Novel administration regimens for oncolytic adenovirus vectors

Abstract

According to one aspect of the present invention, an oncolytic adenovirus vector for cancer treatment encoding at least TNFα and / or IL-2 is provided. The adenovirus vector is administered to a subject at least three times, preferably at least five, six, or seven times, in an active treatment cycle, with at least one administration, preferably the first, being systemic, and the active treatment cycle preferably does not include the administration of adoptive cell therapeutic compositions containing TIL or the administration of immune checkpoint inhibitors.

Description

[Technical Field]

[0001] This invention generally relates to virology, immunology, and medicine. In particular, this invention relates to a drug regimen for oncolytic adenovirus in cancer treatment, including formulation, route of administration, dosage, administration interval, and duration of treatment. [Background technology]

[0002] International Publication WO2014170389 relates to oncolytic adenovirus vectors used alone or in combination with therapeutic compositions, particularly adoptive cell compositions, for therapeutic applications and methods of cancer. Following years of development, oncolytic viruses are now beginning to be used as cancer treatments, particularly in combination with other anticancer agents. In clinical trials, oncolytic viruses have shown a favorable safety profile and promising efficacy. However, there is still room for improvement in the response, especially in patients with a high metastatic burden. This invention provides novel administration regimens of specific oncolytic adenoviruses that demonstrate, surprisingly, that monotherapy approaches may be effective in cancer patients. [Overview of the project]

[0003] The object of the present invention is to provide a dosing regimen to overcome the problems of inefficient and unpredictable cancer treatment. More specifically, the present invention provides a novel approach in oncolytic viral vector therapy. The object of the present invention is achieved by viral vectors, methods, and configurations having the features described in the appended independent claims. Some specific embodiments are defined in the dependent claims.

[0004] According to one aspect of the present invention, an oncolytic adenovirus vector for cancer treatment encoding at least TNFα and / or IL-2 is provided. The adenovirus vector is administered to a subject at least three times, preferably at least five, six, or seven times, in an active treatment cycle, with at least one administration, preferably the first, being systemic, and the active treatment cycle preferably does not include the administration of adoptive cell therapeutic compositions containing TIL or the administration of immune checkpoint inhibitors.

[0005] The drawings illustrate the present invention according to at least some embodiments of the present invention. [Brief explanation of the drawing]

[0006] [Figure 1] This describes the TILT-123 treatment regimen in humans. Trial participants were screened for eligibility up to two weeks prior to initiating TILT-123 treatment. Eligible patients were scheduled to receive intravenous TILT-123 (day 1) and multiple intratumoral doses (days 8, 22, 36, 50, and 64). Participants entering the treatment extension period (from day 78 onward) continued intratumoral or intravenous doses every three weeks. Positron emission tomography (PET) combined with radiographic computed tomography (CT) image analysis was performed on patients at baseline, day 78, and approximately every three months during the treatment extension period. [Figure 2]This study demonstrates the therapeutic effect of TILT-123 on tumor lesions in patients with advanced lung cancer, using positron emission tomography (PET) combined with X-ray computed tomography (X-ray) image analysis. Patients with stage IV non-small cell lung cancer received six doses of TILT-123 (one intravenous dose followed by multiple intratumor administrations). Representative PET-CT images of tumor lesions treated with TILT-123 (left lower neck / shoulder (lymph nodes)) and untreated tumor lesions (left axilla (lymph nodes)) are shown before and after treatment. Metabolic activity was quantified and measured as the maximum normalized uptake (SUVmax). The percentage change was calculated by comparing the SUVmax value before treatment with the value after TILT-123 administration. Data cutoff: March 1, 2023. [Figure 3] This study demonstrates the therapeutic effect of TILT-123 on tumor lesions in thyroid cancer patients using positron emission tomography (PET) combined with X-ray computed tomography (CT) image analysis. Patients with anaplastic thyroid cancer received 10 doses of TILT-123 (one intravenous dose followed by multiple intratumor doses). Representative PET-CT images of the tumor lesion treated with TILT-123 (lower abdomen) and two untreated tumor lesions (mesenteric lymph nodes and a solitary left lower lobe lung lesion) are shown before and after treatment (days 78 and 167). Metabolic activity was quantified as maximum normalized uptake (SUVmax) and measured at the lesion site. The computed tomography values ​​are shown in the table from TL1. Percentage changes were calculated by comparing pre-treatment SUVmax or CT values ​​with values ​​after TILT-123 administration. Data cutoff: March 1, 2023. [Figure 4]This shows the efficacy of TILT-123 treatment. A: Response evaluation in all injected lesions by CT. B: Response evaluation in all injected lesions by PET. C: Response evaluation in all imaged non-injected lesions by CT. D: Response evaluation in all imaged non-injected lesions by PET. In A-D, the best response is shown when the patient progresses to the extension period. E: Overall survival in the trial. F: Progression-free survival in the trial. G: Progression-free survival in the trial. In E-G, disease control was defined by PET-based criteria, and the comparison between disease control and non-disease control was evaluated by the Mantel-Cox log-rank test. ***=p<0.001, ****=p<0.0001. The dataset used to create the graphs includes data from efficacy cases 1 and 2. [Figure 5] This graph shows baseline neutralizing antibody levels and their changes over time. A: Neutralizing antibody levels against TILT-123 at baseline for all patients. B: Presence of neutralizing antibody levels at day 78 compared to disease control. Disease control is defined as SMD or higher at day 78. Non-disease control is defined as PMD or NA at day 78. Group comparisons were performed using Fisher's exact test. C: Comparison of baseline neutralizing antibody levels with overall survival across the study. Group comparisons were performed using the MaxCombo log-rank test. D: Overall neutralizing antibody titers across the entire treatment cohort. Baseline is defined as the pre-treatment value on day 1, and for each day from day 1 to day 64, the highest antibody titer (pre-treatment or post-treatment) for that day is shown. The dataset used to create the graphs includes data from efficacy cases 1 and 2. [Figure 6] This graph shows neutralizing antibodies and antitumor responses. A: Neutralizing antibodies across the entire study for patients showing the best RECIST 1.1 response. B: Neutralizing antibodies across the entire study for patients showing the best PET reference response. C: Neutralizing antibodies across the entire study for patients showing the longest survival time. The dataset used to create the graph includes data from efficacy cases 1 and 2. [Modes for carrying out the invention]

[0007] Oncolytic virus

[0008] In a preferred embodiment, the oncolytic virus of the present disclosure is an oncolytic adenovirus.

[0009] In this specification, “oncolytic adenovirus vector” refers to an adenovirus vector capable of infecting and killing cancer cells by selective replication between tumor cells and normal cells. Therefore, in this specification, “adenovirus vector” may refer to an adenovirus or adenovirus particle capable of transplanting nucleic acid into cells, or the transplanted nucleic acid itself. International Publication WO2014170389 discloses an oncolytic adenovirus vector encoding TNFα and / or IL-2 as a transgene usable in the present invention.

[0010] The vector may be modified by any method known to those skilled in the art. For example, it may be modified by deletion, insertion, mutagenesis, or modification of any viral region. The vector is prepared to be tumor-specific with respect to replication. For example, the adenovirus vector may include modifications in E1, E3, and / or E4, such as insertion of a tumor-specific promoter (e.g., for driving E1), deletion of regions (e.g., constant region 2 of E1 used in "D24", E3 / gp19k, E3 / 6.7k), and insertion of transgenes. Furthermore, the fiber knob region of the vector may also be modified. In one embodiment of the present invention, the adenovirus vector is an Ad5 / 3 comprising an Ad5 nucleic acid backbone and an Ad3 fiber knob or an Ad5 / 3 chimeric fiber knob.

[0011] In this specification, "adenovirus serotype 5 (Ad5) nucleic acid backbone" refers to the genome of Ad5.

