Novel dosing regimen for oncolytic adenoviral vectors

Abstract

In accordance with example aspects of the invention, there is provided the use of an oncolytic adenoviral vector encoding at least TNF [alpha] and / or IL-2 for the treatment of cancer wherein the adenoviral vector is administered to a subject at least three times, preferably at least 5, 6 or 7 times over a positive treatment cycle; and wherein at least one, preferably the first, administration is systemic administration wherein the positive treatment cycle preferably does not comprise administration of an adoptive cell therapeutic composition comprising TIL or does not comprise administration of an immune checkpoint inhibitor.

Description

Technical Field

[0001] This invention relates generally to virology, immunology, and medicine. Specifically, this invention relates to an oncolytic adenovirus administration regimen for the treatment of cancer, comprising a formulation, route of administration, dosage, dosing interval, and duration of treatment. Background Technology

[0002] WO2014170389 relates to the therapeutic use and methods of oncolytic adenovirus vectors, alone or in combination with therapeutic compositions, particularly adoptive cell compositions, for cancer treatment. After years of research and development, oncolytic viruses are now being used as cancer therapeutic agents, especially in combination with other cancer drugs. In clinical trials, oncolytic viruses have shown good safety and promising efficacy. However, there is still room for improvement in response, particularly in patients with significant metastatic burden. This invention provides a novel dosing regimen for a specific oncolytic adenovirus that unexpectedly demonstrates efficacy as monotherapy in cancer patients. Summary of the Invention

[0003] Therefore, the object of the present invention is to provide a drug delivery regimen for overcoming the problems of inefficient and unpredictable cancer therapies. More specifically, the present invention provides a novel method for oncolytic virus vector therapy. The object of the invention is achieved by viral vectors, methods, and arrangements, as characterized by the contents of the appended independent claims. Some specific embodiments are defined in the dependent claims.

[0004] According to an aspect of the invention, there is provided the use of an oncolytic adenovirus vector encoding at least TNFα and / or IL-2 for the treatment of cancer, wherein the adenovirus vector is administered to a subject at least three times, preferably at least five, six or seven times, during an active treatment cycle; and wherein at least one, preferably the first, administration is systemic, wherein the active treatment cycle preferably does not include administration of an adoptive cell therapy composition comprising TIL or does not include administration of an immune checkpoint inhibitor. Attached Figure Description

[0005] The accompanying drawings illustrate the invention in accordance with at least some of its embodiments.

[0006] Figure 1- Human TILT-123 Treatment Regimen. Trial participants were screened up to 2 weeks prior to initiating TILT-123 treatment to determine eligibility. Eligible patients were scheduled to receive intravenous administration (day 1) and multiple intratumoral administrations of TILT-123 (days 8, 22, 36, 50, and 64). Trial participants entering the treatment extension phase (after day 78) continued intratumoral or intravenous administration every 3 weeks. Patients underwent the following imaging at baseline, day 78, and approximately every 3 months during the treatment extension phase: positron emission tomography (PET) combined with X-ray computed tomography (CT).

[0007] Figure 2 - The therapeutic effect of TILT-123 on tumor lesions in patients with advanced lung cancer was demonstrated using positron emission tomography (PET) combined with X-ray computed tomography. Patients with stage IV non-small cell lung cancer received six doses of TILT-123—one intravenous administration followed by multiple intratumoral administrations. Representative PET-CT images of TILT-123-injected tumor lesions (left lower neck / shoulder (lymph nodes)) and uninjected tumor lesions (left axillary (lymph nodes)) are shown before and after treatment. Metabolic activity was measured using maximum normalized uptake value (SUVmax). Percentage change was calculated by comparing SUVmax values ​​before and after TILT-123 treatment. Data cutoff date is March 1, 2023.

[0008] Figure 3 - Imaging using positron emission tomography (PET) combined with X-ray computed tomography (CT) to demonstrate the therapeutic effect of TILT-123 on tumor lesions in patients with thyroid cancer. Patients with anaplastic thyroid carcinoma received 10 doses of TILT-123—one intravenous administration followed by multiple intratumoral administrations. Representative PET-CT images of the TILT-123-injected tumor lesion (lower abdomen) and two non-injected tumor lesions (mesenteric lymph nodes and a solitary lesion in the lower lobe of the left lung) are shown before and after treatment (days 78 and 167). Metabolic activity in the lesions was measured using maximum normalized uptake value (SUVmax). Measurements of computed tomography values ​​are shown in tables from TL1. Percentage changes were calculated by comparing SUVmax or CT values ​​before and after TILT-123 treatment. Data cutoff date is March 1, 2023.

[0009] Figure 4 - Efficacy of TILT-123 treatment. A. Response evaluation of all injected lesions as assessed by CT. B. Response evaluation of all injected lesions as assessed by PET. C. Response evaluation of all non-injected lesions as assessed by CT. D. Response evaluation of all non-injected lesions as assessed by PET. AD indicates the best response if the patient continues to the extended phase. E. Overall survival in the trial. F. Progression-free survival in the trial. G. Time to disease progression in the trial. For EG, disease control was defined according to PET-based criteria, and the Mantel-Cox log-rank test was used to compare disease control with no disease control. *** = p < 0.001, **** = p < 0.0001. The dataset used to generate the charts includes data from efficacy examples #1 and #2.

[0010] Figure 5 - Neutralizing Antibodies and Their Development A. Neutralizing antibodies against TILT-123 at baseline in all patients. B. Comparison of neutralizing antibody presence with disease control at day 78. Disease control was defined as SMD or better at day 78. No disease control was defined as PMD or NA at day 78. Fisher's exact probability method was used for intergroup comparisons. C. Comparison of baseline neutralizing antibody presence with overall survival throughout the trial. Max Combo log-rank test was used for intergroup comparisons. D. Neutralizing antibody titers in all dose cohorts during the trial. Baseline is defined as the value before treatment on day 1, and the highest daily titer (before or after treatment) is shown for days 1–64. The datasets used to generate the charts included data from efficacy examples #1 and #2.

[0011] Figure 6 - Antibodies and Antitumor Responses. A. Neutralizing antibodies in patients with the best RECIST 1.1 response during the trial. B. Neutralizing antibodies in patients with the best PET standard response during the trial. C. Neutralizing antibodies in patients with the longest survival during the trial. The datasets used to generate these charts include data from efficacy examples #1 and #2. Detailed Implementation

[0012] Oncolytic virus

[0013] In a preferred embodiment, the oncolytic virus disclosed herein is an oncolytic adenovirus.

