IFNβ as pharmacodynamic marker in vsv-IFNβ-nis oncolytic therapy

Abstract

To provide real-time measurement and monitoring in a dynamic clinical environment, and treatment decisions based on individual responses and changing circumstances in each patient.SOLUTION: The present invention relates to methods of determining the likelihood that a cancerous tissue in a subject having the cancerous tissue will respond to administration of a cancer therapy regimen. The methods generally comprise (a) administering, intratumorally to the cancerous tissue, a subtherapeutic diagnostic dose of an oncolytic virus probe that comprises a nucleic acid that codes for soluble interferon beta (IFNβ), and (b) measuring the circulating level of IFNβ in the subject after administration of the oncolytic virus to determine if the cancerous tissue is a strong responder, an intermediate responder, a low responder or a non-responder.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application Serial No. 62 / 825,482, filed March 28, 2019. The disclosure of the prior application is hereby incorporated by reference into the present application.

[0002] Statement of Government Interest This invention was made with Government support under CA015083 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0003] The present invention relates generally to pharmacokinetic and pharmacodynamic markers for therapeutic regimens and methods of treating cancer.

[0004] Cancer remains the leading cause of death worldwide. In 2015, an estimated 1,658,370 new cases of cancer were diagnosed in the United States, resulting in 589,430 cancer deaths. The 5-year relative survival rate for all cancer diagnoses in 2004-2010 was only 68%. Furthermore, some cancers have particularly poor prognoses, with 5-year relative survival rates of 7% for pancreatic cancer and less than 20% for liver, lung, and esophageal cancer; survival rates for advanced malignancies with distant metastases range from 2% for pancreatic cancer to 55% for thyroid cancer.

[0005] Chemotherapy is the standard treatment option for the majority of patients with metastatic and / or advanced cancer. Unfortunately, for many patients, chemotherapy is not curative and their disease becomes resistant to the therapy. Patients with resistant metastatic solid tumors have few treatment options.

[0006] Cancer immunotherapy is a rapidly evolving treatment class that offers the potential for clinical benefit when chemotherapy is ineffective. Over the past decade, immune checkpoint inhibitors such as ipilimumab, pembrolizumab, atezolizumab, and nivolumab have been approved. These approvals were initially for melanoma but have more recently been extended to other disease types, and additional agents such as avelumab and durvalumab have recently been approved. These agents have spurred a resurgence of immunotherapy in the clinical pipeline. Numerous agents are in development, including oncolytic virus therapy.

[0007] Oncolytic virus therapy is a promising alternative to chemotherapy, especially in patients with resistant or recurrent disease who have already failed two or more lines of cancer therapy. The therapeutic efficacy of oncolytic viruses is determined by their ability to cause a multifaceted attack. Oncolytic viruses selectively replicate in cancer cells, inducing proinflammatory cell lysis and exposure of tumor-associated antigens while reversing microenvironmental immune suppression and reactivating host effector cells to help promote systemic and sustained anticancer immunity.

[0008] In 2015, the first oncolytic virotherapy, Imlygic (talimogene laherparepvec), was approved for use in patients with locally advanced melanoma. To further understand their safety and efficacy, oncolytic viruses must be evaluated in patients with refractory solid tumors. Recently, T-Vec, an oncolytic herpes simplex virus type 1 encoding granulocyte-macrophage colony-stimulating factor, was approved by the FDA for the treatment of surgically unresectable melanoma, becoming the first-in-class approved in the United States (Andtbacka 2015). Three other phase III trials testing oncolytic virotherapy are ongoing: intratumoral administration of an oncolytic vaccinia virus encoding GMCSF (Pexa-Vec) for the treatment of hepatocellular carcinoma, intravesical administration of an adenovirus (CG0070), also encoding GMCSF, for the treatment of bladder cancer, and IV treatment of reovirus (Reolysin) for head and neck cancer. Among other oncolytic virus clinical trials, a phase 1 trial using intratumoral administration of an IFNβ-expressing (but not cotransporter-expressing) oncolytic VSV for the treatment of hepatocellular carcinoma is open and recruiting.

[0009] Oncolytic virus therapy can also be combined with other cancer therapies, such as chemotherapy or immunotherapy. Emerging data suggest that the use of checkpoint inhibitors in combination with oncolytic viruses can enhance antitumor immune responses through the release of neoantigens, resulting in durable objective responses in a greater proportion of patients than would be expected with checkpoint inhibitors alone. Although some studies suggest that the combination of checkpoint inhibitors and oncolytic viruses may be useful, there are currently no studies that have examined combination therapy consisting of checkpoint inhibitors and oncolytic viruses for metastatic colon cancer in humans.

[0010] Oncolytic virus therapy can be optimized or customized. For example, cancer cells with antiviral deficiency can be identified based on the presence of gene expression signatures of virus therapy tolerance. Such a set of markers is shown in WO2017218757A1. The gene expression signature of tumors provides actionable information. However, it is static and cannot take into account the changes in conditions that may occur during treatment. Furthermore, gene expression signatures cannot take into account tumor load.

[0011] Thus, there is a need for real-time measurement and monitoring in a dynamic clinical environment and for applying treatment decisions based on each patient's individual response and changing circumstances. Summary of the Invention

[0012] The present invention generally relates to methods of diagnosis. In certain embodiments, the present invention relates to a method for determining the likelihood that a cancerous tissue in a subject having cancerous tissue will respond to administration of a cancer therapy regimen. The method generally includes: (a) administering intratumorally to the cancerous tissue a sub-therapeutic diagnostic dose of an oncolytic virus probe comprising a nucleic acid encoding soluble interferon beta (IFNβ); and (b) measuring circulating levels of IFNβ in the subject after administration of the oncolytic virus to determine whether the cancerous tissue is a strong responder, intermediate responder, poor responder, or non-responder.