[0012] The "Ad5 / 3 vector" refers to a chimeric vector having parts of both the Ad5 and Ad3 vectors. In certain embodiments of the present invention, the capsid modification of the vector is Ad5 / 3 chimerism. As used herein, the "Ad5 / 3 chimeric fiber knob" refers to a chimerism in which the knob portion of the fiber is derived from Ad serotype 3 and the remaining portion of the fiber is derived from Ad serotype 5. Specifically, in one embodiment, the construct has a fiber knob derived from Ad3 and the remaining portion of the genome is derived from Ad5 (SEQ ID NO: 5).

[0013] One approach to generating a tumor-specific oncolytic adenovirus is to design a 24-base pair deletion (D24) that affects the constant region 2 (CR2) of E1 (SEQ ID NO: 4). In wild-type adenovirus, CR2 binds to the cellular Rb tumor suppressor / cell cycle regulatory protein and plays a role in inducing the synthesis phase (S phase), i.e., the DNA synthesis or replication phase. The interaction between Rb and E1A requires 8 amino acids (121 - 127) in the conserved region of the E1A protein, which are deleted in this vector. The vector of the present invention contains a nucleotide deletion corresponding to amino acids 122 - 129 of the vector by Heise C. et al. (2000). Viruses having D24 have a reduced ability to overcome the G1-S checkpoint and are known to replicate efficiently only in cells where this interaction is unnecessary, such as tumor cells with a defect in the Rb-p16 pathway. Almost all human tumors are included in this.

[0014] It is also possible to replace the E1A endogenous viral promoter with, for example, a tumor-specific promoter. In certain embodiments of the present invention, the hTERT or E2F promoter is utilized in place of the E1A endogenous viral promoter.

[0015] In certain embodiments, the E1B19K gene (SEQ ID NO: 1), which is generally known to support the replication of adenoviral vectors, has a deletion (inactivating deletion) dE1B19K (SEQ ID NO: 2) that causes it to lose its function in this vector. Deletion of E1B19K is known to make cancer cells sensitive to TNFα and to promote apoptosis.

[0016] The sequence of the wild-type E1B19K gene is as follows (the deletable region is underlined): atggaggctt gggagtgttt ggaagatttt tctgctgtgc gtaacttgct ggaacag agc tctaacagta cctcttggtt ttggaggttt ctgtggggct catcccaggc aaagttagtc tgcagaatta aggaggatta caagtgggaa tttgaagagc ttttgaaatc ctgtggtgag ctgtttgatt ctttgaat ct gggtcaccag gcgcttttcc aagagaaggt catcaagact ttggattttt ccacaccggg gcgcgctgcg gctgctgttg cttttttgag ttttataaag gataaatgga gcgaagaaac ccatctgagc ggggggtacc tgctggattt tctggccatg catctgtgga gagcggttgt gagacacaag aatcgcctgc tactgttgtc ttccgtccgc ccggcgataa taccgacgga ggagcagcag cagcagcagg aggaagccag gcggcggcgg caggagcaga gcccatggaa cccgagagcc ggcctggacc ctcgggaatg a(SEQ ID NO: 1)[[ID=⑨]] [[ID=⑩]]

[0017] [[ID=⑪]] [[ID=⑫]]Therefore, in one embodiment, the sequence of dE1B19K in this viral vector is as follows: [[ID=⑬]] atggaggctt gggagtgttt ggaagatttt tctgctgtgc gtaacttgct ggaacagctg ggtcaccagg cgcttttcca agagaaggtc atcaagactt tggatttttc cacaccgggg cgcgctgcgg ctgctgttgc ttttttgagt tttataagg ataaatggag cgaagaaacc catctgagcg gggggtacct gctggatttt ctggccatgc atctgtggag agcggttgtg agacacaaga atcgcctgct actgttgtct tccgtccgcc cggcgataat accgacggag gagcagcagc agcagcagga ggaagccagg cggcggcggc aggagcagag cccatggaac ccgagagccg gcctggaccc tcgggaatga (Sequence code 2)

[0018] The E3 region is not essential for viral replication outside the body, but the E3 protein plays a crucial role in regulating the host immune response, specifically in suppressing both innate and specific (adaptive) immune responses. The gp19k / 6.7K deletion in E3 refers to the deletion of 965 base pairs from the adenovirus E3A region. The resulting adenovirus construct lacks both the gp19k and 6.7K genes (Kanerva A et al., 2005). The gp19k gene product is known to bind and segregate major histocompatibility complex type I (MHC I, HLA1 in humans) molecules within the endoplasmic reticulum, preventing infected cells from being recognized by cytotoxic T lymphocytes. Since many tumors are HLA1 / MHC1 deficient, the deletion of gp19k enhances the virus's tumor selectivity (the virus is eliminated more rapidly in normal cells than wild-type viruses, but there is no difference in tumor cells). The 6.7K protein is expressed on the cell surface and is involved in the downregulation of TNF-related apoptosis-inducing ligand (TRAIL) receptor 2.

[0019] Both deletions have merits. For example, in adoptive T cells, gp19k protein expression is counterproductive to restoring HLA / MHC expression for tumor epitope presentation; in fact, gp19k deletion is necessary for upregulation of HLA / MHC. As for 6.7k, one embodiment of the present invention is the production of TNFα from a virus, and one of its antitumor activities is a direct antitumor apoptosis-promoting effect (on both transduced and non-transduced bystander cells), so the presence of 6.7k is counterproductive.

[0020] In one embodiment of the present invention, the cytokine transgene is positioned within an E3 region lacking gp19k / 6.7k under the E3 promoter. This restricts transgene expression to tumor cells that allow viral replication and consequently activate the E3 promoter. The E3 promoter may be an exogenous promoter known to those skilled in the art (e.g., CMV promoter or E2F promoter, SEQ ID NO: 3) or an endogenous promoter, particularly an endogenous E3 promoter. The E3 promoter is primarily activated by replication, but some expression occurs when E1 is expressed. Since the selectivity of D24 viruses occurs after E1 expression (when E1 cannot bind to Rb), these viruses also express E1 in transduced normal cells. Therefore, regulating E1 expression is also important to restrict transgene expression via the E3 promoter to tumor cells.

[0021] In certain embodiments of the present invention, the oncolytic adenovirus vector is based on an adenovirus serotype 5 (Ad5) nucleic acid backbone comprising a 5 / 3 chimeric fiber knob and includes: an E2F1 promoter for tumor-specific expression of E1A; a 24-base pair deletion (D24) in the Rb-binding constant region 2 of adenovirus E1; nucleic acid sequence deletions in the leading frames of the viral gp19k and 6.7k; replication-associated transgene expression regulation under the viral E3 promoter by transgene insertion into the deletion region; and a nucleic acid sequence encoding at least one cytokine transgene at the position of the deleted adenovirus gene gp19k / 6.7k in the E3 region. In one embodiment of the present invention, the adenovirus vector is based on a human adenovirus.

[0022] The precise function of the Early Region (E3) protein in adenovirus 3 is unclear. Generally, in adenoviruses, deletion of the E3 protein does not hinder replication but is thought to affect the host's antiviral response to adenovirus. The E3 of the human adenovirus genome has the highest genetic diversity among the six adenovirus species (A-F) found in humans. This diversity in genetic content is mainly due to its location between the highly conserved E3-gp19K and E3-RIDα open reading frames (ORFs), which encode species-specific gene sets.

[0023] Cytotoxic T cell-mediated killing of virus-infected cells is regulated by E3-gp19K. This is achieved by inhibiting the transport of MHC class I to the cell membrane and inhibiting the formation of the TAP-MHC class I complex.

[0024] Therefore, in one embodiment of the present invention, the key molecule E3-gp19K is included in the adenovirus vector, thereby making viral replication more stealthy and allowing more time for tumor lysis and its beneficial effects. Furthermore, by retaining E3-gp19K, the induction of anti-adenovirus cytotoxic T cells is reduced, resulting in the acquisition of more antitumor T cells.