[0014] As used herein, "oncolytic adenovirus vector" refers to an adenovirus vector capable of infecting and killing cancer cells through selective replication in tumors relative to normal cells. Therefore, the term adenovirus vector may refer to an adenovirus or adenovirus particle capable of transferring nucleic acids into cells, or to the transferred nucleic acids themselves. WO2014170389 discloses oncolytic adenovirus vectors encoding TNFα and / or IL-2 as one or more transgenes that can be used in this invention.

[0015] The vector can be modified in any manner known in the art, such as by deletion, insertion, mutation, or modification of any viral region. This makes the vector tumor-specific in terms of replication. For example, an adenovirus vector may include modifications in E1, E3, and / or E4, such as the insertion of a tumor-specific promoter (e.g., to drive E1), the deletion of regions (e.g., constant region 2 of E1, E3 / gp19k, E3 / 6.7k, as used in “D24”), and the insertion of transgenes. Furthermore, the fiber knob region of the vector can be modified. In one embodiment of the invention, the adenovirus vector is Ad5 / 3, comprising an Ad5 nucleic acid backbone and Ad3 fiber knobs or Ad5 / 3 chimeric fiber knobs.

[0016] As used in this article, the expression “adenovirus serotype 5 (Ad5) nucleotide backbone” refers to the genome of Ad5.

[0017] "Ad5 / 3 vector" refers to a chimeric vector having portions of both Ad5 and Ad3 vectors. In a particular embodiment of the invention, the capsid modification of the vector is an Ad5 / 3 chimera. As used herein, "Ad5 / 3 chimeric ciliary projection" refers to a chimera in which the projection portion of the ciliary is derived from Ad serotype 3, and the remainder of the ciliary is derived from Ad serotype 5. In particular, in one embodiment, the construct has ciliary projections derived from Ad3, while the remainder of the genome is derived from Ad5 (SEQ ID NO: 5).

[0018] One method for generating tumor-specific oncolytic adenoviruses is to engineer a 24-base pair deletion (D24) affecting the constant region 2 (CR2) of E1 (SEQ ID NO: 4). In wild-type adenoviruses, CR2 is responsible for binding to cellular Rb tumor repressor / cell cycle regulators to induce the synthetic (S) phase, i.e., the DNA synthesis or replication phase. The interaction between Rb and E1A requires eight amino acids (121 to 127) of the conserved region of the E1A protein, which are deleted in this vector. The vector of the present invention comprises the deletion of nucleotides corresponding to amino acids 122 and 129 of the vector according to Heise C. et al. (2000). Viruses with D24 are known to have reduced ability to overcome the G1-S checkpoint and replicate efficiently only in cells where such interaction is not necessary, such as in tumor cells with Rb-p16 pathway defects, which include most (if not all) human tumors.

[0019] The E1A endogenous viral promoter can also be replaced, for example, by a tumor-specific promoter. In a particular embodiment of the invention, the E1A endogenous viral promoter is replaced by the hTERT or E2F promoter.

[0020] In a particular embodiment, it is generally known that the E1B 19K gene (SEQ ID NO: 1) supporting adenoviral vector replication has a disabling deletion of dE1B 19K (SEQ ID NO: 2) in this vector. It is known that the deletion of E1B 19K sensitizes cancer cells to TNFα and thus promotes apoptosis.

[0021] The sequence of the wild-type E1B 19K gene is as follows (deleted regions are underlined):

[0022]

[0023]

[0024] Therefore, in this embodiment, the sequence of dE1B 19K in the viral vector is

[0025]

[0026] The E3 region is not essential for viral replication in vitro, but the E3 protein plays a crucial role in regulating the host immune response, specifically suppressing both innate and specific immune responses. The gp19k / 6.7K deletion in E3 refers to the removal of 965 base pairs from the adenovirus E3A region. In the resulting adenovirus construct, both the gp19k and 6.7K genes are deleted (Kanerva A et al. 2005). The gp19k gene product is known to bind to and isolate the major histocompatibility complex I (MHC1, known as HLA1 in humans) molecule in the endoplasmic reticulum, preventing cytotoxic T lymphocytes from recognizing infected cells. Because many tumors lack HLA1 / MHC1, the deletion of gp19k increases viral tumor selectivity (the virus is cleared from normal cells faster than wild-type virus, but there is no difference in tumor cells). The 6.7K protein is expressed on the cell surface, and they are involved in downregulating TNF-related apoptosis-inducing ligand (TRAIL) receptor 2.

[0027] Both of these deficiencies offer advantages. For the purpose of restoring HLA / MHC expression to present tumor epitopes, such as to adoptive T cells, gp19k expression is counterproductive, and in fact, HLA / MHC upregulation requires gp19k deficiency. Regarding 6.7k, since embodiments of the invention produce TNFα from a virus, and one of its antitumor activities is a direct antitumor and pro-apoptotic effect (on both transduced and untransduced bystander cells), the presence of 6.7k is counterproductive.

[0028] In one embodiment of the invention, one or more cytokine transgenes are placed in the gp19k / 6.7k-deficient E3 region below the E3 promoter. This restricts transgene expression to tumor cells that allow viral replication and subsequent activation of the E3 promoter. The E3 promoter can be any exogenous (e.g., CMV or E2F promoter, SEQ ID NO: 3) or endogenous promoter known in the art, particularly the endogenous E3 promoter. Although the E3 promoter is primarily activated by replication, some expression occurs when E1 is expressed. Because the selectivity of D24 virus occurs after E1 expression (when E1 cannot bind Rb), these viruses do also express E1 in transduced normal cells. Therefore, it is important to similarly regulate E1 expression to restrict E3 promoter-mediated transgene expression to tumor cells.

[0029] In a specific embodiment of the invention, the oncolytic adenovirus vector is based on an adenovirus serotype 5 (Ad5) nucleic acid backbone, including a 5 / 3 chimeric ciliated protrusion, and comprises: an E2F1 promoter for tumor-specific expression of E1A, a 24 bp deletion (D24) in the Rb-binding constant region 2 of adenovirus E1, deletions of viral gp19k and 6.7k reading frames, transgene insertion into the deleted regions, resulting in replication-related control of transgene expression under the viral E3 promoter, and a nucleic acid sequence encoding at least one cytokine transgene replacing the adenovirus gene gp19k / 6.7K missing in the E3 region. In one embodiment of the invention, the adenovirus vector is based on human adenovirus.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] Specific embodiments of the present invention include a viral vector encoding at least one cytokine. In a specific embodiment 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, as well as other cytokines, preferably selected from the group consisting of: 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, CCL1 8. CCL19, CCL2, CCL20, CCL21, CCL22, CCL23-1, CCL23-2, CCL24, CCL25-1, CCL25-2, CCL26, CCL27, CCL 28. CCL3, CCL3L1, CCL4, CCL4L1, CCL5, CCL6, CCL7, CCL8, CCL9, CCR10, CCR2, CCR5, CCR6, CCR7, CCR8, C CRL1, CCRL2, CX3CL1, CX3CR, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCR1, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7, and XCL2.