[0013] The present invention also relates to a method of treating a subject diagnosed with cancer, comprising (a) administering to the subject a first dose of an oncolytic virus cancer therapy regimen that includes a nucleic acid encoding interferon beta (IFNβ), and (b) administering at least a second dose of the oncolytic virus cancer therapy regimen if the subject has been identified as a strong or intermediate responder to the oncolytic virus cancer therapy regimen. [Brief description of the drawings]

[0014] [Figure 1] Figure 1 shows that intratumoral injected Voyager-V1 virus concentration correlates with response. IFNβ levels are predictive of patient response to Voyager-V1. [Diagram 2] Figure 2 shows plasma IFNβ levels in patients receiving dose levels (DL) 1, 2, or 3 of Voyager-V1. DL1, DL2, and DL3 correspond to 5x109, 1.7x1010, and 5x1010 TCID50, respectively. SD means stable disease. PR means partial response. [Diagram 3] FIG. 3 shows a plot of plasma IFNβ levels on day 2 (24 hours post-dose) against anti-VSV antibody titers on day 29 (28 days post-dose). [Figure 4] Figures 4A-4F show a comparison of relative IFNβ and IFNα trends in patients. Figures 4A-4C show that IFNβ levels (thick line) increase at 24 hours post-dose. Figures 4D-4F show that IFNα levels (thick line) decrease at 24 hours post-dose. The data indicate that IFNβ transgene levels may serve as a biomarker for viral infection. [Diagram 5] Figures 5A-5F show that circulating levels of IFNβ detected in serum are indicative of variability in Voyager-V1 infection and transmission in individual patients. In particular, Figures 5A-5C show that circulating levels of IFNβ can be detected in patients injected intratumorally with doses ranging from approximately 106 to 108 TCID50. [Figure 6] FIG. 6 shows a representation of the construct of Voyager-V1 (VSV-IFNβ-NIS, VV1). [Figure 7] FIG. 7 shows a flow chart overview of the methods used in the Voyager-V1 systemic virotherapy trial provided in Example 1. [Figure 8]Figures 8A and 8B show the clinical activity of Voyager-V1 after a single intravenous administration. Specifically, Figure 8A shows CT scans of a subject with endometrial cancer before treatment and 3 months after Voyager-V1 treatment. Overall tumor reduction is 16.5% in diameter on day 29. Figure 8B shows that a subject with T-cell lymphoma shows a 75% reduction in tumor diameter. [Figure 9] Figures 9A and 9B show that NIS imaging confirms tumor infection by Voyager-V1 in two subjects, subject 105-021 (Figure 9A) and subject 105-020 (Figure 9B). [Figure 10] Figures 10A and 10B show that Voyager-V1 treatment increases CD8 tumor-infiltrating cells one month after intravenous (subject 6, Figure 10A) or intratumoral injection (subject 103-014, Figure 10B). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Detailed Description of the Invention The present invention generally relates to methods of diagnosis. In certain embodiments, the present invention relates to a method for determining the likelihood that a cancerous tissue in a subject having cancerous tissue will respond to administration of a cancer therapy regimen. The method generally includes: (a) administering intratumorally to the cancerous tissue a sub-therapeutic diagnostic dose of an oncolytic virus probe comprising a nucleic acid encoding soluble interferon beta (IFNβ); and (b) measuring circulating levels of IFNβ in the subject after administration of the oncolytic virus to determine whether the cancerous tissue is a strong responder, intermediate responder, poor responder, or non-responder.

[0016] In certain embodiments, the cancer therapy regimen of the method comprises an oncolytic viral probe administered intratumorally in (a). In certain embodiments, the cancer therapy regimen of the method comprises a different oncolytic viral probe than that administered intratumorally in (a). In certain embodiments, the cancer therapy regimen is an immune oncolytic therapy. In certain embodiments, the cancer therapy regimen is an antibody or small molecule anti-cancer therapy.

[0017] In certain embodiments, the oncolytic viral probe is administered at a non-toxic and non-therapeutic dose. In certain embodiments, the non-therapeutic and non-toxic dose is about 10 5 TCID 50 ~Approx. 3×10 9 TCID 50 In certain embodiments, the non-therapeutic and non-toxic doses are about 10 8 TCID 50 ~Approx. 5×10 8 TCID 50 It is.

[0018] In other embodiments, the oncolytic viral probe can be any GMP grade virus.In certain embodiments, the oncolytic viral probe is vesicular stomatitis virus (VSV).In certain embodiments, the oncolytic viral probe further comprises a nucleic acid encoding sodium iodide symporter (NIS).In certain embodiments, the oncolytic viral probe has the construct of NPM-IFNβ-G-NIS-L.

[0019] In certain embodiments, the circulating level of IFNβ is measured in the subject between about 12 hours and about 45 days after administration of the oncolytic virus. In certain embodiments, the circulating level of IFNβ is measured in the subject between about 12 hours and about 3 days after administration of the oncolytic virus. In certain embodiments, the circulating level of IFNβ is measured in the subject about 48 hours after administration of the oncolytic virus. In certain embodiments, the circulating level of IFNβ is measured in the subject about 24 hours after administration of the oncolytic virus.