[0025] Cytokines are involved in immune responses through various mechanisms, including the recruitment of T cells to tumors. The nucleotide sequences encoding cytokine transgenes may originate from any animal, such as humans, apes, rats, mice, hamsters, dogs, or cats, but are particularly encoded by human sequences. The nucleotide sequences encoding transgenes may be modified to improve their effects, or they may remain unmodified, i.e., wild-type.

[0026] Certain embodiments of the present invention include a viral vector encoding at least one cytokine. In certain embodiments of the present invention, the cytokine is IL-2 or TNFα, and preferably the viral vector encodes both cytokines. In one embodiment of the present invention, the viral vector encodes IL-2 and / or TNFα, preferably another cytokine selected from the following group: interferon α, interferon β, interferon γ, complement C5a, CD40L, IL12, IL23, IL15, IL17, 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, CCL3, CCL3L1, CCL4, CCL4L1, CCL5, CCL6, CCL7, CCL8, CCL9, CCR10, CCR2, CCR5, CCR6, CCR7, CCR8, CCRL1, CCRL2, CX3CL1, CX3CR, CXCL1, C XCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCR1, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7, and XCL2.

[0027] The cytokine TNFα (tumor necrosis factor α) functions by attracting and activating T cells and reducing tumor immunosuppression, while IL-2 (interleukin 2) induces T cell proliferation.

[0028] In one embodiment of the present invention, the viral vector includes an internal ribosome entry site (IRES) or optionally a ribosomal shunt site 2A between two transgenes. Therefore, the IRES or ribosomal shunt site 2A may be between any cytokine, such as IL-2 and any other cytokine preferably selected from the cytokine group described above. In this specification, "IRES" refers to a nucleotide sequence that enables the initiation of translation midway through a messenger RNA sequence during protein synthesis. The IRES may be derived from any virus, but in one embodiment of the present invention, it is derived from encephalomyocarditis virus (EMCV). In this specification, "ribosomal shunt site 2A" refers to a translation initiation site where the ribosome physically bypasses a portion of the 5′ untranslated region to reach the start codon. Both the IRES and A2 allow the virus to express two transgenes from a single promoter (E3 promoter).

[0029] Detailed structural examples of oncolytic adenovirus vectors encoding TNFα and / or IL-2 as transgenes are disclosed in International Publication WO2014170389.

[0030] In summary, the main advantages of the present invention, which uses a viral vector containing at least one cytokine transgene, are as follows: i) Cytokines and the virus itself generate danger signals, mobilizing T cells and other immune cells to the tumor. ii) Cytokines induce T cell proliferation in tumors and local lymphoid tissues. iii) Cytokines and the virus itself promote the intratumoral proliferation of T cells (innate immune antitumor T cells), iv) Cytokines and / or viruses induce upregulation of antigen-presenting molecules (HLA) in cancer cells, conferring sensitivity to recognition and killing by T cells, and v) Cytokine and viral replication improve the tumor microenvironment by reducing immunosuppression and cellular anergy.

[0031] The viral vector used in this invention may include modifications other than those described above. Additional components and modifications are optional and not essential to this invention.

[0032] Insertion of exogenous elements can enhance the effect of the vector in target cells. The use of exogenous tissue-specific or tumor-specific promoters in recombinant vectors is common, and these are also applicable in this invention.

[0033] In summary, the replication of oncolytic viruses reduces immunosuppression and cellular anergy by recruiting T cells and inducing danger signals in tumors. These effects are mediated through evolutionarily conserved immune induction mechanisms and pathogen-associated molecular pattern recognition receptors that are not affected by tolerance. An additional advantage of the oncolytic platform is that it is replicable within tumors but not in normal cells, i.e., it has the ability to self-amplify in tumors. Furthermore, the oncolytic effect itself may contribute to the overall antitumor effect in humans.

[0034] cancer

[0035] The recombinant vector of the present invention is capable of replicating in tumor cells. In one embodiment of the present invention, the vector is capable of replicating in cells lacking the Rb pathway, particularly the Rb-p16 pathway. These deficient cells include all animal and human tumor cells. In this specification, “Rb pathway deficiency” refers to mutations and / or epigenetic changes in any gene or protein of the pathway. These deficiencies lead to overexpression of E2F in tumor cells, resulting in the elimination of Rb binding by E1A CR2, which is normally required for effective replication. Further selectivity is mediated by an E2F promoter that is activated only in the presence of free E2F, which is observed in Rb / p16 pathway deficient cells. In the absence of free E2F, E1A transcription does not occur, and the virus does not replicate. Incorporating the E2F promoter is important in preventing the expression of E1A in normal tissues, which could cause direct and indirect toxicity by allowing transgene expression from the E3 promoter.

[0036] The present invention relates to an approach for the treatment of cancer in a subject. In one embodiment of the present invention, the subject is a human or mammal, more specifically a mammalian or human patient, and more specifically a human or mammal suffering from cancer.

[0037] This approach can be used to treat any cancer or tumor, including both malignant and benign tumors, and can be applied to both primary and metastatic tumors. In one embodiment of the present invention, the cancer is characterized by tumor-infiltrating lymphocytes (TILs). The means of the present invention are particularly useful for treating metastatic solid tumors having tumor-infiltrating lymphocytes.

[0038] In this specification, “treatment” or “to treat” means administering at least an oncolytic adenovirus vector to a subject, preferably a mammal or human subject, for purposes including not only complete cure but also prevention, improvement, or reduction of cancer or tumor-related disorders or symptoms. The therapeutic effect may be evaluated by monitoring the patient’s symptoms, blood tumor markers, or, for example, tumor size, tumor metabolic activity, or patient survival.

[0039] In other embodiments of the present invention, the cancer or tumor is nasopharyngeal cancer, synovial cancer, hepatocellular carcinoma, renal cancer, connective tissue cancer, melanoma, lung cancer, intestinal cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, pharyngeal cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastric tumor (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's sarcoma, cancer of unknown primary origin, carcinoid, gastrointestinal carcinoid, fibrosarcoma, breast cancer, Paget's disease, cervical cancer, esophageal cancer. The cancers selected are from the group consisting of gallbladder cancer, head cancer, eye cancer, neck cancer, Wilms' tumor, Kaposi's sarcoma, prostate cancer, testicular cancer, Hodgkin's disease, non-Hodgkin 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, choriocarcinoma, 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, nerve cancer, palatine 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 kidney cancer, ovarian cancer, bladder cancer, prostate cancer, breast cancer, colorectal cancer, lung cancer (such as small cell lung cancer, non-small cell lung cancer, and squamous cell non-small cell lung cancer), gastric cancer, classical Hodgkin lymphoma, mesothelioma, and liver cancer. In a more preferred embodiment, the type of cancer or tumor is melanoma, lung cancer (such as small cell lung cancer, non-small cell lung cancer, and squamous cell non-small cell lung cancer), ovarian cancer, head and neck cancer, or thyroid cancer. In another more preferred embodiment, the type of cancer or tumor is soft tissue sarcoma, such as leiomyosarcoma or liposarcoma.

[0040] A clinician may examine a human or animal patient before classifying them as eligible for treatment according to the present invention. Based on results indicating deviations from normal values ​​and the presence of tumors or cancer, the clinician may propose treatment according to the present invention to the patient.

[0041] In one embodiment of the present invention, the subject or patient has failed at least once in previous chemotherapy, radiotherapy, or immunotherapy, such as immune checkpoint inhibitor (CPI) treatment. That is, the patient's cancer is a CPI-resistant tumor. In a preferred embodiment, the present invention relates to the treatment of a CPI-resistant tumor.

[0042] In this specification, "immune checkpoint inhibitor (CPI)" refers to any compound capable of inhibiting the function of an immune checkpoint protein. This inhibition includes not only complete blockade of function but also reduction of function. In particular, the immune checkpoint protein in question is a human checkpoint protein. Therefore, the immune checkpoint inhibitor is preferably an inhibitor of human immune checkpoints.