[0035] 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.

[0036] 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 IL-2 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 start site in which a ribosome physically bypasses a portion of the 5' translation region to reach the start codon. Both IRES and A2 enable the virus to produce two transgenes from a single promoter (E3 promoter).

[0037] Examples of detailed structures encoding TNFα and / or IL-2 as transgenic oncolytic adenovirus vectors are disclosed in WO2014170389.

[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 (naturally occurring anti-tumor T cells) proliferation at the tumor site; iv) the cytokines and / or the virus induce upregulation of antigen-presenting molecules (HLA) on cancer cells, making them sensitive to T cell recognition and killing; and v) 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, oncolytic virus replication can recruit T cells and induce danger signals at the tumor site, thereby reducing immunosuppression and cell dysfunction. These effects are mediated by pathogen-associated molecular pattern recognition receptors, an evolutionarily conserved mechanism for inducing immunity that is unaffected by tolerance. An additional advantage of oncolytic platforms is their ability to replicate within tumors rather than in normal cells, i.e., they can self-amplify at the tumor site. Furthermore, oncolysis itself can enhance overall antitumor activity in humans.

[0042] cancer

[0043] The recombinant vector of the present invention has the ability to replicate 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 E1A CR2 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.

[0044] 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.

[0045] 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.

[0046] As used herein, the terms "treatment" or "treating" refer to the administration of at least an oncolytic adenovirus vector to a subject, preferably a mammalian or human subject, with the aim of not only achieving complete cure but also preventing, improving, or alleviating ailments or symptoms associated with cancer or tumors. The effectiveness of the treatment can be assessed by monitoring the patient's symptoms, tumor markers in the blood, or, for example, tumor size, tumor metabolic activity, or patient survival.

[0047] In another embodiment 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-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, etc. Cancers of the ducts, gallbladder, head, eyes, neck, Wilms' tumor, Kaposi's sarcoma, prostate cancer, testicular cancer, Hodgkin's disease, non-Hodgkin's lymphoma, skin cancer, mesothelioma, multiple myeloma, ovarian cancer, endocrine pancreatic cancer, glucagonoma, parathyroid cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus cancer, thyroid cancer, trophoblastic cancer, 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, nerve cancer, palate cancer, parotid gland cancer, peritoneal cancer, pharyngeal cancer, pleural cancer, salivary gland cancer, tongue cancer, and tonsil cancer. Preferably, the cancer or tumor being 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 non-small cell lung cancer), stomach cancer, classical Hodgkin's lymphoma, mesothelioma, and liver cancer. In a more preferred embodiment, the cancer or tumor type is melanoma, lung cancer (such as small cell lung cancer, non-small cell lung cancer, and squamous non-small cell lung cancer), ovarian cancer, head and neck cancer, or thyroid cancer. In another more preferred embodiment, the cancer or tumor type is a soft tissue sarcoma, such as leiomyosarcoma or liposarcoma.

[0048] 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.

[0049] In embodiments of the invention, the subject or patient has previously failed at least one instance of chemotherapy, radiotherapy, or immunotherapy, such as CPI therapy, meaning the patient's cancer is an immune checkpoint inhibitor (CPI) refractory tumor. In a preferred embodiment, the invention relates to the treatment of CPI-refractory tumors.

[0050] 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.

[0051] 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. In some embodiments, the immune checkpoint inhibitor is an antibody.

[0052] Pharmaceutical Composition

[0053] The pharmaceutical compositions of the present invention comprise at least one type of the viral vector of the present invention. 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 vectors, 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 respective products. The selection of suitable ingredients and appropriate manufacturing methods for formulating the composition is common knowledge to those skilled in the art.

[0054] The pharmaceutical composition may be in any form suitable for administration, such as solid, semi-solid, or liquid. The formulation may be selected from, but is not limited to, solutions, sprays, 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.

[0055] 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 a first formulation; and if used in combination with another active pharmaceutical ingredient, the latter is formulated as a second formulation. In another embodiment of the invention, the first and second formulations are administered to a subject simultaneously or sequentially in any order. In another embodiment, the kit is used for the treatment of cancer or tumors.

[0056] Administration and dosing regimen

[0057] The carrier or pharmaceutical composition of the present invention can be administered to any mammalian subject. In a particular embodiment of the invention, the subject is a human. Mammals can be selected from the group consisting of pets, livestock, and productive animals.

[0058] Any conventional method can be used to administer the carrier or composition 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 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.

[0059] In one embodiment of the invention, the oncolytic virus is administered via intratumoral (it), intraarterial (iv), intrapleural (i.p.), intracystic, intracavitary, or intraperitoneal (ip) injection, or orally or intranasally. In another embodiment of the invention, the oncolytic virus is administered systemically. Systemic administration is a route of administration in which the drug is delivered into the circulatory system, thereby acting on the whole body. Systemic administration can be performed via parenteral administration (i.e., typically by injection or infusion). Any combination of administrations is also possible. Despite local injection, this method can still produce systemic effects.

[0060] 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. Each dose can vary, for example from approximately 1 x 10⁻⁶. 8 One viral particle (VP) to approximately 1x1014 VP, specifically from approximately 5x10 9 VP to approximately 1x10 13 VP, and more specifically from about 3x10 9 VP to approximately 4x10 12 VP. In one implementation, with 1x10 10 -1x10 14 The amount of virus particles administered is an oncolytic adenovirus vector encoding at least IL-2 and / or TNFα. In another embodiment of the invention, the dose is approximately 5 x 10^6 particles. 10 -5x10 11 Within the range of VP.

[0061] In a particular embodiment, the present invention relates to a dosing regimen in which an adenovirus vector is administered to a subject at least three times, preferably at least four, five, six, or seven times, during an active treatment cycle; and wherein at least one, preferably the first, administration is systemic, and wherein the active treatment cycle preferably does not include administration of an adoptive cell therapy composition comprising TILs or administration of immune checkpoint inhibitors. In a preferred embodiment, the duration of the active treatment cycle is at least 60 days, preferably 61-81 days. In another preferred embodiment, the active treatment cycle is performed from the start of the treatment cycle until the last administration of the adenovirus vector as monotherapy for cancer.

