livestock, and productive animals.

[0072] Any conventional method can be used to administer a drug delivery vehicle, checkpoint inhibitor, chemotherapeutic agent, or composition to a subject. The route of administration depends on the formulation or form of the composition, the disease, the location of the tumor, the patient, comorbidities, and other factors. Therefore, the dosage and frequency of administration of each therapeutic agent in the combination depends in part on the specific therapeutic agent, the severity of the cancer being treated, and the patient characteristics. Preferably, the dosing regimen maximizes the amount of each therapeutic agent delivered to the patient, consistent with acceptable levels of side effects. In a preferred embodiment, checkpoint inhibitors are administered at amounts from about 2 mg / kg to 50 mg / kg, more preferably from about 2 mg / kg to 25 mg / kg.

[0073] In other preferred embodiments, the chemotherapy drug is an antimicrotubule agent (such as paclitaxel) administered at a dose range of about 60-300 mg / m² (including, for example, about 80-260 mg / m², such as about 100 mg / m²). In embodiments, the paclitaxel dosage range is 100 mg / m²-260 mg / m². Examples of paclitaxel dosage regimens are as follows (but not limited to): i) prostate cancer: once every 3 weeks; ii) lung cancer: a 21-day cycle, administered on days 1, 8, and 15; and iii) pancreatic cancer: a 28-day cycle, administered on days 1, 8, and 15.

[0074] In other preferred embodiments, the chemotherapeutic agent is an antimetabolite (such as gemcitabine) administered at a dose range of about 60-3000 mg / m² (including, for example, about 80-2600 mg / m², such as about 1000 mg / m²). In certain embodiments, the dose range of gemcitabine is about 1000-1250 mg / m², depending on the cancer indication.

[0075] In other preferred embodiments, the chemotherapeutic agent is an alkylating agent (such as cyclophosphamide, cisplatin, or epirubicin) administered at a dose range of about 20-300 mg / m² (including, for example, about 80-260 mg / m², such as about 100 mg / m²). In certain embodiments, the dose range of epirubicin is about 45-135 mg / m², depending on the cancer indication. In other specific embodiments, the dose range of cyclophosphamide is about 10-50 mg / m², depending on the cancer indication. In other specific embodiments, the dose range of cisplatin is about 20-100 mg / m², depending on the cancer indication.

[0076] In one embodiment of the invention, the subject is given (a) a transgenic oncolytic adenovirus vector encoding CD40L, (b) preferably selectively bound to one or more immune checkpoint inhibitors of PD-L1 or PD-1, and (c) a chemotherapeutic agent, either simultaneously or sequentially, in any order. This means that (a), (b), and (c) can be provided in a single unit dosage form for administration together or as separate entities (e.g., in separate containers) simultaneously or at a certain time difference. This time difference can be between 1 hour and 1 week, preferably between 12 hours and 3 days, more preferably up to 24 or 48 hours. In a preferred embodiment, the first administration of the adenovirus vector is performed before the first administration of the checkpoint inhibitor or the first administration of the chemotherapeutic agent. Alternatively, the virus can be administered via routes other than checkpoint inhibitors or chemotherapeutic agents. In view of this, it may be advantageous to administer the virus and / or checkpoint inhibitor intratumorally and the chemotherapeutic agent systemically or orally. In a particularly preferred embodiment, the virus, checkpoint inhibitor, and / or chemotherapeutic agent are administered intravenously. Preferably, the virus, checkpoint inhibitor, and chemotherapeutic agent are administered as separate compounds. These three medications can also be used simultaneously for treatment.

[0077] As used herein, “monotherapy” or “monotherapy” means the following: (a) CD40L is encoded as a transgenic oncolytic adenovirus vector; (b) preferably selectively binds to PD-L1 or one or more immune checkpoint inhibitors of PD-1; and (c) the chemotherapeutic agents are three different products or compositions that are different from each other.