[0043] 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. The pathways involving LAG3, BTLA, B7-H3, B7-H4, TIM3, and KIR are recognized in the art as constituting immune checkpoint pathways similar to the CTLA-4-dependent and PD-1-dependent pathways. Immune checkpoint inhibitors may 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. In one embodiment, the immune checkpoint inhibitor is an antibody.

[0044] Pharmaceutical composition

[0045] The pharmaceutical composition of the present invention comprises at least one of the viral vectors of the present invention. The present invention also provides the 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 vectors, the pharmaceutical composition may comprise other therapeutic active ingredients, as well as pharmaceutically acceptable carriers, buffers, excipients, adjuvants, additives, preservatives, bactericides, fillers, stabilizers and / or thickeners, and / or any components commonly found in the corresponding product. The selection of appropriate components and appropriate manufacturing methods for formulating the composition is within the general knowledge of those skilled in the art.

[0046] The pharmaceutical composition may be in any form suitable for administration, such as a solid, semi-solid, or liquid. The formulation can be selected from, but is not limited to, solutions, sprays, emulsions, suspensions, tablets, pellets, and capsules. The compositions of the present invention are not limited to any particular formulation and can be formulated into any known pharmaceutically acceptable formulation. The pharmaceutical composition may be manufactured by any conventional method known in the art.

[0047] The pharmaceutical kit of the present invention comprises an oncolytic adenovirus vector encoding TNFα and / or IL-2 as a transgene. In one embodiment, the oncolytic adenovirus vector encoding TNFα and / or IL-2 as a transgene is formulated as the first formulation, and in combination therapy with other active ingredients, the latter is formulated as the second formulation. In another embodiment of the present invention, the first and second formulations are administered to the subject simultaneously or sequentially in any order. In another embodiment, the kit is used for the treatment of cancer or tumors.

[0048] Administration and administration regimens

[0049] The vector or pharmaceutical composition of the present invention can be administered to any mammalian subject. In certain embodiments of the present invention, the subject is human. The mammal may be selected from the group consisting of pets, livestock, and production animals.

[0050] Any conventional method may be used to administer the vector or composition of the present invention to the 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 dose and frequency of administration of each therapeutic agent in combination therapy depend in part on the specific therapeutic agent, the severity of the cancer being treated, and the characteristics of the patient. Preferably, the administration regimen maximizes the amount of each therapeutic agent delivered to the patient within an acceptable level of side effects.

[0051] In one embodiment of the present invention, the oncolytic virus is administered by intratumoral (IT), intra-arterial, intravenous (IV), intrapleural (IPL), intravesical, intra-cavitary, or intraperitoneal (IP) injection, or by oral or nasal administration. In another embodiment of the present invention, the oncolytic virus is administered systemically. Systemic administration is a route through which a drug is delivered to the circulatory system to act systemically. Systemic administration can be carried out by parenteral administration (i.e., generally by injection or infusion). Any combination of administration methods is also possible. This approach can produce systemic efficacy despite being a local injection.

[0052] The effective dose of the vector depends at least on the patient requiring treatment, the type and location of the tumor, and the stage of the tumor. Each dose is, for example, approximately 1 × 10⁻⁶. 8 Approximately 1 x 10⁻¹⁶ particles (VP) 14 Up to VP, specifically about 5 x 10 9 Approximately 1 x 10 from VP 13 More specifically, up to VP, approximately 3 x 10 9 Approximately 4 x 10 from VP 12 It can vary up to VP. In one embodiment, an oncolytic adenovirus vector encoding at least IL-2 and / or TNFα is 1 × 10⁻¹⁶10 ~1×10 14 It is administered in terms of the amount of virus particles. In another embodiment of the present invention, the dosage is about 5×10 10 ~5×10 11 in the range of VP.

[0053] In a particular embodiment, the present invention relates to an administration regimen in which the adenovirus vector is administered to a subject at least 3 times, preferably at least 4 times, 5 times, 6 times, or 7 times in an active treatment cycle, at least one administration, preferably the first administration, is systemic administration, and the active treatment cycle preferably does not include the administration of an adoptive cell therapy composition containing TIL or the administration of an immune checkpoint inhibitor. In a preferred embodiment, the period of the active treatment cycle is at least 60 days, preferably 61 to 81 days. In another preferred embodiment, the active treatment cycle is performed as a monotherapy for cancer using the adenovirus vector from the start of the treatment cycle to the last administration of the adenovirus vector.

[0054] As used herein, "adoptive cell therapy composition" refers to any composition containing cells suitable for adoptive cell transplantation (see, for example, International Publication WO2014170389). Generally, adoptive cell therapy compositions include cell types selected from the group consisting of tumor infiltrating lymphocytes (TIL), TCR (heterologous T cell receptor) modified lymphocytes, and CAR (chimeric antigen receptor) modified lymphocytes. Adoptive cell therapy compositions may include cell types selected from the group consisting of T cells, CD8+ cells, CD4+ cells, NK cells, delta gamma T cells, regulatory T cells, and peripheral blood mononuclear cells. Generally, TIL, T cells, CD8+ cells, CD4+ cells, NK cells, delta gamma T cells, regulatory T cells, or peripheral blood mononuclear cells form an adoptive cell therapy composition. As a specific example, the adoptive cell therapy composition contains T cells. As used herein, "tumor infiltrating lymphocytes (TIL)" refers to white blood cells that have migrated from the bloodstream into the tumor.

[0055] In a preferred embodiment, the systemic administration is parenteral, preferably intravenous.

[0056] In another preferred embodiment, subsequent administrations are performed locally, preferably intratumorally, intraperitoneally, or intrapleurally.

[0057] In another preferred embodiment, the second dose of the adenovirus vector is administered within 10 days, preferably on day 8, after the start of treatment, i.e., after the first dose of the adenovirus vector.

[0058] In another preferred embodiment, further administration of the adenovirus vector is performed within 20 days after the start of treatment, preferably on day 15. In a more preferred embodiment, further administration of the adenovirus vector is performed within 20 days after the start of treatment, so that the previous administration is performed within 10 days after the start of treatment.

[0059] In another preferred embodiment, further administration of the adenovirus vector is performed within 32 days after the start of treatment, preferably on day 22 or day 29. In a more preferred embodiment, further administration of the adenovirus vector is performed within 32 days after the start of treatment, so that the previous administration is performed within 20 days after the start of treatment.

[0060] In another preferred embodiment, further administration of the adenovirus vector is performed within 46 days after the start of treatment, preferably on day 36 or day 43. In a more preferred embodiment, further administration of the adenovirus vector is performed within 46 days after the start of treatment, so that the previous administration is performed within 32 days after the start of treatment.

[0061] In another preferred embodiment, further administration of the adenovirus vector is performed within 60 days after the start of treatment, preferably on day 50 or day 57. In a more preferred embodiment, further administration of the adenovirus vector is performed within 60 days after the start of treatment, so that the previous administration is performed within 46 days after the start of treatment.

[0062] In another preferred embodiment, further administration of the adenovirus vector is performed within 67 days after the start of treatment, preferably on day 64. In a more preferred embodiment, further administration of the adenovirus vector is performed within 67 days after the start of treatment, so that the previous administration is performed within 60 days after the start of treatment.

[0063] In another preferred embodiment, further administration of the adenovirus vector is performed within 81 days after the start of treatment, preferably on day 71 or day 78. In a more preferred embodiment, further administration of the adenovirus vector is performed within 81 days after the start of treatment, so that the previous administration is performed within 67 days after the start of treatment.

[0064] In another preferred embodiment, the second dose of the adenovirus vector is administered after the start of treatment, i.e., within 2 to 6 hours after the first dose of the adenovirus vector, and the administration is preferably entirely intravenous in the treatment.