[0062] As used herein, “adoptive cell therapy composition” means any composition comprising cells suitable for adoptive cell transfer (see, for example, WO2014170389). Typically, adoptive cell therapy compositions comprise cell types selected from the group consisting of tumor-infiltrating lymphocytes (TILs), TCR (i.e., heterologous T cell receptor) modified lymphocytes, and CAR (i.e., chimeric antigen receptor) modified lymphocytes. Adoptive cell therapy compositions may comprise cell types selected from the group consisting of T cells, CD8+ cells, CD4+ cells, NK- cells, δ-γ T cells, regulatory T cells, and peripheral blood mononuclear cells. Commonly, TILs, T cells, CD8+ cells, CD4+ cells, NK- cells, δ-γ T cells, regulatory T cells, or peripheral blood mononuclear cells form adoptive cell therapy compositions. In one particular case, the adoptive cell therapy composition comprises T cells. As used herein, “tumor-infiltrating lymphocytes” (TILs) refers to leukocytes that have left the bloodstream and migrated into the tumor.

[0063] In a preferred embodiment, the systemic administration is administered parenterally, preferably intravenously.

[0064] In another preferred embodiment, the second and subsequent applications are administered locally, preferably intratumorally, intraperitoneally, or intrapleurally.

[0065] In another preferred embodiment, the second administration of the adenovirus vector is administered within 10 days after the start of treatment (i.e., after the first administration of the adenovirus vector), preferably on day 8.

[0066] In another preferred embodiment, the additional administration of the adenovirus vector is administered within 20 days of the start of treatment, preferably on day 15. In a more preferred embodiment, the additional administration of the adenovirus vector is administered within 20 days of the start of treatment, thus the previous administration was administered within 10 days of the start of treatment.

[0067] In another preferred embodiment, the additional administration of the adenovirus vector is administered within 32 days of the start of treatment, preferably on day 22 or day 29. In a more preferred embodiment, the additional administration of the adenovirus vector is administered within 32 days of the start of treatment, thus the previous administration was administered within 20 days of the start of treatment.

[0068] In another preferred embodiment, the additional administration of the adenovirus vector is administered within 46 days of the start of treatment, preferably on day 36 or 43. In a more preferred embodiment, the additional administration of the adenovirus vector is administered within 46 days of the start of treatment, thus the previous administration was administered within 32 days of the start of treatment.

[0069] In another preferred embodiment, the additional administration of the adenovirus vector is administered within 60 days of the start of treatment, preferably on day 50 or 57. In a more preferred embodiment, the additional administration of the adenovirus vector is administered within 60 days of the start of treatment, thus the previous administration was administered within 46 days of the start of treatment.

[0070] In another preferred embodiment, the additional administration of the adenovirus vector is administered within 67 days of the start of treatment, preferably on day 64. In a more preferred embodiment, the additional administration of the adenovirus vector is administered within 67 days of the start of treatment, thus the previous administration was administered within 60 days of the start of treatment.

[0071] In another preferred embodiment, the additional administration of the adenovirus vector is administered within 81 days of the start of treatment, preferably on day 71 or 78. In a more preferred embodiment, the additional administration of the adenovirus vector is administered within 81 days of the start of treatment, thus the previous administration was administered within 67 days of the start of treatment.

[0072] In another preferred embodiment, the second administration of the adenovirus vector is performed within 2-6 hours after the start of treatment (i.e., after the first administration of the adenovirus vector), wherein, in the treatment, the administration is preferably performed intravenously.

[0073] In another preferred embodiment, the two additional administrations of the adenovirus vector (preferably the third and fourth) are administered 2-6 hours apart within 5 days after the start of treatment (preferably on the third day).

[0074] In another preferred embodiment, the two additional administrations of the adenovirus vector (preferably the 5th and 6th) are administered 2-6 hours apart within 10 days after the start of treatment (preferably on day 8). In a more preferred embodiment, the two additional administrations of the adenovirus vector are administered within 10 days after the start of treatment, thus the previous administration was administered within 5 days after the start of treatment.

[0075] In another preferred embodiment, the two additional (preferably the 7th and 8th) administrations of the adenovirus vector are administered 2-6 hours apart within 13 days after the start of treatment (preferably on day 10). In a more preferred embodiment, the two additional administrations of the adenovirus vector are performed within 13 days after the start of treatment, thus the previous administration was administered within 8 or 10 days after the start of treatment.

[0076] In another preferred embodiment, the two additional (preferably the 9th and 10th) administrations of the adenovirus vector are administered 2-6 hours apart within 30 days of the start of treatment (preferably on day 22). In a more preferred embodiment, the two additional administrations of the adenovirus vector are administered within 30 days of the start of treatment, thus the previous administration was administered within 13 days of the start of treatment.

[0077] In another preferred embodiment, the two additional (preferably the 11th and 12th) administrations of the adenovirus vector are administered 2-6 hours apart within 46 days of the start of treatment (preferably on day 43). In a more preferred embodiment, the two additional administrations of the adenovirus vector are administered within 46 days of the start of treatment, thus the previous administration was administered within 30 days of the start of treatment.

[0078] In another preferred embodiment, the two additional (preferably the 13th and 14th) administrations of the adenovirus vector are administered 2-6 hours apart within 67 days after the start of treatment (preferably on day 64). In a more preferred embodiment, the two additional administrations of the adenovirus vector are administered within 67 days after the start of treatment, thus the previous administration was administered within 46 days after the start of treatment.

[0079] In one implementation, the treatment period is extended to more than 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 [link to implementation details]). Figure 1 ).

[0080] In a more preferred embodiment, the adenovirus vector is administered to the subject at least five times, preferably six times, during an active treatment period of at least 60 days; and wherein at least one administration, preferably the first administration, is intravenous, and the remaining administrations are intratumoral.

[0081] Examples of dosing regimens Figure 1 As shown in the image.

[0082] Another example of a dosing regimen is as follows (the preferred viral dose range for each administration is 1 × 10⁻⁶). 11 -2×10 12 between):

[0083] Day 1 - 2x IV administration of oncolytic virus (4 hours + / - 2 hours between each injection)

[0084] Day 3 - 2x IV administration of oncolytic virus (4 hours + / - 2 hours between each injection)

[0085] On day 8 (+ / - 1) - 2x IV administration of oncolytic virus (each injection 4h + / - 2h interval)

[0086] On day 10 (+ / - 3) - 2x IV administration of oncolytic virus (each injection 4h + / - 2h interval).