[0078] A single administration of (a) an oncolytic adenovirus vector encoding CD40L as a transgenic vector, (b) preferably selectively binding to one or more immune checkpoint inhibitors of PD-L1 or PD-1, and (c) a chemotherapeutic agent may have a therapeutic effect. Depending on, for example, the patient and the type, extent, or location of the cancer, any time interval may exist between administrations. In one embodiment of the invention, a time interval of one minute to four weeks, particularly one to ten days, more particularly one to five days, and most particularly up to 24 or 48 hours, and / or several administrations of (a) an oncolytic adenovirus vector encoding CD40L as a transgenic vector, (b) preferably selectively binding to one or more immune checkpoint inhibitors of PD-L1 or PD-1, and (c) a chemotherapeutic agent may exist between administrations of (a) an oncolytic adenovirus vector encoding CD40L as a transgenic vector, (b) preferably selectively binding to one or more immune checkpoint inhibitors of PD-L1 or PD-1, and (c) a chemotherapeutic agent. (a) an oncolytic adenovirus vector encoding CD40L as a transgenic vector, (b) preferably selectively binding to PD-L1 or one or more immune checkpoint inhibitors of PD-1, and (c) the number of chemotherapeutic doses may vary during the treatment period. The oncolytic adenovirus vector, checkpoint inhibitor, or chemotherapeutic agent may be administered, for example, 1 to 10 times, for the first 2 weeks, 4 weeks, monthly, or during the treatment period. In one embodiment of the invention, the vector or any combination is administered three to seven times for the first 2 weeks, then 4 weeks, and then monthly. In a particular embodiment of the invention, the vector is administered four times for the first 2 weeks, then 4 weeks, and then monthly. In other particular embodiments, the adenovirus vector is administered three times during the first four weeks (in one embodiment, the first dose is administered intravenously, and the second and third doses are administered intratumorally) and one or two checkpoint inhibitor / chemotherapeutic agent doses are administered, followed by monthly administration of the viral vector and checkpoint inhibitor / chemotherapeutic agent. The length of the treatment period can vary and may, for example, last from two months to 24 months or longer.

[0079] In a particular embodiment of the invention, (a) CD40L is encoded as a transgenic oncolytic adenovirus vector, (b) preferably selectively binds to PD-L1 or one or more immune checkpoint inhibitors of PD-1, and (c) the chemotherapeutic agent is administered on the same day, and thereafter the oncolytic adenovirus vector is administered weekly, bi-weekly, bi-weekly, or monthly during a treatment period that may last, for example, one to six or 24 months or longer.

[0080] In one embodiment of the invention, oncolytic viruses are administered via intratumoral, intranasal, intra-arterial, intravenous, intrapleural, intracystic, intracavitary, or intraperitoneal injection, or orally. Any combination of administration is also possible. Despite local injection, this method can still produce systemic effects. Checkpoint inhibitors can be administered intravenously or intratumorally. In one embodiment, checkpoint inhibitors are administered via intratumoral, intranasal, intra-arterial, intravenous, intrapleural, intracystic, intracavitary, or intraperitoneal injection, or orally. Chemotherapy agents can be administered intravenously or intratumorally. In one embodiment, chemotherapeutic agents are administered via intratumoral, intranasal, intra-arterial, intravenous, intrapleural, intracystic, intracavitary, or intraperitoneal injection, or orally.

[0081] The effective dose of the carrier depends at least on the subject requiring treatment, the type and location of the tumor, and the stage of the tumor. The dose can vary, for example, from approximately 1 x 10⁻⁶. 8 One viral particle (VP) to approximately 1x10 14 VP, specifically from about 110 9 VP to approximately 1x10 13 VP, and more specifically from about 3x10 9 VP to approximately 5x10 12 VP. In one implementation, with 1x10 10 - 1x10 14 A dose of one viral particle is administered to an oncolytic adenovirus vector encoding at least one cytokine. In other embodiments of the invention, the dose is approximately 5 x 10^6 particles. 10 - 5x10 11 Within the range of VP.

[0082] In addition to the therapies of the present invention, any other treatment or combination of treatments may be used. In certain embodiments, the methods or uses of the present invention further include administering radiotherapy, or other anticancer drugs or interventions (including surgery), simultaneously or sequentially to the subject.

[0083] As used herein, the terms “treatment” or “enhancement” and words stemmed from them do not necessarily imply 100% or complete treatment or enhancement. Rather, there are varying degrees of potential benefit or therapeutic effect that a person skilled in the art would consider to have.

[0084] Other implementation methods The present invention also relates to: (a) an oncolytic adenovirus vector encoding CD40L as a transgenic gene, (b) one or more immune checkpoint inhibitors, and (c) a chemotherapeutic agent, preferably paclitaxel, for use in the treatment of cancer or tumors. Preferably, human cancer or tumors.

[0085] Throughout this specification, the phrase "in one embodiment" or "in an embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the invention. Therefore, the phrases "in one embodiment" or "in an embodiment" appearing in various paragraphs throughout this specification do not necessarily refer to the same embodiment. Where numerical values ​​are referred to using terms such as, for example, about or substantially, precise numerical values ​​are also disclosed.

[0086] The verbs “to comprise” and “to include” are used in this document as open-ended restrictions, neither excluding nor requiring the presence of any unlisted features. Unless otherwise expressly stated, the features recited in the dependent claims may be freely combined with each other. Furthermore, it should be understood that the use of “a” or “an” throughout this document, i.e., the singular form, does not exclude the plural form.

[0087] It will be apparent to those skilled in the art that the inventive concept can be implemented in various ways as technology advances. The invention and its embodiments are not limited to the examples described below, but can be varied within the scope of the claims.