[0065] In another preferred embodiment, two further doses (preferably the third and fourth) of the adenovirus vector are administered at intervals of 2 to 6 hours within 5 days after the start of treatment, preferably on the third day.

[0066] In another preferred embodiment, two further doses (preferably the fifth and sixth doses) of the adenovirus vector are administered within 10 days after the start of treatment, preferably on day 8, at intervals of 2 to 6 hours. In a more preferred embodiment, two further doses of the adenovirus vector are administered within 10 days after the start of treatment, so that the previous dose was administered within 5 days after the start of treatment.

[0067] In another preferred embodiment, two further doses (preferably the seventh and eighth) of the adenovirus vector are administered within 13 days after the start of treatment, preferably on day 10, at intervals of 2 to 6 hours. In a more preferred embodiment, two further doses of the adenovirus vector are administered within 13 days after the start of treatment, so that the previous dose is administered within 8 or 10 days after the start of treatment.

[0068] In another preferred embodiment, two further doses of the adenovirus vector (preferably the ninth and tenth doses) are administered within 30 days of the start of treatment, preferably on day 22, at intervals of 2 to 6 hours. In a more preferred embodiment, two further doses of the adenovirus vector are administered within 30 days of the start of treatment, so that the previous dose is administered within 13 days of the start of treatment.

[0069] In another preferred embodiment, two further doses of the adenovirus vector (preferably the 11th and 12th doses) are administered at intervals of 2 to 6 hours within 46 days after the start of treatment, preferably on day 43. In a more preferred embodiment, two further doses of the adenovirus vector are administered within 46 days after the start of treatment, so that the previous dose is administered within 30 days after the start of treatment.

[0070] In another preferred embodiment, two further doses of the adenovirus vector (preferably the 13th and 14th doses) are administered within 67 days after the start of treatment, preferably on day 64. The administration is performed at intervals of approximately 6 hours. In a more preferred embodiment, two further doses of the adenovirus vector are administered within 67 days of the start of treatment, so that the previous dose is administered within 46 days of the start of treatment.

[0071] In one embodiment, the treatment period is extended to 60–81 days, and at least one additional intravenous, intratumoral, intraperitoneal, or intrapleural administration is performed after the initial 60–81 day treatment period (see Figure 1).

[0072] In a more preferred embodiment, the adenovirus vector is administered to the subject at least five times, preferably six times, in an active treatment cycle of at least 60 days, with at least one administration, preferably the first, being intravenous and the remaining administrations being intratumorally.

[0073] An example of an administration plan is shown in Figure 1.

[0074] Another example of a dosing plan is as follows (preferably, the viral load is 1 × 10⁶ per dose). 11 ~2×10 12 (Within the range). Day 1 (hereinafter the same): Two intravenous injections of oncolytic virus (each injection 4 hours ± 2 hours apart) Day 3: Two intravenous injections of oncolytic virus (each injection 4 hours ± 2 hours apart). Day 8 (±1 day): Two intravenous administrations of oncolytic virus (each injection 4 hours ± 2 hours apart). Day 10 (±3 days): Two intravenous administrations of oncolytic virus (each injection 4 hours ± 2 hours apart). Day 22 (±3 days): Two intravenous administrations of oncolytic virus (each injection 4 hours ± 2 hours apart). Day 43 (±3 days): Two intravenous administrations of oncolytic virus (each injection 4 hours ± 2 hours apart). Day 64 (±3 days): Two intravenous administrations of oncolytic virus (each injection 4 hours ± 2 hours apart).

[0075] Another example of a dosing plan is as follows (preferably, viral load is 3 × 10⁶ per dose). 10 ~2×10 12 (Within the range). Day 1: One intravenous administration of oncolytic virus. Day 8 (±1 day): One intratumoral administration of oncolytic virus. Day 15 (±3 days): One intratumoral administration of oncolytic virus. Day 29 (±3 days): One intratumoral administration of oncolytic virus. Day 43 (±3 days): One intratumoral administration of oncolytic virus. Day 57 (±3 days): One intratumoral administration of oncolytic virus. Day 71 (±3 days): One intratumoral administration of oncolytic virus.

[0076] Another example of a dosing plan is as follows (preferably, viral load is 1 × 10⁶ per dose). 11 ~4×10 12 (Within the range). Day 1: One intravenous administration of oncolytic virus. Day 8 (±1 day): One intratumoral / intraperitoneal administration of oncolytic virus. Day 22 (±3 days): One intratumoral / intraperitoneal administration of oncolytic virus. Day 36 (±3 days): One intratumoral / intraperitoneal administration of oncolytic virus. Day 57 (±3 days): One intratumoral / intraperitoneal administration of oncolytic virus. Day 78 (±3 days): One intratumoral / intraperitoneal administration of oncolytic virus.

[0077] In addition to the treatment methods of the present invention, any other treatment methods or combinations of treatment methods may be used in combination. In specific embodiments, the methods or uses of the present invention further include administering to a subject, either concurrently or sequentially, targeted therapies such as radiotherapy, chemotherapy, anti-angiogenic agents, or other anticancer agents such as alkylating agents, nucleoside analogs, cytoskeletal modifiers, cell proliferation inhibitors, monoclonal antibodies, kinase inhibitors, or other anticancer agents, or interventions (including surgery).

[0078] While we do not wish to be bound by any theory, the experimental results disclosed herein suggest that higher levels of neutralizing antibodies against adenovirus vectors may enhance the efficacy of the treatment according to the present invention in some patients. Therefore, in some embodiments, an immune system stimulatory administration of an adenovirus vector may be performed prior to the active treatment cycle described herein to increase neutralizing antibodies in the patient. The adenovirus vector preferably comprises an adenovirus serotype 5 (Ad5) backbone (optionally including an adenovirus serotype 3 (Ad3) fiber knob), and in more preferred embodiments, the vector is the same as that used in the active treatment cycle.

[0079] The present invention also relates to a method for selecting patients for cancer treatment with oncolytic adenovirus vectors. The method includes the steps of: providing a biological sample of a subject; measuring the level of adenovirus neutralizing antibodies in the sample; and selecting patients based on the level of adenovirus neutralizing antibodies present in the sample. In a preferred embodiment, the biological sample is a blood sample or a sample derived from a blood sample (e.g., a serum sample). In another preferred embodiment, the selection step is performed by comparing the level of adenovirus neutralizing antibodies in the sample with the level of adenovirus neutralizing antibodies in samples obtained from healthy individuals or patients who have benefited from cancer treatment with oncolytic adenovirus vectors.

[0080] In this specification, the terms “treat” or “increase,” and any terms derived therefrom, do not necessarily imply a 100% or complete cure or increase. Rather, the degree to which a person skilled in the art would recognize a potential benefit or therapeutic effect varies.

[0081] In this specification, “administration” refers to an administration session in which one or more injections / infusions of a therapeutic agent are administered to a human or mammal, and in the case of multiple injections / infusions, the injections / infusions are preferably performed within a time frame of 1 to 8 hours and may be performed through one or more routes of administration.

[0082] Other embodiments

[0083] Throughout this specification, any reference to "one embodiment" or "a certain embodiment" means that a particular feature, structure, or characteristic described in relation to that embodiment is included in at least one embodiment of the present invention. Therefore, throughout this specification, expressions such as "in one embodiment" or "in a certain embodiment" do not necessarily refer to the same embodiment. When terms such as "about" or "substantially" are used in relation to numerical values, the exact numerical values ​​are also disclosed.

[0084] It should be understood that the embodiments of the present invention disclosed herein are not limited to any specific structure, process, or material disclosed herein, but also extend to equivalents recognized by those skilled in the art. Furthermore, it should be understood that the terms used herein are for the purpose of describing specific embodiments and are not intended to be restrictive.