[0087] Day 22 (+ / - 3) - 2x IV administration of oncolytic virus (4h + / - 2h interval between each injection)

[0088] Day 43 (+ / - 3 days) - 2x IV administration of oncolytic virus (each injection 4h + / - 2h interval)

[0089] Day 64 (+ / - 3 days) - 2x IV administration of oncolytic virus (each injection 4 hours + / - 2 hours apart).

[0090] Another example of a dosing regimen is as follows (the preferred viral dose range for each administration is 3 × 10⁻⁶). 10 -2×10 12 between):

[0091] Day 1 - 1x IV administration of oncolytic virus

[0092] On day 8 (+ / - 1 day) - 1x oncolytic virus administration

[0093] Day 15 (+ / - 3 days) - 1x oncolytic virus administration

[0094] Day 29 (+ / - 3 days) - 1x oncolytic virus administration

[0095] Day 43 (+ / - 3 days) - 1x oncolytic virus administration

[0096] Day 57 (+ / - 3 days) - 1x oncolytic virus administration

[0097] Day 71 (+ / - 3 days) - 1x oncolytic virus administration

[0098] Another example of a dosing regimen could be the following (the viral dose range for each administration is preferably 1 × 10⁻⁶). 11 -4×10 12 between):

[0099] Day 1 - 1x IV administration of oncolytic virus

[0100] Oncolytic virus administration on day 8 (+ / - 1 day) - 1 hour (it / ip)

[0101] Oncolytic virus administration at 22 (+ / - 3 days) - 1x it / ip

[0102] Oncolytic virus administration at 36 (+ / - 3 days) - 1x it / ip

[0103] Oncolytic virus administration at 57 (+ / - 3 days) - 1x it / ip

[0104] Oncolytic virus administration at 78 (+ / - 3 days) - 1x IT / IP

[0105] In addition to the therapies of the present invention, any other treatments or combinations of treatments may be used. In certain embodiments, the methods or uses of the present invention further include administering, in parallel or sequentially, radiotherapy, chemotherapy, anti-angiogenic agents, or targeted therapies such as alkylating agents, nucleoside analogs, cytoskeleton modifiers, cytostatic agents, monoclonal antibodies, kinase inhibitors, or other anticancer drugs or interventions (including surgery) to a subject.

[0106] Unintentionally bound by any theory, the experimental results disclosed herein demonstrate that, in some patients, higher levels of neutralizing antibodies against adenovirus vectors can improve the efficacy of the treatment according to the invention. Accordingly, in embodiments, prior to the active treatment cycle described herein, an immune system-stimulating dose of the adenovirus vector may be administered to increase the patient's neutralizing antibody levels, wherein the adenovirus vector preferably comprises an adenovirus serotype 5 (Ad5) backbone (optionally including ciliated projections of adenovirus serotype 3 (Ad3)), and wherein, in a more preferred embodiment, the vector is the same as that used in the active treatment cycle.

[0107] The present invention also relates to a method for selecting patients for cancer treatment using an oncolytic adenovirus vector, the method comprising the steps of: providing a biological sample of a subject, measuring the level of adenovirus neutralizing antibodies in the sample, and selecting a patient based on the presence level of the adenovirus neutralizing antibodies 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 a sample obtained from a healthy subject or a patient who has benefited from cancer treatment using an oncolytic adenovirus vector.

[0108] As used herein, the terms “treatment” or “enhancement” and words stemmed therein 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.

[0109] As used herein, the term "administration" refers to a drug administration process in which a therapeutic agent can be administered to a human or mammal once or more times by injection / infusion; and in the case of more than one injection / infusion, the injection / infusion is preferably performed within a time frame of 1 to 8 hours and can be performed via one or more routes of administration.

[0110] Other implementation methods

[0111] Throughout this specification, references to "one embodiment" or "implementation" mean that a particular feature, structure, or characteristic described in connection with that 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 the specification do not necessarily refer to the same embodiment. Where numerical values ​​are referenced using terms such as, for example, about or substantially, precise numerical values ​​are also disclosed.

[0112] It should be understood that the embodiments of the present invention disclosed herein are not limited to the specific structures, processes, or materials disclosed herein, but can be extended to equivalents that will be recognized by those skilled in the art. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0113] Throughout this specification, references to "one embodiment" or "implementation" mean that a particular feature, structure, or characteristic described in connection with that 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.

[0114] As used herein, for convenience, multiple items, structural elements, constituent elements, and / or materials may be presented in a common list. However, these lists should be interpreted as if each member in the list were individually identified as a separate and unique member. Therefore, no single member in such a list should be construed as being factually equivalent to any other member in the same list, based solely on their performance within the common group, without any indication to the contrary. Furthermore, various embodiments and examples of the invention, along with alternatives to its various components, may be mentioned herein. It should be understood that such embodiments, examples, and alternatives should not be construed as factual equivalents of each other, but should be regarded as separate and autonomous representations of the invention.

[0115] Furthermore, the described features, structures, or characteristics can be combined in any suitable manner in one or more embodiments. Numerous specific details, such as examples of length, width, shape, etc., are provided in the following description to provide a thorough understanding of embodiments of the invention. However, those skilled in the art will recognize that the invention can be practiced without one or more specific details, or using other methods, components, materials, etc. In other instances, well-known structures, materials, or operations have not been shown or described in detail to avoid obscuring various aspects of the invention.

[0116] While the foregoing examples illustrate the principles of the invention in one or more specific applications, it will be apparent to those skilled in the art that various modifications can be made in detail of form, use, and implementation without the application of inventive capability and without departing from the principles and concept of the invention. Therefore, the invention is not intended to be limited except for the claims set forth below.

[0117] The verbs “to comprise” and “to include” are used in this document as open-ended restrictions, neither excluding nor requiring nor listing any 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.

[0118] Table 1. Sequences listed in the attached sequence list.

[0119] SEQ ID NO: name: 1 E1B 19K 2 dE1B 19K 3 E2F 4 D24 5 5 / 3 protrusion modification

[0120] Example Section

[0121] Example 1

[0122] Materials and methods

[0123] Trial participants

[0124] Female or male participants over 18 years of age with pathologically confirmed refractory or recurrent cancer who had failed prior standard therapy were enrolled in this trial. Additional inclusion criteria included the presence of an evaluable disease (without meeting the RECIST 1.1 criteria) and at least one injectable tumor suitable for injection and biopsy. Participants had adequate liver and kidney function values ​​and a WHO / Eastern Cooperative Oncology Group (ECOG) performance status score of 0 to 1. Participants were ineligible for this trial if they had received anticancer therapy or immunosuppressive therapy (with some exceptions) within 30 days prior to their first treatment injection, or if they met other exclusion criteria. Among the trial participants, one patient with non-small cell lung cancer (Efficacy Example #1; data cutoff March 1, 2023) and one patient with anaplastic thyroid carcinoma (Efficacy Example #2; data cutoff March 1, 2023) participated in this trial.