[0088] Example Section Example 1 Materials and methods Adenovirus vector The Ad3-hTERT-CMV-hCD40L virus was generated from the pWEA-Ad3-GFP vector (Zafar et al., 2017). A 13.2 kb fragment from pWEA-Ad3-GFP, digested with FseI / NotI, was inserted into a modified pBluescript KS(-) vector. The CMV-GFP-PA transgenic cassette and the 3' end of the Ad3 genome were removed using EcoRI / FseI digestion. A 1.1 kb fragment containing the Ad3 5' end, the hTERT promoter, and part of the Ad3 E1 region was amplified from pKSB2-hTERT. The portion containing the first 800 bp of the Ad3 genome was replaced by ligating a PmeI / NheI-digested PCR fragment. The GFP gene in the construct was then replaced with the hCD40L gene (pWEA-Ad3-3' end-CMV-hCD40L). The final construct was formed by ligating a 13.4 kb fragment containing the 5' end of the Ad3 genome and hTERT, and a 6–5 kb fragment containing the 3' end of the Ad3 genome and CMV-hCD40L (pWEA-Ad3-3' end-CMV-hCD40L). The ligation product was packaged into phages using Gigapack III Plus packaged extract (Stratagene, La Jolla, CA) and amplified. The Ad3-hTERT-CMV-hCD40L viral genome was released via FseI digestion and transfected into 293 cells using the standard calcium phosphate method. HeLA cells were then infected with cell lysates containing the Ad3-hTERT-CMV-hCD40L virus. Subsequent viral amplification was performed on A549 cells.

[0089] Cell lines: The human bladder cancer cell line UM-UC-3 was purchased from Sigma-Aldrich (Missouri, USA) and cultured in RPMI 1640 (BioWest, Nuallie, France) supplemented with 10% fetal bovine serum, 1% L-glutamine, 1% penicillin and 1% streptomycin.

[0090] Patient sample processing: Single-cell tumor digests of patient tumors (bladder cancer) were prepared using a previously described protocol (Taipale et al., 2018). In brief, after surgical removal of the patient's tumor, it was cut into smaller fragments and placed at 37°C in RPMI 1640 supplemented with 1% L-glutamine, 1% penicillin / streptomycin, type I collagenase, type IV collagenase, DNase I, and elastase (Worthington Biochemcial, New Jersey, USA) for 2 hours. After digestion, the sample was filtered through a 70 μM filter membrane, and ACK lysis buffer (Sigma-Aldrich, Missouri, USA) was added to remove residual red blood cells. The sample was then frozen in freezing medium (90% FBS, 10% DMSO) and stored at -180°C until future use.

[0091] Cell viability assay: UM-UC-3 cells were seeded at a density of 1E4 cells / well in RPMI 1640 (2% FBS) in 96-well plates and incubated at 37°C for 24 hours. For patient samples, cells were seeded at a density of 1E5 cells / well in RPMI 160 (10% FBS). Approximately 24 hours after seeding, cells were treated alone or in combination with TILT-234 (250 VP), paclitaxel (Fresenius Kabi, Germany) (0.01 μM), or anti-PD-1 (20 μg / ml) (Merck & Co, New Jersey, USA). After 72 hours of treatment, CellTiter 96 AQueous One Solution proliferation assay (Promega, Wisconsin, USA) was added, and cells were incubated at 37°C for 2 hours. For human patient samples, CellTiter 96 AQueous One Solution proliferation assay was added 144 hours (six days) after treatment. After two hours of incubation, the absorbance of each well was measured at 490 nm using a Hidex Sense plate reader.

[0092] PBMC amplification: HLA-A 02:01 PBMCs obtained from Cellular Technology Limited (CTL, Ohio, USA) were thawed and washed with CTL-Anti-Aggregate Wash (CTL, Ohio, USA). PBMC amplification was performed according to a modified “rapid amplification” protocol described elsewhere (Jin et al., 2012). 1E7 cells were seeded in 30 mL of TIL medium in 6-well G-rex plates (Wilson Wolf, Minnesota, USA) supplemented with 20% FBS, 1% L-glutamine, 1% P / S, 15 mM HEPES, 1 mM Na pyruvate, 50 μM 2-mercaptoethanol, 100 IU / ml recombinant human IL-2 (Peprotech, New Jersey, USA), and 50 ng / ml anti-human CD3 (Thermofisher, Massachusetts, USA). PBMCs were incubated at 37°C for 3 days. Irradiated PBMCs from multiple donors were then added to the PBMCs at a ratio of 1:200, with a 1:1 TIL:REM medium. Rapid expansion medium (REM) consisted of RPMI 1640 supplemented with 20% FBS, 1% L-glutamine, 1% P / S, and 100 IU / ml recombinant hIL-2. Cell counting was performed after 2 days of incubation, and the cell density was maintained at 5E6 cells / well until day 14 when cells were collected, washed with basal medium, and stored at -180°C until use.