[0020] In certain embodiments, circulating levels of IFNβ are measured by an immunological assay.

[0021] In certain embodiments, the cancer tissue is solid tumor or hematological malignancy.In certain embodiments, the cancer tissue is head and neck cancer, colon cancer, rectal cancer, pancreatic cancer, bladder cancer, breast cancer, hepatocellular carcinoma, lung cancer, medulloblastoma, atypical teratoid / rhabdoid tumor, leukemia, lymphoma, or myeloma.

[0022] The present invention also relates to a method of treating a subject diagnosed with cancer, comprising (a) administering to the subject a first dose of an oncolytic virus cancer therapy regimen that includes a nucleic acid encoding interferon beta (IFNβ), and (b) administering at least a second dose of the oncolytic virus cancer therapy regimen if the subject has been identified as a strong or intermediate responder to the oncolytic virus cancer therapy regimen.

[0023] In certain embodiments, the cancer therapy regimen comprises the administration of two or more anti-cancer compositions. In certain embodiments, the cancer tissue is a solid tumor, and the cancer therapy regimen is an oncolytic virus administered intratumorally at a dose based on the number of viral particles per unit volume of the tumor.

[0024] In certain embodiments, the therapeutic dose of the intratumorally administered oncolytic virus is administered within the standard dose range. In certain embodiments, the cancer therapy regimen is an intravenously administered oncolytic virus.

[0025] In certain embodiments, the first dose of the oncolytic virus cancer therapy regimen is administered intravenously.In certain embodiments, the first dose of the oncolytic virus cancer therapy regimen is administered intratumorally.In certain embodiments, the first dose of the oncolytic virus cancer therapy regimen is a non-therapeutic dose and a non-toxic dose of the oncolytic virus cancer therapy regimen.In certain embodiments, the administration of the second dose of the oncolytic virus cancer therapy regimen is administered intravenously or intratumorally.

[0026] In certain embodiments, the oncolytic virus cancer therapy regimen comprises a nucleic acid encoding sodium iodide symporter (NIS). In certain embodiments, the oncolytic virus is an RNA virus. In certain embodiments, the oncolytic virus is a vesicular stomatitis virus (VSV). In certain embodiments, the VSV has the construct of NPM-IFNβ-G-NIS-L.

[0027] In certain embodiments, the method of treatment further comprises administering to the subject one or more additional immuno-oncology therapeutic agents if the subject is identified as an intermediate responder to the oncolytic virus cancer therapy regimen.

[0028] In certain embodiments, the method of treatment further comprises administering to the subject a Janus kinase inhibitor (JAK inhibitor) if the subject is identified as a strong responder to the oncolytic virus cancer therapy regimen. In certain embodiments, the JAK inhibitor is ruxolitinib.

[0029] In certain embodiments, the level of IFNβ is assessed between about 0.5-45 days after administration of the first dose of the oncolytic viral cancer therapy regimen. In certain embodiments, the level of IFNβ is assessed between about 0.5-3 days after administration of the first dose of the oncolytic viral cancer therapy regimen. In certain embodiments, the second dose of the oncolytic viral cancer therapy regimen is administered within about 1-10 days after administration of the first dose of the oncolytic viral cancer therapy regimen. In certain embodiments, the circulating level of IFNβ is assessed within about 12-24 hours after administration of the first dose of the oncolytic viral cancer therapy regimen. In certain embodiments, the circulating level of IFNβ is assessed by immunological assay.

[0030] In certain embodiments, cancer is solid tumor or hematological malignancy.In certain embodiments, solid tumor is head and neck cancer, colon cancer, rectal cancer, pancreatic cancer, bladder cancer, breast cancer, hepatocellular carcinoma, lung cancer, medulloblastoma or atypical teratoid / rhabdoid tumor.In certain embodiments, hematological malignancy is leukemia, lymphoma or myeloma.

[0031] In certain embodiments, the second administration of the oncolytic virus cancer therapy regimen is by intratumoral injection.In certain embodiments, the second intratumoral injection is administered to the subject based on the number of virus particles per unit volume of tumor.In certain embodiments, the second intratumoral injection is administered to the subject within a standard dose range.In certain embodiments, the therapeutic dose of oncolytic virus is administered intravenously.

[0032] The present invention generally relates to methods for diagnosing and treating cancer. In certain embodiments, the present invention provides methods for early assessment of individual patient response to cancer therapy and adapting treatment decisions based on each patient's individual response and changing circumstances. In certain embodiments, the present invention provides methods for investigating the microenvironment of cancer tissues and the potential immune response of cancer therapeutic agents in individual patients. Such methods can inform the selection of the most effective treatment regimen tailored to a particular individual.

[0033] A "sample", "test sample" or "biological sample", as used interchangeably herein, is of biological origin, such as from a mammal, in certain embodiments. In certain instances, the sample is a tissue or bodily fluid obtained from a subject. In other specific instances, the sample is a human or animal sample. Non-limiting sources of samples include blood, plasma, serum, urine, spinal fluid, lymphatic fluid, synovial fluid, cerebrospinal fluid, tears, saliva, milk, mucosal secretions, exudates, sweat, biopsy aspirates, peritoneal fluid or fluid extracts. In specific examples, the sample is a liquid sample. In specific examples, the sample is a cancer tissue. In some embodiments, the sample is derived from a subject (e.g., a human), including the different sample sources described herein. In some embodiments, the sample is subjected to further processing. Exemplary procedures for processing samples are provided throughout this application, for example, in the Examples section.