[0085] Throughout this specification, any reference to “one embodiment” or “a certain embodiment” means that a particular feature, structure, or characteristic described in relation to that embodiment is included in at least one embodiment of the present invention. Therefore, throughout this specification, expressions such as “in one embodiment” or “in a certain embodiment” do not necessarily refer to the same embodiment.

[0086] In this specification, multiple items, structural elements, compositional elements, and / or materials may be presented as common lists for convenience. However, these lists should be interpreted as if each element of the list were individually identified as a distinct and unique element. Therefore, individual elements of such lists should not be interpreted as being substantially equivalent to other elements simply because they are presented as a common group, unless otherwise indicated. Furthermore, various embodiments and examples of the present invention may be referred to herein along with alternative examples of their respective components. It should be understood that such embodiments, examples, and alternative examples should not be interpreted as being substantially equivalent to one another, but rather as distinct and independent expressions of the present invention.

[0087] Furthermore, the described features, structures, or properties may be combined in any suitable manner in one or more embodiments. The following description provides many specific details, such as examples of length, width, and shape, for a full understanding of the embodiments of the invention. However, those skilled in the art will recognize that the invention can be implemented without some or all of these specific details, or using other methods, components, materials, etc. In other examples, well-known structures, materials, or operations are not illustrated or described in detail to avoid obscuring the features of the invention.

[0088] The examples described above illustrate the principles of the present invention in one or more specific applications. As will be apparent to those skilled in the art, various modifications to the form, use, and details of the embodiments may be made without exercising inventive ability and without departing from the principles and concepts of the present invention. Therefore, the present invention is not limited except as defined by the claims described below.

[0089] In this specification, the verbs “comprise” and “include” are used as open limitations, neither excluding nor requiring the existence of features not described. Features described in dependent claims may be freely combined with each other unless otherwise specified. Furthermore, it should be understood that the use of “a” or “an,” i.e., the singular form, throughout this specification does not exclude the plural.

[0090] [Table 1] [Example of experiment] [Examples]

[0091] material and method Test participants This study enrolled males and females aged 18 years or older with pathologically confirmed refractory or recurrent cancer and a history of previous failure to standard treatment. Additional inclusion criteria included having an evaluable disease (meeting the response criteria for solid tumors (RECIST 1.1) was not required) and having at least one injectable tumor that could be injected and biopsied. Participants had to have adequate liver and kidney function and a World Health Organization (WHO) / East Coast Cancer Clinical Group (ECOG) performance status of 0-1. Participants were excluded from this study if they had received anticancer treatment or immunosuppressant treatment (with some exceptions) within 30 days prior to the initial treatment injection, or if they met any other exclusion criteria. Among the subjects, one patient with non-small cell lung cancer (Efficacy Case 1, data cutoff: March 1, 2023) and one patient with anaplastic thyroid cancer (Efficacy Case 2, data cutoff: March 1, 2023) participated in the study.

[0092] Study design and treatment This study was an open-label, dose-escalation phase 1 clinical trial conducted at two different clinical sites in Helsinki, Finland. The treatment duration was 64 days, with the possibility of extension if enrolled patients showed signs of benefit.

[0093] During the study period, patients received a total of six doses of the oncolytic vector Ad5 / 3-E2F-D24-TNFα-IRES-IL2 (TILT-123) during the first 64 days of treatment (see Figure 1). Treatment with TILT-123 began with an intravenous dose on day 1, followed by intratumoral doses on days 8, 22, 36, 50, 64, and every three weeks thereafter if the patient entered extended treatment. A tolerance of ±1 to 3 days was allowed for all other study visits except for the visit on day 1. Patient Efficacy Case 1 received 3 × 10⁶ doses of TILT-123 via both intravenous and intratumoral administration. 9 In patients who received viral particles (VP) and showed efficacy in case 2, 3 × 10¹⁶ doses were administered intravenously. 11 VP, 1 × 10⁶ in intratumoral administration 11 VP was administered.

[0094] For intratumoral administration, priority was given to injecting the virus into 10 sites with a total volume of 4 mL per administration, preferably into 10 different lesions (i.e., one injection per lesion). If there were fewer than 10 lesions suitable for intratumoral injection, multiple injections into a single tumor were also possible. Multiple injections into large lesions were preferred over injections into small lesions.

[0095] Test evaluation The primary endpoint of this study was safety up to day 85, assessed based on adverse events, serious adverse events, vital signs, electrocardiogram (ECG), and safety laboratory results. Secondary endpoints included, but were not limited to, tumor response according to RECIST1.1 immune(i)RECIST and PET-based criteria, measured by whole-body computed tomography (CT) and positron emission tomography (PET) scans at screening and day 78. CT scans provide material for measuring the actual size of tumor lesions, while PET image analysis measures tumor metabolic activity via the maximum normalized uptake (SUVmax) of a radiotrace (fluorodeoxyglucose (FDG)).

[0096] result Effectiveness Example 1 Subjects with advanced stage IV non-small cell lung cancer were enrolled in this study and, as mentioned above, received six doses of TILT-123 by day 64 (see Figure 2). On day 78 post-treatment, image analysis of target lesion 3 showed a 61% decrease in metabolic activity (SUVmax) in the lesion treated with TILT-123 compared to baseline (Figure 2). A similar phenomenon was observed in target lesion 4, where SUVmax decreased by 54% compared to baseline. Notably, the latter lesion had not received TILT-123. This demonstrates the antitumor effect of TILT-123 on distant lesions.

[0097] Effectiveness Example 2 Patients with advanced stage IV anaplastic thyroid cancer were enrolled in this study and, as described above, received 10 doses of TILT-123 by day 167 (see Figure 3). After TILT-123 treatment, the tumor size of target lesion 1, as measured by CT scan, decreased by up to 69% by day 167. In target lesion 1, the metabolic activity of the tumor completely disappeared by day 167, confirming further antitumor effects. Other non-target lesions present in the patients before TILT-123 treatment became negative by day 78 (non-target lesion 2) and day 167 (non-target lesion 4). This demonstrates that TILT-123 treatment induces antitumor effects even in non-injectable lesions, reaffirming the systemic efficacy of this treatment. [Examples]

[0098] material and method Patients and Methods Between February 18, 2021, and July 13, 2023, 20 patients were enrolled in this study. Inclusion criteria included having a cancer that had failed standard treatment or for which no standard treatment exists, having at least one tumor suitable for intratumoral injection, and having adequate hematological function (hemoglobin > 100 g / L, white blood cell count > 3.0 E9 / L, platelet count > 75,000 / mm³). 3 The criteria included liver function (AST, ALT < 3 × upper limit of normal (ULN), bilirubin < 1.5 × ULN), renal function (GFR > 60 ml / min), a WHO / ECOG performance score of 0-1 at screening, and a life expectancy of 3 months or more. Exclusion criteria included the use of immunosuppressants (corticosteroids or autoimmune disease medications), anti-cancer treatment within the past 30 days, a history of severe liver disease or coagulation disorders, uncontrolled cardiovascular disease, and a history of treatment for oncolytic viruses.

[0099] All patients provided written informed consent. The study protocol and ethics were reviewed by the Finnish Pharmaceuticals Agency (FIMEA) and the Ethics Committee of Helsinki University Hospital (HUS) (Approval No. 49 / 2020, Statement No. HUS / 1804 / 2020).

[0100] Manufacturing of TILT-123 TILT-123 was manufactured in accordance with GMP (Good Manufacturing Practice) using A549 cells. Prior to administration, TILT-123 was suspended in 0.9% physiological saline and administered in volumes of 1.0–5.0 mL for intratumoral injection and 10.0–40.0 mL for intravenous injection, depending on the dose cohort.

[0101] treatment Patients received multiple doses of TILT-123. Intravenous administration was given on day 1, and intratumoral administration was given on days 8, 22, 36, 50, and 64. Patients deemed to be likely to respond to treatment were allowed to continue receiving additional doses of TILT-123 beyond the primary endpoint. The intravenous dose was 3 × 10⁶. 9 ~4×10 12 Viral particles (VP), intratumoral dose: 3 × 10 9 ~5×10 11 In VP, a dose escalation plan was followed.