[0093] DC generation: DCs were generated from HLA-A 02:01 PBMCs using a previously established protocol (Zafar et al., 2017). Briefly, HLA-A 02:01 PBMCs (CTL, Ohio, USA) were thawed, and CD14+ cells were isolated using human CD14+ microbeads (Miltenyi Biotec, North Rhine-Westphalia, Germany) according to the manufacturer's instructions. The CD14+ cells were then cultured in T75 flasks containing 10 mL of RPMI supplemented with 1000 U / ml GMCSF (Peprotech, New Jersey, USA) and 20 ng / ml IL-4 (Peprotech, New Jersey, USA). After 5 days of incubation, 50 μg / ml of UM-UC-3 cell lysate was added. The next day, 100 ng / ml of lipopolysaccharide (Sigma-Aldrich, Missouri, USA) was added for overnight culture, and the mature DCs were used for animal experiments the following day.

[0094] Animal experiments: 2E6 UM-UC-3 cells / animal were transplanted into the lower left flank of 56 immunodeficient females (4-5 weeks old) of NOD.Cg-PrkdcscidIl2rgtm1Sug / JicTac (Taconic Biosciences GmbH, Leverkusen, Germany). 1E7 partially HLA-matched PBMCs and 2E3 DCs were administered intraperitoneally. The following day (Day 1), mice were treated with 1E9 VP of TILT-234 via tail vein (iv), administered every 3 days for 6 rounds. Paclitaxel (Fresenius Kabi, Germany) (2.5 mg / kg) and aPD-1 (Merck&Co, New Jersey, USA) (5 mg / kg) were administered intraperitoneally (ip), every 6 days for 3 rounds. Tumor weight was measured and recorded every other day until Day 6, and then daily until Day 19, after which the animals were euthanized. The tumor volume is calculated using the formula (length × width 2) / 2, and the percentage of tumor growth is calculated by normalizing the measurement to the tumor volume on day 0.

[0095] result The combination of paclitaxel and TILT-234 exhibited the highest cytotoxicity in both UM-UC-3 cells and in vitro tumor cultures. Injury effect To assess the ability of combination therapies to induce cell killing, UM-UC-3 cells and ex vivo samples surgically removed from patients with urothelial carcinoma were treated with TILT-234 and paclitaxel. In the commercial cell line (UM-UC-3), we observed a statistically significant enhancement in cell killing in the paclitaxel plus TILT-234 combination therapy group compared to monotherapy alone (p<0.05) (Fig. 1a). When assessing the cytotoxic effects of the combination therapy in patient samples, some differences were observed between patient samples, likely due to cell population heterogeneity present in each individual tumor (Fig. 1b, c). Despite this difference, patient sample #1 showed a trend toward enhanced cell killing in the combination therapy group, but this did not reach statistical significance (p=0.0887) (Fig. 1b). In contrast, patient sample #2 showed decreased susceptibility to TILT-234 treatment alone; however, the combination with paclitaxel resulted in the lowest cell viability among all treatment groups (Fig. 1c).

[0096] The triple therapy of anti-PD1, paclitaxel and TILT-234 improved tumor control in a humanized mouse model.

[0097] like Figure 2 As described, humanized mice were treated with TILT-234 every 3 days, followed by paclitaxel / aPD-1 every 6 days for a total of 18 days. Mice treated with TILT-234, paclitaxel, and aPD-1 alone showed significantly reduced tumor growth compared to the virus, paclitaxel, or aPD-1 alone (p<0.01). Figure 3 Comparing dual combination therapies including TILT-234 with chemotherapy or antibody immunotherapy, the combination of virus with aPD-1 and paclitaxel (triple therapy) showed a statistically significant advantage.

[0098] Example 2 Materials and methods Patient sample processing After surgical resection of the patient's urothelial tumor (bladder cancer), the tumor was cut into smaller fragments and placed at 37°C in RPMI 1640 supplemented with 1% L-glutamine, 1% penicillin / streptomycin, type I collagenase, type IV collagenase, DNase I, and elastase (Worthington Biochemical, NJ, USA) for 2 hours. Following digestion, the sample was filtered through a 70 μM filter membrane, and ACK lysis buffer (Sigma-Aldrich, MI, USA) was added to remove residual red blood cells, yielding a single-cell suspension. The sample was then frozen in freezing medium (90% FBS, 10% DMSO) and stored at -180°C until future use.

[0099] Cell viability assay Single-cell suspensions isolated from patient urothelial tumor (bladder cancer) samples were seeded at a density of 1E5 cells / well in RPMI 160 (10% FBS). Approximately 24 hours after seeding, cells were treated with TILT-234 (250 VP), paclitaxel (0.01 μM), and anti-PD1 (20 μg / ml), alone or in combination. After 144 hours (six days) of treatment, CellTiter 96 AQueous One Solution proliferation assay reagent was added, and the cells were incubated at 37°C for two hours. The absorbance of each well was measured at 490 nm using a HidexSense plate reader (Hidex, Turku, Finland).