[0034] The term "subject" refers to any animal, e.g., mammals, including but not limited to, humans and non-human primates, that will be the recipient of a particular treatment.

[0035] As used herein, sub-therapeutic dose refers to a dose level or range that is lower than the dose level or range that is normally administered for a particular indication or for a particular individual.In certain embodiments, sub-therapeutic dose is a dose level or range that is lower than that listed on the label of the agent, for example, any cancer therapeutic agent.In certain embodiments, sub-therapeutic dose refers to a dose level or range that does not induce an adverse or therapeutic response in the subject.In certain embodiments, sub-therapeutic dose is a non-adverse and non-therapeutic dose.

[0036] Oncolytic virus, as used herein, refers to a virus that infects and kills cancer cells through normal viral replication and life cycle, but does not infect and kill normal cells.In some instances, oncolytic virus therapy can make it easier for other cancer therapies, such as chemotherapy and radiation therapy, to kill tumor cells.Oncolytic virus therapy is a type of targeted therapy.It is also referred to as oncolytic virotherapy, viral therapy, and virotherapy, which are used interchangeably herein.

[0037] Oncolytic virus probe, as used herein, refers to an oncolytic virus that is used at a lower dose than when used as a therapeutic agent to examine cancer tissue, e.g., tumor, for specific characteristics of cancer tissue, e.g., immune response to the virus, tissue or tumor microenvironment, or defense capacity of cancer tissue. In some embodiments, the oncolytic virus probe is used to examine an individual subject who has been diagnosed with cancer. The oncolytic virus probe can be any GMP grade virus. In certain embodiments, the oncolytic virus probe is a vesicular stomatitis virus (VSV). In certain embodiments, the oncolytic virus probe further comprises a nucleic acid that codes for sodium iodine symporter (NIS). In some embodiments, the probe is a virus that is therapeutic when provided in sufficient dose.

[0038] In certain embodiments, the sub-therapeutic dose of the oncolytic virus probe is about 10 5 TCID 50 ~Approx. 3×10 9 TCID 50 In certain embodiments, the sub-therapeutic dose is about 10 8 TCID 50 ~Approx. 5×10 8 TCID 50In certain embodiments, the sub-therapeutic dose of the oncolytic viral probe can be calculated by any person skilled in the art using standard methods.

[0039] In a specific embodiment, the oncolytic viral probe has the construct NPM-IFNβ-G-NIS-L. In a specific embodiment, the non-therapeutic and non-toxic dose of the oncolytic viral probe is about 10 5 TCID 50 ~Approx. 3×10 9 TCID 50 In certain embodiments, the non-therapeutic and non-toxic doses are about 10 8 TCID 50 ~Approx. 5×10 8 TCID 50 It is.

[0040] The term "circulating level" refers to the amount or concentration of a marker present in the circulating fluid. Circulating level can be expressed, for example, in absolute amount, concentration, amount per unit mass of subject, or as a relative amount. The level of a marker can be a relative amount, such as, but not limited to, compared to an internal standard, or a baseline level, or can be expressed as a range of minimum and / or maximum amount, average amount, median amount, or the presence or absence of a marker.

[0041] In certain embodiments, the circulating level of IFNβ is measured in the subject prior to administration of the oncolytic virus. The oncolytic virus can be a viral probe or a virotherapy administered at a sub-therapeutic dose. In certain embodiments, the circulating level of IFNβ is measured in the subject between about 12 hours and about 45 days after administration of the oncolytic virus. In certain embodiments, the circulating level of IFNβ is measured in the subject between about 12 hours and about 3 days after administration of the oncolytic virus. In certain embodiments, the circulating level of IFNβ is measured in the subject about 48 hours after administration of the oncolytic virus. In certain embodiments, the circulating level of IFNβ is measured in the subject about 24 hours after administration of the oncolytic virus. The level of circulating IFNβ in the subject identifies the subject as a strong responder, intermediate responder, poor responder, or non-responder to administration of the oncolytic virus.

[0042] The level of circulating IFNβ in strong responder, intermediate responder, low responder, or non-responder may be determined by two or more factors and may overlap.For example, the actual amount of IFNβ produced in a subject depends on the type of viral vector used, the marker gene or protein carried by the vector, the initial dose administered, individual tumor microenvironment, and individual immune defense mechanism.Marker gene or protein as used herein means a gene or protein whose level, i.e., circulating level or expression level, can be detected by common techniques.In some embodiments, it is soluble IFNβ.In some embodiments, it is NIS.

[0043] For soluble IFNβ expressed by VSV viruses such as Voyager-V1, circulating IFNβ levels of 0-100 pg / mL may be considered low depending on the initial dose of probe, identifying the subject as a poor or non-responder. In some embodiments, circulating IFNβ levels of 10 pg / mL or higher may be considered high depending on the initial dose of probe, identifying the subject as a strong responder. However, different initial doses induce different high and low ranges.