[0125] Experimental Design and Treatment

[0126] This study is an open-label, dose-escalation phase 1 clinical trial conducted at two different clinical centers in Helsinki, Finland. The treatment duration is 64 days, with the option to extend treatment if potential benefits are observed in enrolled patients.

[0127] During the trial period, patients received a total of 6 doses of the oncolytic vector Ad5 / 3-E2F-D24-TNFα-IRES-IL2(TILT-123) during the first 64 days of treatment. Figure 1 Treatment with TILT-123 began with intravenous administration on day 1, followed by intratumoral administration on days 8, 22, 36, 50, and 64; and if the patient entered the extended treatment phase, it was administered every 3 weeks thereafter. Except for the day 1 visit, all other trial visits were allowed an error of + / - 1 to 3 days. Patients in efficacy example #1 received 3 x 10 [units of medication] with each intravenous and intratumoral administration. 9 The viral particle (VP) TILT-123, while patients in efficacy example #2 received 3x10 intravenous doses. 11 VP, 1x10 inside the tumor 11 VP.

[0128] For intratumoral administration, each dose should preferably be injected into 10 viral deposition sites in a total volume of 4 mL, ideally at 10 different lesions (i.e., one injection per lesion). If there are fewer than 10 lesions suitable for intratumoral injection, multiple injections can be performed on each tumor. In this case, multiple injections should be given to larger lesions rather than smaller lesions.

[0129] Research Evaluation

[0130] Based on adverse events, serious adverse events, vital signs, electrocardiogram (ECG) results, and safety laboratory findings, the primary endpoint of this trial was safety at day 85. Secondary endpoints included (but were not limited to) tumor response assessed according to RECIST 1.1, immune(i) (RECIST), and PET-based criteria, measured at screening and on day 78 using whole-body X-ray computed tomography (CT) and positron emission tomography (PET). CT scans provided a measurement of the actual size of tumor lesions, while PET imaging provided a measurement of tumor metabolic activity via the maximum normalized uptake (SUVmax) of the radiotracer (fluorodeoxyglucose, FDG).

[0131] result

[0132] Efficacy Example #1

[0133] Subjects with advanced stage IV non-small cell lung cancer were enrolled in this trial and received six TILT-123 administrations over the period up to day 64, as described above. Figure 2 On day 78 post-treatment, imaging of target lesion 3 showed a 61% reduction in metabolic activity in terms of SUVmax value in lesions treated with TILT-123 compared to baseline. Figure 2 A similar situation was observed in target lesion 4, where its SUVmax value decreased by 54% compared to baseline. Notably, the latter had not received TILT-123 administration, suggesting that TILT-123 has an anti-tumor effect on distant lesions.

[0134] Efficacy Example #2

[0135] A subject with advanced stage IV anaplastic thyroid carcinoma was enrolled in this trial and received 10 doses of TILT-123 over the period up to day 167, as described above. Figure 3Following TILT-123 treatment, CT scans showed that by day 167, the tumor size of target lesion 1 had shrunk by up to 69%. Furthermore, by day 167, tumor metabolic activity in target lesion 1 had completely disappeared, further demonstrating its anti-tumor effect. Other non-target lesions present in the patient prior to TILT-123 treatment became negative on day 78 (non-target lesion 2) and on day 167 (non-target lesion 4). The induction of anti-tumor effects by TILT-123 treatment in untreated lesions further confirms the systemic effects of this treatment method.

[0136] Example 2

[0137] Materials and methods

[0138] Patients and methods

[0139] Between February 18, 2021 and July 13, 2023, a total of 20 patients were enrolled in this trial. Inclusion criteria included: failure of standard therapy or absence of standard therapy for cancer; at least one tumor suitable for intratumoral injection; and qualified hematological results (hemoglobin >100 g / L, WBC >3.0 E9 / L, platelets >75,000 / mm³). 3 The following criteria were considered for eligibility: liver function (AST, ALT < 3 x ULN, and bilirubin < 1.5 x ULN) and kidney function (GFR > 60 ml / min), with a WHO / ECOG performance score of 0-1 at screening; and life expectancy exceeding 3 months. Exclusion criteria included: use of immunosuppressive drugs (corticosteroids or drugs used for autoimmune diseases); prior treatment with anticancer therapies within 30 days; history of severe liver disease or coagulation disorders; uncontrolled heart or vascular disease; or prior oncolytic virus therapy.

[0140] All patients provided written informed consent. The trial protocol and ethics were reviewed by the Finnish Medical Agency (FIMEA) and the ethics committee of Helsinki University Hospital (HUS) (Approval No. 49 / 2020 and Statement HUS / 1804 / 2020).

[0141] Production of TILT-123

[0142] TILT-123 is manufactured in A549 cells according to Good Manufacturing Practices (GMP). Prior to administration, TILT-123 is resuspended in 0.9% saline and administered in intratumoral volumes of 1.0–5.0 mL and intravenous volumes of 10.0–40.0 mL, depending on the dose cohort.

[0143] treat

[0144] Patients received multiple doses of TILT-123: intravenous on day 1, and intratumoral on days 8, 22, 36, 50, and 64. Patients assessed as potentially benefiting could continue with additional courses of TILT-123 beyond the primary endpoint. The intravenous dose range was 3 x 10⁻⁶ doses, following the dose escalation regimen. 9 Up to 4x10 12 One viral particle (VP), and an intratumoral dose range of 3x10 9 Up to 5x10 11 VP.

[0145] Intratumoral injection was performed under ultrasound guidance using a 21-gauge needle. During intratumoral administration, at least one tumor was injected, and the drug was evenly distributed at multiple sites within each injected tumor.

[0146] Assessment of antitumor efficacy, survival time and progression-free survival

[0147] On day 78, enhanced contrast computed tomography (CT) imaging and... 18 Positron emission tomography (PET) with F-FDG was used to assess antitumor efficacy. The maximum tumor diameter and SUVmax were read from the images by a qualified radiologist. Tumor response was evaluated using RECIST 1.1, iRECIST, and the PET-based criteria in Koski et al. 2013. For single lesion analysis, CT disease control was defined as an increase in lesion size of less than 20%. For single lesion PET analysis, metabolic disease control was defined as an increase in lesion SUVmax of less than 30%.