[0100] Flow cytometry - Mouse tumors Mice were euthanized on day 19 of the treatment regimen (see Example 1, Materials and Methods, Experimental Animals), tumors were harvested, mechanically processed into single-cell suspensions, and frozen at -80°C. 1E6 cells / well were seeded for subsequent flow cytometry staining. For intracellular staining, cells were permeabilized using the BD Cytofix / Cytoperm Plus fixation / permeabilization kit (BD Biosciences, NJ, USA) according to the manufacturer's instructions and stained using the antibodies listed below. Cells were collected using a Novocyte Quanteon flow cytometer (Agilent, CA, USA), with 100k events collected per well. Cell gating and data processing were performed in FlowJo v10.7.1 (FlowJo LLC, OR, USA). A list of all antibodies used in this analysis can be found in Table 1.

[0101] Table 1

[0102] result Triple therapy enhances tumor cell killing in ex vivo tumor tissue cultures. When the cytotoxic effects of triple therapy were compared with other treatment regimens, triple therapy resulted in the lowest cell viability, highlighting the cytotoxic capabilities of this combination in the patient sample. Figure 4 ).

[0103] In mice treated with triple therapy, the expression of T-cell granzyme B and IFNG was enhanced in tumors. Flow cytometry analysis of tumors isolated from humanized mice treated with different treatment combinations revealed a significant increase in the activation of both CD4+ and CD8+ T cells in the triple therapy group. Specifically, compared with other treatment groups, the median fluorescence intensity (MFI) of granzyme B and IFNG in both CD4+ and CD8+ T cells was significantly enhanced (Figure 5). Statistical analysis revealed significant differences in the intensity of CD4+ and CD8+ granzyme B between triple therapy and TILT-234 monotherapy and TILT-234 + PTX combination therapy. Furthermore, the CD4+ IFNG intensity was significantly higher in the triple therapy group compared with TILT-234 monotherapy. However, no significant difference in CD8+ IFNG intensity was observed among the treatment groups.

[0104] References Citing non-patent documents: Jin J, Sabatino M, Somerville R, et al. Simplified method of thegrowth of human tumor infiltrating lymphocytes in gas-permeable flasks tonumbers needed for patient treatment. J Immunother. 2012;35(3):283-292. doi:10.1097 / CJI.0b013e31824e801f Knowles MA, Hurst CD. Molecular biology of bladder cancer: new insights into pathogenesis and clinical diversity. Nat Rev Cancer 2015 151.2014;15(1):25-41. doi:10.1038 / nrc3817 Kyo S, Takakura M, Fujiwara T, Inoue M. Understanding and exploiting hTERT promoter regulation for diagnosis and treatment of human cancers. Cancer Sci. 2008 Aug;99(8):1528-38. Taipale K, Tähtinen S, Havunen R, et al. Interleukin 8 activityinfluences the efficacy of adenoviral oncolytic immunotherapy in cancerpatients. Oncotarget. 2018;9(5):6320-6335. doi:10.18632 / oncotarget.23967 Tilsed CM, Scott AF, Nowak AK, Lake RA, Lesterhuis WJ. Cancerchemotherapy: insights into cellular and tumor microenvironmental mechanismsof action. Front. Oncol. 29 July 2022; Vol. 12, doi:10.3389 / fonc.2022.960317 Zafar S, Parviainen S, Siurala M, et al. Intravenously usable fullyserotype 3 oncolytic adenovirus coding for CD40L as an enabler of dendriticcell therapy. Oncoimmunology. 2017;6(2). doi:10.1080 / 2162402X.2016.1265717 Cited patent documents: WO 2014170389。

Claims

1. A method for treating cancer in a subject who requires such treatment, comprising administering to the subject an effective amount of: (a) an oncolytic adenovirus vector encoding CD40L as a transgenic vector, (b) preferably selectively binding to PD-L1 or one or more immune checkpoint inhibitors of PD-1, and (c) a chemotherapeutic agent.

2. The method according to claim 1, wherein, The one or more immune checkpoint inhibitors selectively bind to PD-L1 or PD-1.

3. The method according to claim 1, wherein, The chemotherapeutic agent is an antimicrotubule agent.

4. The method according to claim 3, wherein, The antimicrotubule agent is paclitaxel.

5. The method according to claim 1, wherein, The oncolytic adenovirus vector can be administered intratumorally, intravenously, intra-arterially, or intraperitoneally.

6. The method according to claim 5, wherein, The oncolytic adenovirus vector was administered intravenously.

7. The method according to claim 2, wherein, The immune checkpoint inhibitor is a monoclonal antibody that selectively binds to PD-L1.

8. The method according to claim 7, wherein, The monoclonal antibody that selectively binds to PD-L1 is selected from the group consisting of: BMS-936559, LY3300054, atezolizumab, durvalumab, and averumab.

9. The method according to claim 2, wherein, The immune checkpoint inhibitor is a monoclonal antibody that selectively binds to PD-1.