[0044] The term "cancer" has its usual meaning in the art. In general, cancer is a term for diseases in which abnormal cells divide uncontrollably and can invade nearby tissues. There are several main types of cancer. For example, carcinoma is a cancer that begins in the skin, or tissues that line or cover internal organs. Sarcoma is a cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supporting tissues. Leukemia is a cancer that begins in blood-forming tissues, such as bone marrow, where large numbers of abnormal blood cells are produced and enter the blood. Lymphoma and multiple myeloma are cancers that begin in cells of the immune system. Central nervous system cancer is a cancer that begins in the tissues of the brain and spinal cord. Also referred to as malignant tumors. Cancer as used herein includes any type of cancer, whether it is a solid tumor or a blood cancer, and regardless of the origin of the cancer. In some embodiments, the cancer is head and neck cancer, colon cancer, rectal cancer, pancreatic cancer, bladder cancer, breast cancer, hepatocellular carcinoma, lung cancer, medulloblastoma, atypical teratoid / rhabdoid tumor, leukemia, lymphoma, or myeloma.

[0045] Cancerous tissue refers to tissue that has identifiable cancer cells. In some embodiments, the cancerous tissue is a solid tumor.

[0046] As used herein, administration includes any method of providing a drug to a subject, including but not limited to intratumoral administration and intravenous administration.Intravenous (IV) injection or infusion means that a drug is delivered directly into a subject's vein using a needle or tube.In some embodiments, a thin plastic tube called an IV catheter is inserted into a vein.Intratumoral administration means that a drug is delivered directly into tumor or cancer tissue.

[0047] The present invention also relates to pharmacodynamic (PD) markers for therapeutic regimens and methods of treating cancer, comprising administering to a subject a recombinant vesicular stomatitis virus engineered to express interferon beta and a sodium iodide symporter (e.g., VSV-IFNβ-NIS). In the present invention, the terms subject and patient are used interchangeably.

[0048] Wild-type VSV infection in humans is usually asymptomatic but can cause an acute, febrile, flu-like illness lasting 3-6 days, characterized by fever, chills, nausea, vomiting, headache, retrobulbar pain, myalgia, substernal pain, malaise, pharyngitis, conjunctivitis, and lymphadenitis. Complications are not commonly observed in humans infected with wild-type VSV, and no fatalities have been recorded, although a published case of nonfatal meningoencephalitis in a 3-year-old Panamanian child was attributed to VSV infection. Modified Indiana strain VSV has been used in Ebola vaccination programs in over 17,000 healthy volunteers, leading researchers to conclude that the safety profile is considered acceptable in healthy adults. VSV-based vaccines are generally well tolerated, with few reported vaccine-related adverse events. Common adverse events include headache, fever, fatigue, and myalgia, most of which are mild to moderate in severity and generally short-lived. Neither shedding of live virus nor person-to-person transmission has been observed.

[0049] Vesicular stomatitis virus is a member of the Rhabdoviridae family.VSV genome is a single molecule of minus-strand RNA that encodes five major polypeptides: nucleocapsid (N) polypeptide, phosphoprotein (P) polypeptide, matrix (M) polypeptide, glycoprotein (G) polypeptide and viral polymerase (L) polypeptide.Vesicular stomatitis virus nucleic acid sequence provided herein that encodes VSV N polypeptide, VSV P polypeptide, VSV M polypeptide, VSV G polypeptide and VSV L polypeptide can be from VSV Indiana strain shown in GenBank accession number NC_001560 (GI No.9627229) or from VSV New Jersey strain.

[0050] In one embodiment, the methods and regimens of the present invention include administration of Voyager-V1 (VSV-IFNβ-NIS, VV1). VSV-IFNβ-NIS is a live virus designed to express both the human interferon β (hIFNβ) gene and the thyroid sodium iodide symporter (NIS). This virus was constructed by inserting the hIFNβ gene downstream of the M gene with respect to the G protein and the NIS gene downstream of that gene (cDNA) into a full-length infectious molecular clone of the Indiana strain of Vesicular Stomatitis Virus (VSV). VSV-IFNβ-NIS is described in PCT / US2011 / 050227, which is incorporated herein by reference. An illustration of the construction of Voyager-V1 is shown in FIG. 6. EXAMPLES

[0051] Example 1. Voyager-V1 systemic viral therapy Voyager-V1 (VSV-IFNβ-NIS, VV1) is an armed, traceable, oncolytic vesicular stomatitis virus (VSV) designed to selectively destroy tumor cells via direct oncolysis and immune activation. VV1 expresses human interferon beta (IFNβ) and the NIS sodium iodine symporter. During testing, it was discovered that IFNβ could also serve as a soluble biomarker for monitoring viral replication in vivo. We report here the novel use of virally encoded IFNβ using correlative data from three Phase 1 trials of Voyager-V1 in patients (n=51) with resistant cancers, and a case study illustrates the mechanism of action (MOA) of Voyager-V1. A description of the Voyager-V1 construct is shown in Figure 6.

[0052] The primary objective of the study includes the safety and tolerability of Voyager-V1 following intratumoral (IT) or intravenous (IV) administration in patients with relapsed or recurrent hematologic malignancies or solid tumors.

[0053] Secondary objectives of this study include establishing proof of concept (e.g., by NIS imaging, immune activation, and tumor selectivity), PK and PD of Voyager-V1, viral shedding, immune response, and response rate. A schematic flow chart of the study design is shown in Figure 7.

[0054] 51 patients had a 3 × 10 6 ~5×10 10 TCID 50 Patients received a single dose of Voyager-V1 either IT or IV at doses ranging from 0.1 mg / kg to 1.5 mg / kg.