[0148] Survival and progression-free survival (PFS) data were extracted from the electronic clinical trial system. The data cutoff date for survival and PFS was November 26, 2023. Patients who did not report death or progression by the data cutoff date were considered to be alive or progression-free before the data cutoff date in the analysis.

[0149] Neutralizing antibody detection and analysis

[0150] The measurement of anti-adenovirus antibodies using a neutralizing antibody assay was previously described in more detail by Hemminki et al 2002, with a titer of 1:64 being the lowest detectable titer.

[0151] Statistical analysis

[0152] For overall survival and group analyses, as illustrated in the attached figures, the Mantell-Cox log-rank test or Max Combo test was used to compare groups. For categorical analyses, Fisher's exact test was used to compare groups. Statistical analyses were performed using GraphPadPrism 9.4.1 and the R package "nph".

[0153] result

[0154] effect

[0155] Antitumor efficacy was observed in both injected and uninjected tumors. In injected lesions, disease control was achieved in 9 out of 19 lesions detected by CT and 11 out of 17 lesions detected by PET (Figures 4A and 4B, including patients discussed in Example 1). In uninjected lesions, disease control was achieved in 9 out of 13 lesions detected by CT and 11 out of 14 lesions detected by PET (Figures 4C and 4D, including patients discussed in Example 1). The median overall survival for all enrolled patients was 124.5 days (Figure 4E, including patients discussed in Example 1). The median overall survival was 213.5 days for patients showing disease control and 109 days for those not showing disease control (p = 0.165, Figure 4E, including patients discussed in Example 1). The median progression-free survival (PFS) for all enrolled patients was 87.5 days (Figure 4F, including patients discussed in Example 1). For patients who showed disease control by day 78, the median PFS was significantly longer at 181 days, compared to 65 days for patients who did not show disease control by day 78 (Figure 4F, p<0.0001).

[0156] The median time to progression (TTP) for all patients was 97 days (Figure 4G; including patients discussed in Example 1). Patients showing disease control at day 78 had a significantly longer median TTP of 518 days compared to patients without disease control, whose median TTP was 83 days (Figure 4G, p = 0.0002). Notably, four patients showed significantly prolonged survival, with two of them surviving for more than 600 days after enrollment (Figure 4F).

[0157] Four patients who survived for more than one year after enrollment included one patient with myxoid liposarcoma (20204), one patient with anaplastic thyroid carcinoma (20103, i.e., efficacy example #1 in Example 1 results), one patient with leiomyosarcoma (20212), and one patient with nodular melanoma (20211). Notably, three of these four patients had received extensive prior treatment and were resistant to other therapies. The patient with myxoid liposarcoma (20204) had previously received 10 cancer therapies, consisting of neoadjuvant radiotherapy, 3 surgeries, and 6 lines of chemotherapy. This patient completed the trial and received an additional 4 rounds of intratumoral injections of TILT-123 after the trial, ultimately passing away from the disease 821 days after enrollment. This patient received only palliative radiotherapy for the groin metastases after the trial and did not receive any other cancer therapies. Therefore, this patient survived for more than 600 days without receiving any further systemic treatment after the trial.

[0158] The patient with leiomyosarcoma in 20212 had previously received six cancer treatment regimens, consisting of two radiation therapy regimens and four lines of chemotherapy. This patient completed the trial and was assessed as having disease progression on day 78. The patient received palliative doxorubicin and pazopanib after the trial and was still alive at the data cutoff, 654 days after enrollment.

[0159] Another patient with a longer survival was patient 20211, who had nodular melanoma. This patient was significantly resistant to treatment, having previously received four rounds of surgery, two lines of nivolumab, paclitaxel combined with carboplatin, and the experimental CTLA-4-targeting immune checkpoint inhibitor BMS-986218. The patient was assessed as having disease progression on day 78 and subsequently received palliative temozolomide and radiation therapy. The patient survived for 295 days after the final dose of TILT-123.

[0160] Immune stimulation

[0161] At baseline, 9 out of 20 patients had low or no neutralizing antibody titers against TILT-123 (defined as titers less than 1:64), while 11 out of 20 patients had detectable titers (Figure 5A, including patients discussed in Example 1). No patients had high titers at baseline, consistent with the absence of 5 / 3 chimeric adenovirus in nature. Baseline neutralizing antibody levels were not correlated with disease control (Figure 5B, including patients discussed in Example 1), but a long-term survivor population emerged among patients with baseline neutralizing antibodies, although this was not statistically significant due to the small sample size (p = 0.258, Figure 5C, including patients discussed in Example 1).

[0162] Furthermore, the two best responders (20103 and 20108) assessed according to RECIST 1.1 criteria both produced detectable highest neutralizing titers (Figure 6A, including efficacy example #1 in the results of Example 1). Similarly, the best PET responder and two of the four longest survivors both produced detectable highest neutralizing titers (Figures 6B and 6C, including the patients discussed in Example 1). Neutralizing antibody titers increased across all dose cohorts and were not significantly correlated with dose (Figure 5D, including the patients discussed in Example 1).

[0163] References

[0164] Non-patent literature

[0165] Heise C et al., 2000, Nature Med 6, 1134-1139.

[0166] Kanerva A et al., 2005, Gene Therapy 12, 87-94.

[0167] Koski A et al., 2013, Hum Gene Ther 24(12), 1029-41.

[0168] Hemminki A et al., 2002, Hum Gene Ther 13(12), 1505-14.

[0169] Patent documents

[0170] WO2014170389.

Claims

1. The use of an oncolytic adenovirus vector encoding at least TNFα and / or IL-2 for the treatment of cancer. in, The adenovirus vector will be administered to the subject at least three times, preferably at least five, six, or seven times, during the active treatment period; and The treatment includes at least one application, preferably the first application being a systemic application, wherein the active treatment cycle preferably does not include the application of an adoptive cell therapy composition comprising TIL or the application of an immune checkpoint inhibitor.

2. The oncolytic adenovirus vector according to claim 1, wherein, The systemic administration is delivered parenterally, preferably intravenously.

3. The oncolytic adenovirus vector according to claim 1 or 2, wherein, The second and subsequent administrations are given locally, preferably intratumorally, intraperitoneally, and / or intrapleurally.

4. The oncolytic adenovirus vector according to any one of claims 1-3, wherein, The additional administration of the adenovirus vector is performed after the start of treatment, i.e. within 10 days after the first administration of the adenovirus vector, preferably on day 8.