10. The method according to claim 9, wherein, The monoclonal antibody that selectively binds to PD-1 is selected from the group consisting of: pembrolizumab (MK-3475), nivolumab (BMS-936558), cimiprimab, dotalimab, toripalimab, and refulimab.

11. The method according to claim 1, wherein, The virus was divided into approximately 10 6 -10 14 VP, 10 6 -10 12 VP, 10 8 -10 14 VP, 10 8 -10 12 VP, 10 10 -10 12 VP or 10 8 -10 10 VP dosage.

12. The method according to claim 1, wherein, The checkpoint inhibitor was administered at a dose of approximately 2 mg / kg to 25 mg / kg.

13. The method according to claim 1, wherein, The chemotherapeutic agent is an antimicrotubule agent selected from the group consisting of paclitaxel and docetaxel, preferably at a concentration of about 60-300 mg / m². 2 Administer within the dosage range.

14. The method according to claim 1, wherein, The subjects had cancers selected from the following: nasopharyngeal carcinoma, synovial carcinoma, hepatocellular carcinoma, kidney cancer, connective tissue cancer, melanoma, lung cancer, intestinal cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, laryngeal cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, Ewing sarcoma, cancer of unknown primary site, carcinoid tumor, gastrointestinal carcinoid tumor, fibrosarcoma, breast cancer, Paget's disease, cervical cancer, esophageal cancer, gallbladder cancer, head cancer, etc. Cervical cancer, eye cancer, kidney 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, thymic cancer, thyroid cancer, trophoblastic cancer, vesicular birthmark, uterine cancer, endometrial cancer, vaginal cancer, vulvar cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gingival cancer, heart cancer, lip cancer, meningeal cancer, oral cancer, nerve cancer, palate cancer, parotid gland cancer, peritoneal cancer, pharyngeal cancer, pleural cancer, salivary gland cancer, tongue cancer, and tonsil cancer.

15. The method according to claim 14, wherein, The subjects had urothelial carcinoma, breast cancer, prostate cancer, or bladder cancer.

16. The method according to claim 1, wherein, The treatment was the subject's first cancer treatment.

17. The method according to claim 1, wherein, The subjects had at least one previous failure of chemotherapy or immunotherapy, such as treatment with immune checkpoint inhibitors.

18. The method of claim 1, further comprising the following steps: The subject was treated with one or more additional therapies selected from radiotherapy, chemotherapy, anti-angiogenic agents, or targeted therapies, such as alkylating agents, nucleoside analogs, cytoskeleton modifiers, cell inhibitors, monoclonal antibodies, and kinase inhibitors.

19. The method according to claim 18, wherein, The chemotherapy agent is cisplatin, epirubicin, cyclophosphamide, or gemcitabine.

20. The method according to claim 1, wherein, The subjects were human.

21. The method according to claim 1, wherein, The subject was simultaneously administered a first dose of the oncolytic adenovirus vector and a first dose of the immune checkpoint inhibitor.

22. The method according to claim 1, wherein, The first dose of the oncolytic adenovirus vector and the first dose of the chemotherapeutic drug were simultaneously administered to the subject.

23. The method according to claim 1, wherein, The first dose of the checkpoint inhibitor and the first dose of the chemotherapy drug were administered simultaneously to the subject.

24. The method according to claim 1, wherein, The oncolytic adenovirus vector, the immune checkpoint inhibitor, and the chemotherapeutic drug are administered sequentially in a first dose.

25. The method according to claim 1, wherein, The oncolytic adenovirus vector encoding CD40L encodes one or more other cytokines selected from the group consisting of: interferon α, interferon β, interferon γ, complement C5a, IL-2, TNFα, IL-12, IL-23, IL-15, IL-17, CCL1, CCL11, CCL12, CCL13, CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL17, CCL18, CCL19, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23-1, CCL23-2, CCL24, CCL25-1, CCL25-2, CCL26, CCL2 7. CCL28, CCL3, CCL3L1, CCL4, CCL4L1, CCL5, 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, CXCL9, CXCR1, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7, and XCL2.

26. The method according to claim 1, wherein, The CD40L-encoded oncolytic adenovirus vector includes - Deletion in the E3 region and a tumor-specific promoter for expressing the transgene at the location of the E3 deletion region. - hTERT promoter for E1A tumor-specific expression - The nucleic acid sequence encoding CD40L in the E3 region leads to replication-associated control of transgene expression under the viral E3 promoter.

27. The method according to claim 26, wherein, The E3 region includes the absence of one or more regions selected from E3 9-kDa, E3 10.2 kDa, E3 15.2 kDa, and E3 15.3 kDa.

28. The method according to claim 26 or 27, wherein, The tumor-specific promoter used to express the transgene is CMV or E2F.

29. The method according to any one of claims 24-27, wherein, The oncolytic adenovirus vector is a serotype 3 (Ad3) oncolytic adenovirus vector.