[0055] Blood was collected before administration of virus (both IV and IT), 4 hours after injection (IV), on day 2 (24 hours; both IT and IV), on days 3, 8, and 15 (both IT and IV), on day 22 (IV only), and on day 29 (IT only). IFNβ levels were measured using a standard ELISA kit specific for human IFNβ (PBL Assay Science, NJ). Cytokine levels were tested using a multiple cytokine assay kit (R&D Systems, MN). Exemplary protocols are shown in Examples 3 and 4 below.

[0056] The efficacy of Voyager-V1 systemic virotherapy is illustrated in Figures 8A, 8B, 9A, 9B, 10A, and 10B. Specifically, Figure 8A shows CT scans in a subject with endometrial cancer before treatment and 3 months after Voyager-V1 treatment. Overall tumor reduction is 16.5% in diameter at day 29. Figure 8B shows that a subject with T-cell lymphoma shows a 75% reduction in tumor diameter. Figures 9A and 9B show that NIS imaging confirms infection of tumors by Voyager-V1 in two subjects, subject 105-021 (Figure 9A) and subject 105-020 (Figure 9B). Figures 10A and 10B show that Voyager-V1 treatment increases CD8 tumor-infiltrating cells one month after intravenous (subject 6, Figure 10A) or intratumoral (subject 103-014, Figure 10B) injection.

[0057] Example 2. Viral concentration predicts response Patients with various solid tumor indications were injected intratumorally with Voyager-V1. The dose of Voyager-V1 was 3×10 6 ~3×10 9 TCID 50 The injected virus concentration ranged from 7.5 × 10 to 7.5 × 10 for n = 27 patients, and the injected volume ranged from 0.5 to 4.0 mL, depending on the size of the injected lesion. 5 ~1.5×10 9 TCID 50 / mL and contained some interferon beta in the injection volume (clinical product was 8 × 10 5 ~1.2×10 6 The IFNβ serum levels were assessed at each time point, and peak serum interferon beta levels for all patients with detectable (>1.2 pg / mL) interferon beta were plotted against the concentration of injected virus for each patient (n=18). Peak serum IFNβ levels followed a bell-shaped curve with respect to the concentration of injected virus.

[0058] The highest IFNβ reading was observed at 1 × 10 8 ~2.5×10 8 TCID 50 / mL concentration range (two-tailed Student's t test evaluating peak interferon beta levels in patients treated within this concentration range (n = 8) versus all other patients (n = 19), P = 0.031).

[0059] 78% of patients with stable disease (SD) had a 1 × 10 8 ~2.5×10 8 TCID 50 Nine patients showed SD at 6 weeks after Voyager-V1 therapy. Of these patients, 7 / 9 (78%) were treated with a concentration range of 1 × 10 8 ~2.5×10 8 TCID 50 / mL concentration range).

[0060] Increasing the concentration of IFNβ in the virus preparation can be inhibitory to virus replication. The mean serum interferon beta level measured 24 hours after Voyager-V1 administration was 7.5 × 10 6 TCID 50 2.0 pg / mL IFNβ to 2.5 × 108 TCID 50 / mL (mean), above which peak IFNβ levels began to decline (5 × 10 8 TCID 50 77 pg / mL IFNβ in 10 / mL; 7.5 × 10 8 TCID 50 / mL, 23 pg / mL IFNβ, and 1 × 10 9 TCID 50 / mL and above 11 pg / mL IFNβ). Higher virus concentrations imply higher IFNβ concentrations in the injected virus preparation, which may inhibit viral growth and spread.

[0061] As shown in Figure 1, the concentration of intratumorally injected Voyager-V1 virus correlates with patient response to treatment at day 2 (24 hours after administration). IFNβ levels are predictive of patient response to Voyager-V1. Patients with detectable levels of IFNβ tend to have stable disease. Additionally, Figure 2 shows plasma IFNβ levels at day 2 (24 hours) in patients who received a single intravenous dose of Voyager-V1. Dose levels (DL) 1, 2, or 3 of Voyager-V1. DL1, DL2, and DL3 are 5 x 10 of virus administered by IV route to each subject, respectively. 9 , 1.7×10 10 , and 5 × 10 10 TCID 50 SD means stable disease. PR means partial response. Each diamond represents a single treated subject.

[0062] VSV infection appears to result in an adaptive host immune response, producing neutralizing antiviral antibodies (Figure 3). Peak IFNβ levels (day 2 shown in Figure 3) correlated with anti-VSV antibody titers, indicating that IFNβ levels early (24 hours) after therapeutic virus injection may be a good indicator of Voyager-V1 virus replication and infection as well as tumor permissiveness to viral therapy.

[0063] The kinetics of IFNβ (increase) and IFNα (decrease) indicate that day 2 may be a suitable time point to measure IFNβ as a pharmacodynamic (PD) marker of Voyager-V1 infection in tumors. In particular, Figures 4A-4F show a comparison of the relative IFNβ and IFNα trends in patients. Figures 4A-4C show that IFNβ levels (thick line) increase at 24 hours post-dose. Figures 4D-4F show that IFNα levels (thick line) decrease at 24 hours post-dose in the same patients. The data indicate that IFNβ transgene levels may serve as a PD marker of viral infection in tumors.

[0064] In conclusion, Voyager-V1 was administered to 51 subjects by IT or IV route. No viral shedding was observed in buccal swabs or urine. Plasma levels of IFNβ are a good early indicator of viral replication and may be a good PD marker for tumor susceptibility to Voyager-V1.