[0102] Intratumoral injections were performed using a 21-gauge needle under ultrasound guidance. During intratumoral administration, at least one tumor was injected, and the administered drug was evenly distributed to multiple sites within each injected tumor.

[0103] Evaluation of antitumor effect, survival time, and progression-free survival. The antitumor effect was observed on day 78 by contrast-enhanced CT (computed tomography) and 18 The lesions were evaluated by PET (positron emission tomography) using F-FDG. The maximum tumor diameter and SUVmax values ​​were obtained from images by a specialist radiologist. Tumor response was evaluated according to PET-based criteria defined by RECIST 1.1, iRECIST, and Koski et al. (2013). In individual lesion analyses, disease control by CT was defined as an increase in lesion size of less than 20%. In PET analyses of individual lesions, metabolic disease control was defined as an increase in lesion SUVmax of less than 30%.

[0104] Survival and progression-free survival data were obtained from the electronic clinical trial system. The data cutoff for survival and progression-free survival was November 26, 2023. Patients who had not reported death or progression at the data cutoff were included in the analysis as surviving or progression-free up to the data cutoff date.

[0105] Neutralizing antibody assay analysis Anti-adenovirus antibodies were measured by a neutralizing antibody assay described in detail by Hemminki et al. (2002), with the lowest titer being 1:64.

[0106] statistical analysis For overall survival and group-specific analyses, the Mantel-Cox log-rank test or MaxCombo test was used for group comparisons (see figure caption). For categorical analyses, Fisher's exact test was used for group comparisons. Statistical analysis was performed using GraphPad Prism 9.4.1 and the R package "nph".

[0107] result Effectiveness Antitumor effects were observed in both injected and non-injected tumors. In injected lesions, disease control was observed in 9 out of 19 lesions by CT and in 11 out of 17 lesions by PET (Figures 4A and 4B, including patients from Example 1). In non-injected lesions, disease control was observed in 9 out of 13 lesions by CT and in 11 out of 14 lesions by PET (Figures 4C and 4D, including patients from Example 1). The median survival time for all patients enrolled in this study was 124.5 days (Figure 4E, including patients from Example 1). The median survival time for patients showing disease control was 213.5 days, while the median survival time for patients not showing disease control was 109 days (p=0.165, Figure 4E, including patients from Example 1). The median progression-free survival (PFS) for all patients enrolled in this study was 87.5 days (Figure 4F, including patients from Example 1). The median PFS for patients who showed disease control on day 78 was 181 days, which was significantly longer than the 65 days for patients who did not show disease control on day 78 (Figure 4F, p<0.0001).

[0108] The median time to progression (TTP) for all patients was 97 days (Figure 4G, including patients from Example 1). The median TTP for patients who showed disease control on day 78 was 518 days, which was significantly longer than the median TTP of 83 days for patients who did not show disease control (Figure 4G, p=0.0002). Notably, four patients showed long-term survival, two of whom survived for more than 600 days after enrollment (Figure 4F).

[0109] The four patients who survived for more than one year after enrollment included a patient with myxoid liposarcoma (20204), a patient with undifferentiated thyroid cancer (20103, efficacy example 1 of Case 1), a patient with leiomyosarcoma (20212), and a patient with nodular melanoma (20211). Notably, three of these four patients had a significant history of prior treatment and were resistant to other therapies. Patient 20204 with myxoid liposarcoma had received a total of 10 cancer treatments, consisting of neoadjuvant radiotherapy, three surgeries, and six lines of chemotherapy. This patient completed the trial and received four additional intratumoral doses of TILT-123 before finally dying 821 days after trial enrollment. After the trial, this patient received no other cancer treatments except for palliative radiotherapy for inguinal metastases. Therefore, this patient survived for more than 600 days after the trial without receiving additional systemic therapy.

[0110] Patient 20212 with leiomyosarcoma had received six previous cancer treatments, consisting of two regimens of radiotherapy and four lines of chemotherapy. The patient completed the trial and was assessed as having progressive disease on day 78. Post-trial, the patient received palliative doxorubicin and pazopanib and was alive at 654 days post-enrollment at the data cutoff.

[0111] Another patient who achieved long-term survival was patient 20211 with nodular melanoma. This patient was markedly refractory to treatment, having undergone four surgeries, two lines of nivolumab, paclitaxel and carboplatin combination therapy, and the experimental CTLA-4-targeted immune checkpoint inhibitor BMS-986218. This patient was assessed as having progressive disease on day 78 and received palliative temozolomide and radiotherapy after the trial. This patient survived 295 days after the last dose of TILT-123.

[0112] Immune-boosting effect At baseline, 9 out of 20 patients had low or undetectable neutralizing antibody titers against TILT-123 (defined as antibody titer <1:64), while 11 out of 20 patients had detectable antibody titers (Figure 5A, including patients from Example 1). None of the patients showed high antibody titers at baseline, which is consistent with the absence of 5 / 3 chimeric adenovirus in nature. Baseline neutralizing antibody levels did not correlate with disease control (Figure 5B, including patients from Example 1), but patients with neutralizing antibodies at baseline showed a tendency toward long-term survival. However, statistical significance could not be obtained due to the small sample size (p=0.258, Figure 5C, including patients from Example 1).

[0113] Furthermore, the two best-responding patients (20103 and 20108) based on RECIST 1.1 both showed the highest measurable neutralizing antibody titers (Figure 6A, including Efficacy Example 1 of Example 1). Similarly, the best PET-responding patients and two of the four longest-surviving patients also showed the highest measurable neutralizing antibody titers (Figures 6B and 6C, including the patient from Example 1). Neutralizing antibody titers increased throughout the study period in all dose cohorts, although no clear correlation was found with the dose (Figure 5D, including the patient from Example 1). [Prior art documents] [Non-patent literature]

[0114] Heise C. et al., 2000, Nature Med 6, 1134-1139. Kanerva A. et al., 2005, Gene Therapy, 12, 87-94. Koski A et al., 2013, Hum Gene Ther 24(12), 1029-41 Hemminki A et al., 2002, Hum Gene Ther 13(12), 1505-14 [Patent Documents]

[0115] International release WO2014170389

Claims

1. A cancer-treating oncolytic adenovirus vector encoding at least TNFα and / or IL-2, The adenovirus vector is administered to the subject at least three times, preferably at least five, six, or seven times, during an active treatment cycle. An oncolytic adenovirus vector comprising at least one dose, preferably the first dose, which is systemic, and the active treatment cycle preferably does not include the administration of a adoptive cell therapeutic composition containing TIL or the administration of an immune checkpoint inhibitor.

2. The oncolytic adenovirus vector for the use described in claim 1, wherein the systemic administration is performed parenterally, preferably intravenously.

3. A tumor-lytic adenovirus vector for the use described in claim 1 or 2, wherein subsequent administrations are performed locally, preferably intratumor, intraperitoneal, and / or intrapleural.

4. The oncolytic adenovirus vector for the use described in any one of claims 1 to 3, wherein further administration of the adenovirus vector is performed after the start of treatment, i.e., within 10 days, preferably on the 8th day, after the first administration of the adenovirus vector.

5. The oncolytic adenovirus vector for the use described in claim 4, wherein the further administration of the adenovirus vector is performed within 20 days after the start of treatment, preferably on the 15th day, and the previous administration is preferably performed within 10 days after the start of treatment.

6. The oncolytic adenovirus vector for use according to claim 4 or 5, wherein further administration of the adenovirus vector is performed within 32 days after the start of treatment, preferably on the 22nd or 29th day, and the previous administration is preferably performed within 10 or 20 days after the start of treatment.