30. The method according to any one of claims 1-29, wherein, The oncolytic adenovirus vector encodes only one transgene, which is CD40L.

31. A pharmaceutical composition comprising: (a) an oncolytic adenovirus vector encoding CD40L as a transgene, preferably as the only transgene in the vector, (b) preferably selectively binding to PD-L1 or one or more immune checkpoint inhibitors of PD-1, and (c) a chemotherapeutic agent.

32. The composition according to claim 31, wherein, The one or more immune checkpoint inhibitors selectively bind to PD-L1 or PD-1.

33. The pharmaceutical composition according to claim 31, wherein, The CD40L-encoded oncolytic adenovirus vector includes - Deletion in the E3 region and tumor-specific promoters for expressing CD40L at the location of the E3 deletion region. - hTERT promoter for E1A tumor-specific expression - The E3 region encodes at least CD40L as one or more transgenic nucleic acid sequences, resulting in replication-associated control of transgenic expression under the viral E3 promoter, and - CMV or E2F promoters for tumor-specific expression of CD40L.

34. The pharmaceutical composition according to claim 31, wherein, The chemotherapeutic agent is taxane, preferably selected from the group consisting of paclitaxel and docetaxel.

35. A reagent kit comprising a first container, a second container, a third container, and a packaging instruction manual, wherein, The first container contains at least one dose of a pharmaceutical composition comprising an oncolytic adenovirus vector encoding CD40L as a transgene, preferably as the sole transgene in the vector; the second container contains at least one dose of a pharmaceutical composition comprising a checkpoint inhibitor; the third container contains a chemotherapeutic agent, preferably taxane; and the package insert includes instructions for treating an individual with cancer using one or more of the pharmaceutical compositions.

36. The kit according to claim 35, wherein, The CD40L-encoded oncolytic adenovirus vector includes - Deletion in the E3 region and tumor-specific promoters for expressing CD40L at the location of the E3 deletion region. - hTERT promoter for E1A tumor-specific expression - The nucleic acid sequence encoding CD40L in the E3 region, and - CMV or E2F promoters for tumor-specific expression of CD40L.

37. Use of an oncolytic adenovirus vector encoding CD40L as a transgenic vector, in conjunction with an immune checkpoint inhibitor and a chemotherapeutic agent, for the treatment of cancer or tumors in a subject.

38. The oncolytic adenovirus vector for the use according to claim 37, wherein, The immune checkpoint inhibitors selectively bind to PD-L1 or PD-1.

39. The oncolytic adenovirus vector for the use according to claim 37 or 38, wherein, The chemotherapeutic agent is an antimicrotubule agent.

40. The oncolytic adenovirus vector according to any one of claims 37-39, wherein, The antimicrotubule agent is paclitaxel.

41. The oncolytic adenovirus vector according to any one of claims 37-40, wherein, The oncolytic adenovirus vector can be administered intranasally, intratumorally, intravenously, intra-arterially, or intraperitoneally.

42. The oncolytic adenovirus vector according to any one of claims 37-41, wherein, The oncolytic adenovirus vector was administered intravenously.

43. The oncolytic adenovirus vector according to any one of claims 37-42, wherein, The immune checkpoint inhibitor is a monoclonal antibody that selectively binds to PD-L1.

44. The oncolytic adenovirus vector for the use according to claim 43, wherein, The monoclonal antibody that selectively binds to PD-L1 is selected from the group consisting of: BMS-936559, LY3300054, atezolizumab, durvalumab, and averumab.

45. The oncolytic adenovirus vector according to any one of claims 37-42, wherein, The immune checkpoint inhibitor is a monoclonal antibody that selectively binds to PD-1.

46. ​​The oncolytic adenovirus for the use according to claim 45, wherein, The monoclonal antibody that selectively binds to PD-1 is selected from the group consisting of: pembrolizumab (MK-3475), nivolumab (BMS-936558), cimiprimab, dotalimab, and refulimab.

47. The oncolytic adenovirus vector according to any one of claims 37-46, wherein, The virus is approximately 10 6 -10 14 VP, 10 6 -10 12 VP, 10 8 -10 14 VP, 10 8 -10 12 VP, 10 10 -10 12 VP or 10 8 -10 10 VP dosage.

48. The oncolytic adenovirus vector according to any one of claims 37-47, wherein, The checkpoint inhibitor was administered at a dose of approximately 2 mg / kg to 25 mg / kg.

49. The oncolytic adenovirus vector for use according to any one of claims 37-48, wherein, The chemotherapeutic agent is taxane selected from the group consisting of paclitaxel and docetaxel, and preferably administered in a dose range of about 60-300 mg / m².