[0065] Example 3. Sub-therapeutic doses of IT administration of virus for diagnostic testing in cancer therapy There is a long-felt need for early assessment of individual patient response to cancer therapy and to tailor treatment decisions based on each patient's individual response and changing circumstances. Understanding an individual patient's tumor microenvironment and immune response to cancer therapeutic agents can inform the selection of the most effective treatment regimen tailored to a particular individual.

[0066] Furthermore, as shown above in Example 2, plasma levels of IFNβ are a good early indicator of viral replication and a good PD marker for tumor susceptibility to Voyager-V1. For this reason, it is important to know the lowest dose of Voyager-V1 that can produce a detectable signal of IFNβ from readily available samples, such as blood, serum, or plasma.

[0067] Various doses of Voyager-V1 were administered intratumorally to patients with various solid tumors. The doses tested were 3×10 6 ~3×10 9 TCID 50 Circulating levels of IFNβ in serum ranged from approximately 3 × 10 7 TCID 50 It can be detected even in patients who received intratumoral administration of a low, subtherapeutic, non-toxic dose of 1×10. See, for example, Figures 5A-5C. Furthermore, from DL4 onwards (1×10 8 TCID 50 ) showed both an increase in the frequency of detectable circulating IFNβ levels and an increase in the levels of circulating IFNβ with increasing dose levels. See, for example, Figures 5B-5E. Thus, low, non-toxic, non-therapeutic doses of Voyager-V1 can be used to identify cancer tissues in patients that may be responsive to administration of a cancer therapy regimen.

[0068] Furthermore, this method can be used not only with Voyager-V1, but with any oncolytic virus probe, especially GMP-grade viruses, that contains a nucleic acid encoding soluble IFNβ. In the examples shown above, it was established that circulating IFNβ levels can be a good indicator of the variability of viral infection and transmission in individual patients. For Voyager-V1, the subtherapeutic exploration dose is approximately 10 6 TCID 50 ~about 10 8 TCID 50 The dose can be as low as 100 mg / kg and can be administered intratumorally (as shown in Figures 5A-5F) or, more conveniently, intravenously.

[0069] Example 4. Sample collection and preparation Samples from patients can be collected using any suitable protocol available in the art. Exemplary sample collection procedures used in the studies are provided herein.

[0070] Blood (1 x 1.5 mL) was collected in one 5 mL red top tube. Samples were collected at the following intervals: pre-treatment day 1, 2, 3, 4 (IT+IV patients only), 8 and 15. If day 15 is positive, samples should only be collected on days 22 and 43.

[0071] Samples were processed according to the following protocol: Gently invert the tube 5 times. Allow the sample to sit for 30-60 minutes. Then spun down at 2200-2500 RPM for 15 minutes. Transfer 1-2 mL of serum (supernatant) to a 2 mL plastic cryovial. Samples should be transferred to a -80°C freezer. Samples were then stored and transported to the facility for testing. When preparing samples for shipment, it is important to keep all samples completely frozen. For temporary storage / manipulation of samples outside of the -80°C freezer, polystyrene containers containing dry ice can be used.

[0072] Example 5. Assay for IFNβ IFN-β levels from patient samples were assessed by standard ELISA assay using the VeriKine-HS™ Human IFN Beta Serum ELISA Kit (Cat. No. 41415-1, PBL Assay Science, Piscataway Township, NJ) according to the manufacturer's instructions provided in Protocol A (Enhanced Protocol for Improved Performance in Serum Assessment).

[0073] An exemplary protocol is shown below. To each well, add sequentially: 50 μl sample buffer, 50 μl diluted antibody, and 50 μl test sample, IFN-β standard, or blank. Incubate for 2 hours with shaking at 450 rpm. Aspirate and wash 3 times. Add 100 μl diluted HRP solution. Incubate for 30 minutes with shaking at 450 rpm. Aspirate and wash 4 times. Then add 100 μl TMB substrate. Incubate for 60 minutes in the dark. Do not seal, shake, or wash. Add 100 μl stop solution. Read plate within 5 minutes at 450 nm. All incubations are performed at room temperature (22° C.-25° C.). Total assay time is approximately 3 hours and 30 minutes.

[0074] A standard curve was generated according to the following protocol: a) Label eight polypropylene tubes (S1-S8). b) Add the indicated amount of standard diluent or sample matrix to the labeled tubes according to the manufacturer's instructions provided in Protocol A. c) Using a polypropylene tip, add 10 μL of IFN standard to 90 μL of standard diluent or sample matrix. Set the volume to 80 μL and mix well by pipetting up and down 10 times using a 100 μL or 200 μL pipette. d) Add 7.5 μL of 1:10 pre-diluted standard to S8, mix well, and recover all material stuck to the inside of the pipette tip. e) Using a pipette set to 250 μL, mix S8 well by pipetting up and down 10 times. Transfer 250 μL of S8 to S7 and mix well by pipetting up and down 5 times. Repeat to complete the series for S1. f) Set aside until use in step 1 of the assay procedure.

[0075] Example 6. Treating cancer patients who are likely to be responders to viral therapy Following administration of a first therapeutic or sub-therapeutic dose of Voyager-V1 in a subject diagnosed with cancer, circulating IFNβ levels can be detected from a sample obtained from the subject using the methods set forth above.