5. The oncolytic adenovirus vector according to claim 4, wherein, The additional administration of the adenovirus vector is administered within 20 days after the start of treatment, preferably on day 15, and wherein the prior administration is preferably administered within 10 days after the start of treatment.

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

7. The oncolytic adenovirus vector for the use according to claim 6, wherein, The additional administration of the adenovirus vector is administered within 46 days after the start of treatment, preferably on day 36 or 43, and wherein the prior administration is preferably administered within 32 days after the start of treatment.

8. The oncolytic adenovirus vector for use according to claim 7, wherein, The additional administration of the adenovirus vector is administered within 60 days after the start of treatment, preferably on day 50 or 57, and wherein the prior administration is preferably administered within 46 days after the start of treatment.

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

10. The oncolytic adenovirus vector for use according to claim 8, wherein, The additional administration of the adenovirus vector is administered within 81 days after the start of treatment, preferably on day 71 or 78, and wherein the prior administration is preferably administered within 67 days after the start of treatment.

11. The oncolytic adenovirus vector according to any one of claims 4-10, wherein, Each application is administered intratumorally, intraperitoneally, or intrapleurally.

12. The oncolytic adenovirus vector for use according to any one of claims 1-2, wherein, The second administration of the adenovirus vector is administered after the start of treatment, i.e., within 2-6 hours of the first administration of the adenovirus vector.

13. The oncolytic adenovirus vector for the use according to claim 12, wherein, The adenovirus vector was administered twice, 2-6 hours apart, within 5 days after the start of treatment, preferably on day 3.

14. The oncolytic adenovirus vector for the use according to claim 13, wherein, The adenovirus vector is administered twice, 2-6 hours apart, within 10 days after the start of treatment, preferably on day 8, wherein the previous administration is preferably administered within 5 days after the start of treatment.

15. The oncolytic adenovirus vector for the use according to claim 14, wherein, The adenovirus vector is administered twice, 2-6 hours apart, within 13 days after the start of treatment, preferably on day 10, wherein the previous administration is preferably administered within 8 or 10 days after the start of treatment.

16. The oncolytic adenovirus vector for the use according to claim 15, wherein, The adenovirus vector is administered twice, 2-6 hours apart, within 30 days after the start of treatment, preferably on day 22, wherein the previous administration is preferably administered within 13 days after the start of treatment.

17. The oncolytic adenovirus vector for the use according to claim 16, wherein, The adenovirus vector is administered twice, 2-6 hours apart, within 46 days after the start of treatment, preferably on day 43, wherein the previous administration is preferably within 30 days after the start of treatment.

18. The oncolytic adenovirus vector for the use according to claim 17, wherein, The adenovirus vector is administered twice, 2-6 hours apart, within 67 days after the start of treatment, preferably on day 64, wherein the previous administration is preferably within 46 days after the start of treatment.

19. The oncolytic adenovirus vector for use according to any one of claims 12-18, wherein, All administrations during the treatment period are systemic, preferably parenteral, and more preferably intravenous.

20. An oncolytic adenovirus vector for the use according to any one of the preceding claims, wherein, In each application, the oncolytic adenovirus vector is administered at a concentration of 1x102 9 -1x10 13 The amount of each virus particle is preferably 3 x 10⁻⁶. 9 -4x10 12 The amount of virus particles applied between each dose.

21. An oncolytic adenovirus vector for the use according to any one of the preceding claims, wherein, The cancers or tumors mentioned are 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-Lindau disease, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumors, osteochondroma, chondrosarcoma, Ewing sarcoma, cancer of unknown primary site, carcinoid tumors, gastrointestinal carcinoid tumors, fibrosarcoma, breast cancer, Paget's disease, cervical cancer, esophageal cancer, gallbladder cancer. Cancer, head cancer, eye cancer, neck 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, nerve cancer, palate 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 according to 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 according to claim 21, wherein, The soft tissue sarcoma is preferably a leiomyosarcoma or a liposarcoma.

24. The oncolytic adenovirus vector for use according to any one of claims 1-23, wherein, The subjects had at least one previous cancer treatment, such as chemotherapy or radiation therapy, that had failed.

25. An oncolytic adenovirus vector for the use according to any one of the preceding claims, wherein, The oncolytic adenovirus vector comprises the backbone of adenovirus serotype 5 (Ad5) and the ciliated processes of adenovirus serotype 3 (Ad3).

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

27. The oncolytic adenovirus vector for the use according to claim 26, wherein, The deletion of the nucleic acid sequence in the E3 region refers to the deletion of the viral gp19k and 6.7k reading frames.

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

29. The oncolytic adenovirus vector for use according to any one of claims 1-28, wherein, Prior to the active treatment cycle, an immune system-stimulating dose of adenovirus vector is administered to increase the subject's adenovirus neutralizing antibodies; wherein the adenovirus vector preferably comprises an adenovirus serotype 5 (Ad5) backbone, and more preferably the same vector used in the active treatment cycle.

30. The oncolytic adenovirus vector for use according to any one of claims 1-29, wherein, The active treatment cycle, from the start of the treatment cycle until the final administration of the adenovirus vector, is implemented using the adenovirus vector as a monotherapy for cancer.

31. The oncolytic adenovirus vector according to any one of claims 1-30, wherein, The subjects being treated were screened based on the levels of adenovirus neutralizing antibodies present in the subjects prior to or after administration of an immune system-stimulating dose of the adenovirus vector prior to the treatment.

32. A method for treating cancer in a subject, the method comprising the following steps: During the active treatment period, the subject was administered an oncolytic adenovirus vector encoding at least TNFα and / or IL-2 at least three times, preferably at least five, six, or seven times; and The treatment includes at least one application, preferably the first application being a systemic application, wherein the active treatment cycle preferably does not include the application of an adoptive cell therapy composition comprising TIL or the application of an immune checkpoint inhibitor.

33. Use of an oncolytic adenovirus vector encoding at least TNFα and / or IL-2 in the preparation of a medicament, said medicament being administered at least three times, preferably at least five, six, or seven times, during an active treatment cycle; and in, At least one administration of the drug, preferably the first administration, is systemic administration, wherein the active treatment cycle preferably does not include administration of adoptive cell therapy compositions comprising TILs or administration of immune checkpoint inhibitors.

34. A method for selecting patients for cancer treatment using an oncolytic adenovirus vector, the method comprising the following steps: Biological samples of the subjects are provided, the levels of adenovirus neutralizing antibodies in the samples are measured, and patients are selected based on the levels of the adenovirus neutralizing antibodies present in the samples.

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