50. The oncolytic adenovirus vector for use according to any one of claims 37-49, wherein, The subjects had cancer selected from the following groups: nasopharyngeal carcinoma, synovial carcinoma, hepatocellular carcinoma, kidney cancer, connective tissue cancer, melanoma, lung cancer, intestinal cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, laryngeal cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, Ewing sarcoma, cancer of unknown primary site, carcinoid tumor, gastrointestinal carcinoid tumor, fibrosarcoma, breast cancer, Paget's disease, cervical cancer, esophageal cancer, gallbladder cancer. Cancer, including head and neck cancer, eye cancer, kidney 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, thymic cancer, thyroid cancer, trophoblastic cell carcinoma, vesicular mass, uterine cancer, endometrial cancer, vaginal cancer, vulvar cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gingival cancer, heart cancer, lip cancer, meningeal cancer, oral cancer, nerve cancer, palate cancer, parotid gland cancer, peritoneal cancer, pharyngeal cancer, pleural cancer, salivary gland cancer, tongue cancer, and tonsil cancer.

51. The oncolytic adenovirus vector for the use according to claim 50, wherein, The subjects had urothelial carcinoma, breast cancer, prostate cancer, or bladder cancer.

52. The oncolytic adenovirus vector according to any one of claims 37-51, wherein, The treatment was the subject's first cancer treatment.

53. The oncolytic adenovirus vector according to any one of claims 37-51, wherein, The subjects had at least one previous failure of chemotherapy or immunotherapy, such as treatment with immune checkpoint inhibitors.

54. The oncolytic adenovirus vector for the use according to claim 53, wherein, The subjects had anti-PD-1 refractory cancer, preferably urothelial carcinoma, breast cancer, prostate cancer, or bladder cancer.

55. The oncolytic adenovirus vector for any of the uses described in any one of claims 37-54, comprising administering to the subject one or more additional therapies selected from: radiotherapy, chemotherapy, anti-angiogenic agents or targeted therapies, such as alkylating agents, nucleoside analogs, cytoskeleton modifiers, cell inhibitors, monoclonal antibodies, kinase inhibitors.

56. The oncolytic adenovirus vector for the use according to claim 55, wherein, The chemotherapeutic agents in the additional chemotherapy are cisplatin, cyclophosphamide, epirubicin, or gemcitabine, preferably used in conjunction with an antimicrotubule agent.

57. The oncolytic adenovirus vector according to any one of claims 37-56, wherein, The subjects were human.

58. The oncolytic adenovirus vector according to any one of claims 37-57, wherein, The subject was simultaneously administered a first dose of the oncolytic adenovirus vector and a first dose of the immune checkpoint inhibitor.

59. The oncolytic adenovirus vector according to any one of claims 37-57, wherein, The first dose of the oncolytic adenovirus vector and the first dose of the chemotherapy drug were simultaneously administered to the subject.

60. The oncolytic adenovirus vector for use according to any one of claims 37-57, wherein, The first dose of the checkpoint inhibitor and the first dose of the chemotherapy drug were administered simultaneously to the subject.

61. The oncolytic adenovirus vector for use according to any one of claims 37-57, wherein, The oncolytic adenovirus vector, the immune checkpoint inhibitor, and the chemotherapeutic drug are administered sequentially in a first dose.

62. The oncolytic adenovirus vector according to any one of claims 37-61, wherein, The oncolytic adenovirus vector encoding CD40L encodes one or more other cytokines selected from the group consisting of: interferon α, interferon β, interferon γ, complement C5a, IL-2, TNFα, IL-12, IL-23, IL-15, IL-17, CCL1, CCL11, CCL12, CCL13, CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL17, CCL18, CCL19, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23-1, CCL23-2, CCL24, CCL25-1, CCL25-2, CCL26, CCL2 7. CCL28, CCL3, CCL3L1, CCL4, CCL4L1, CCL5, 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, CXCL9, CXCR1, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7, and XCL2.

63. The oncolytic adenovirus vector for use according to any one of claims 37-62, wherein, The CD40L-encoded oncolytic adenovirus vector includes - Deletion in the E3 region and tumor-specific promoters for expressing CD40L at the location of the E3 deletion region. - hTERT promoter for E1A tumor-specific expression - The nucleic acid sequence encoding CD40L in the E3 region, and - CMV or E2F promoters for tumor-specific expression of CD40L.

64. The oncolytic adenovirus vector for the use according to claim 63, wherein, The E3 region includes the absence of one or more regions selected from E3 9-kDa, E3 10.2 kDa, E3 15.2 kDa, and E3 15.3 kDa.

65. The oncolytic adenovirus vector for the use according to claim 63 or 64, wherein, The tumor-specific promoter used to express the transgene is CMV or E2F.

66. The oncolytic adenovirus vector according to any one of claims 37-65, wherein, The oncolytic adenovirus vector is a serotype 3 (Ad3) oncolytic adenovirus vector.

67. The oncolytic adenovirus vector for use according to any one of claims 37-66, wherein, The oncolytic adenovirus vector encodes only one transgene, which is CD40L.

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