7. The oncolytic adenovirus vector for the use described in claim 6, wherein further administration of the adenovirus vector is performed within 46 days after the start of treatment, preferably on the 36th or 43rd day, and the previous administration is preferably performed within 32 days after the start of treatment.

8. The oncolytic adenovirus vector for the use described in claim 7, wherein the further administration of the adenovirus vector is performed within 60 days after the start of treatment, preferably on the 50th or 57th day, and the previous administration is preferably performed within 46 days after the start of treatment.

9. The oncolytic adenovirus vector for use according to claim 7 or 8, wherein the further administration of the adenovirus vector is performed within 67 days after the start of treatment, preferably on the 64th day, and the previous administration is preferably performed within 46 or 60 days after the start of treatment.

10. The oncolytic adenovirus vector for the use described in claim 8, wherein the further administration of the adenovirus vector is performed within 81 days after the start of treatment, preferably on day 71 or day 78, and the previous administration is preferably performed within 67 days after the start of treatment.

11. An oncolytic adenovirus vector for the use described in any one of claims 4 to 10, wherein each administration is either intratumor, intraperitoneal, or intrapleural.

12. The oncolytic adenovirus vector for the use described in claim 1 or 2, wherein the second administration of the adenovirus vector is performed after the start of treatment, i.e., within 2 to 6 hours after the first administration of the adenovirus vector.

13. The oncolytic adenovirus vector for the use described in claim 12, wherein two further doses of the adenovirus vector are administered within five days after the start of treatment, preferably on the third day, at intervals of 2 to 6 hours.

14. The oncolytic adenovirus vector for the use described in claim 13, wherein two further doses of the adenovirus vector are administered at intervals of 2 to 6 hours within 10 days after the start of treatment, preferably on the 8th day, with the previous dose preferably administered within 5 days after the start of treatment.

15. The oncolytic adenovirus vector for the use described in claim 14, wherein two further doses of the adenovirus vector are administered at intervals of 2 to 6 hours within 13 days, preferably on the 10th day, after the start of treatment, with the previous dose preferably administered within 8 or 10 days after the start of treatment.

16. The oncolytic adenovirus vector for the use described in claim 15, wherein two further doses of the adenovirus vector are administered at intervals of 2 to 6 hours within 30 days, preferably on day 22, after the start of treatment, with the previous dose preferably administered within 13 days after the start of treatment.

17. The oncolytic adenovirus vector for the use described in claim 16, wherein two further doses of the adenovirus vector are administered at intervals of 2 to 6 hours within 46 days, preferably on the 43rd day, after the start of treatment, with the previous dose preferably administered within 30 days after the start of treatment.

18. The oncolytic adenovirus vector for the use described in claim 17, wherein two further doses of the adenovirus vector are administered at intervals of 2 to 6 hours within 67 days, preferably on the 64th day, after the start of treatment, with the previous dose preferably administered within 46 days after the start of treatment.

19. An oncolytic adenovirus vector for the use described in any one of claims 12 to 18, wherein all administrations during treatment are systemic, preferably parenteral, and more preferably intravenous.

20. In each administration, the oncolytic adenovirus vector is 1 × 10⁶ ９ ~1 x 10 １３ It is administered in the amount of viral particles, preferably 3 × 10⁻⁶. ９ ~4 x 10 １２ An oncolytic adenovirus vector for the use described in any one of the prior claims, administered in viral particle quantities.

21. The aforementioned cancers or tumors include nasopharyngeal cancer, synovial cancer, hepatocellular carcinoma, kidney cancer, connective tissue cancer, melanoma, lung cancer, intestinal cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, pharyngeal cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastric tumor (gastrinoma), pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, von Hippel-Lindau disease, Zolinger-Ellison syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary origin, carcinoid, gastrointestinal carcinoid, fibrosarcoma, breast cancer, Paget's disease, cervical cancer, esophageal cancer, gallbladder cancer, head cancer, eye cancer, cervical cancer, and Wilm's disease. An oncolytic adenovirus vector for the use described in any one of the prior claims, selected from the group consisting of tumors, Kaposi's sarcoma, prostate cancer, testicular cancer, Hodgkin's disease, non-Hodgkin 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, gastric cancer, thymic cancer, thyroid cancer, choriocarcinoma, hydatidiform mole, uterine cancer, endometrial cancer, vaginal cancer, vulvar cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gingival cancer, cardiac cancer, lip cancer, meningeal cancer, nerve cancer, palatine cancer, parotid gland cancer, peritoneal cancer, pharyngeal cancer, pleural cancer, salivary gland cancer, tongue cancer, and tonsil cancer.

22. The oncolytic adenovirus vector for the use described in claim 21, wherein the tumor is melanoma, lung cancer, head and neck cancer, ovarian cancer, or thyroid cancer.

23. The oncolytic adenovirus vector for the use described in claim 21, wherein the soft tissue sarcoma is preferably a leiomyosarcoma or liposarcoma.

24. The subject has failed at least once in previous cancer treatment, such as chemotherapy or radiotherapy, an oncolytic adenovirus vector for the use described in any one of claims 1 to 23.

25. The oncolytic adenovirus vector for the use described in any one of the prior claims comprises an adenovirus serotype 5 (Ad5) backbone equipped with an adenovirus serotype 3 (Ad3) fiber knob.

26. The oncolytic adenovirus vector for the use described in claim 25, wherein the nucleic acid sequence encoding TNFα and / or IL-2 is located at the position of the deletion nucleic acid sequence in the E3 region of the oncolytic adenovirus vector.

27. The oncolytic adenovirus vector for the use described in claim 26, wherein the deletion of the nucleic acid sequence in the E3 region is a deletion of the viral gp19k and 6.7k leading frames.

28. The oncolytic adenovirus vector for the use described in any one of claims 25 to 27, wherein the vector comprises a 24-base pair deletion (Δ24) in the adenovirus E1 sequence of the oncolytic adenovirus vector.

29. An oncolytic adenovirus vector for use according to any one of claims 1 to 28, wherein, prior to the active treatment cycle, an immune system stimulatory administration of an adenovirus vector is performed in the subject for the purpose of increasing adenovirus neutralizing antibodies, the adenovirus vector preferably comprises an adenovirus serotype 5 (Ad5) backbone, and more preferably the same vector as the one used in the active treatment cycle.

30. The oncolytic adenovirus vector for the use described in any one of claims 1 to 29, wherein the aggressive treatment cycle is performed as a monotherapy for cancer using the adenovirus vector from the start of the treatment cycle until the final administration of the adenovirus vector.

31. A subject receiving treatment is selected for the treatment based on the subject's adenovirus neutralizing antibody level before the treatment or after immune system stimulation with an adenovirus vector prior to the treatment, using an oncolytic adenovirus vector for the application described in any one of claims 1 to 30.

32. A method for treating cancer in a subject, comprising the step of administering to the subject at least three times, preferably at least five, six, or seven times, an oncolytic adenovirus vector encoding at least TNFα and / or IL-2 in an active treatment cycle. A method comprising at least one administration, preferably the first administration, which is systemic, and wherein the active treatment cycle preferably does not include the administration of a adoptive cell therapeutic composition containing TIL or the administration of an immune checkpoint inhibitor.

33. An application for using an oncolytic adenovirus vector encoding at least TNFα and / or IL-2 in the manufacture of a pharmaceutical product administered at least three times, preferably at least five, six, or seven times, in an active therapeutic cycle, An application in which at least one administration of the pharmaceutical agent, preferably the first administration, is systemic, and the active treatment cycle preferably does not include the administration of a adoptive cell therapeutic composition containing TIL or the administration of an immune checkpoint inhibitor.

34. A method for selecting patients for cancer treatment using oncolytic adenovirus vectors, The process of providing a biological sample from the subject, A step of measuring the level of adenovirus neutralizing antibodies in the aforementioned sample, A method comprising the step of selecting a patient based on the level of adenovirus neutralizing antibody present in the sample.

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