[0076] Subjects with plasma IFNβ levels above about 1000 pg / mL have tumors that are highly sensitive to virotherapy. These subjects can be identified as strong responders and can be administered, for example, within one week, an additional therapeutic dose of Voyager-V1 or another oncolytic virus. Subjects with plasma IFNβ levels between about 10 pg / mL and about 1000 pg / mL have tumors that are immunologically infected by the virus at the time of measurement. These subjects are identified as moderate responders at this dose and should be administered an additional therapeutic dose of Voyager-V1, or another oncolytic virus, in combination with other cancer therapeutics. Subjects with plasma IFNβ levels below about 10 pg / mL have tumors that do not respond to virotherapy. These subjects can be identified as poor responders and should be administered other cancer therapeutics or boosters. The other cancer therapeutics can be, for example, immunotherapy, chemotherapy, radiation therapy, hormone therapy, etc. The immunotherapy can be an immune checkpoint inhibitor, for example, a PD-L1 inhibitor.

[0077] Circulating IFN-β levels can be assessed any time between 12 hours and 10 days after administration of the first therapeutic or sub-therapeutic dose of Voyager-V1. For example, circulating IFN-β levels can be assessed at about 12 to 24 hours after administration, or about 24 to 48 hours after administration.

[0078] Within about 12-48 hours after the first administration of Voyager-V1, if circulating levels of IFNβ are too high, e.g., above 10,000 pg / mL, the patient is administered one or more therapeutic doses of a Janus kinase inhibitor (JAK inhibitor). The JAK inhibitor can be, for example, ruxolitinib, or any commonly used JAK inhibitor.

Claims

1. 1. A method for determining the likelihood that cancerous tissue in a subject having cancerous tissue will respond to administration of a cancer therapy regimen, the method comprising: (a) intratumorally administering to the cancer tissue a sub-therapeutic diagnostic dose of an oncolytic viral probe comprising a nucleic acid encoding soluble interferon beta (IFNβ); and (b) measuring circulating levels of IFNβ in the subject following administration of the oncolytic virus and determining whether the cancer tissue is a strong responder, intermediate responder, poor responder, or non-responder; A method comprising:

2. The method of claim 1 , wherein the cancer therapy regimen comprises an oncolytic viral probe administered intratumorally in (a).

3. The method of claim 1, wherein the cancer therapy regimen comprises an oncolytic viral probe different from that administered intratumorally in (a).

4. The method of claim 1 , wherein the cancer therapy regimen is an immuno-oncolytic therapy.

5. 10. The method of claim 1, wherein the cancer therapy regimen is an antibody or small molecule anti-cancer therapy.

6. The method of claim 1 , wherein the oncolytic viral probe is administered in a non-toxic and non-therapeutic dose.

7. The non-therapeutic and non-toxic doses are about 10 ５ T.C.I.D. ５０ ~Approx. 3×10 ９ T.C.I.D. ５０ The method of claim 1, wherein

8. The non-therapeutic and non-toxic doses are about 10 ８ T.C.I.D. ５０ ~Approx. 5×10 ８ T.C.I.D. ５０ The method according to claim 7, wherein

9. The method of claim 1 , wherein the oncolytic viral probe is a GMP grade virus.

10. The method according to any one of claims 1 to 9, wherein the oncolytic viral probe is vesicular stomatitis virus (VSV).

11. The method of claim 10, wherein the oncolytic viral probe further comprises a nucleic acid encoding a sodium iodide symporter (NIS).

12. The method of claim 12, wherein the oncolytic viral probe has the construct N-P-M-IFNβ-G-NIS-L.

13. The method of any one of claims 1 to 12, wherein the circulating level of IFNβ is measured in the subject between about 12 hours and about 45 days after administration of an oncolytic virus.

14. The method of claim 13, wherein the circulating level of IFNβ is measured in the subject between about 12 hours and about 3 days after administration of the oncolytic virus.

15. The method of claim 14, wherein the circulating level of IFNβ is measured in the subject about 48 hours after administration of the oncolytic virus.

16. The method of claim 14, wherein the circulating level of IFNβ is measured in the subject about 24 hours after administration of the oncolytic virus.

17. The method of any one of claims 1 to 16, wherein the circulating levels of IFNβ are measured by an immunological assay.

18. The method of any one of claims 1 to 17, wherein the cancer tissue is a solid tumor or a hematological malignancy.

19. 19. The method of any one of claims 1 to 18, wherein the cancer tissue is head and neck cancer, colon cancer, rectal cancer, pancreatic cancer, bladder cancer, breast cancer, hepatocellular carcinoma, lung cancer, medulloblastoma, atypical teratoid / rhabdoid tumor, leukemia, lymphoma, or myeloma.

20. 1. A method of treating cancer in a subject, comprising: (a) identifying in a subject having cancerous tissue the likelihood that the cancerous tissue will respond to administration of a cancer therapy regimen according to the method of any one of claims 1 to 19; and (b) administering a cancer therapy regimen if the cancer tissue is determined to be a strong or moderate responder. A method comprising:

21. 21. The method of claim 20, wherein the cancer therapy regimen comprises administration of two or more anti-cancer compositions.

22. 21. The method of claim 20, wherein the cancer tissue is a solid tumor and the cancer therapy regimen is an oncolytic virus administered intratumorally at a dose based on the number of viral particles per unit volume of tumor.

23. 23. The method of claim 22, wherein the therapeutic dose of the oncolytic virus administered intratumorally is administered in a standard dose range.

24. The method of any one of claims 19 to 23, wherein the cancer therapy regimen is an oncolytic virus administered intravenously.

25. 1. A method of treating a subject diagnosed with cancer, the method comprising: administering to a subject a first dose of an oncolytic virus cancer therapy regimen that includes a nucleic acid encoding interferon beta (IFNβ), and if the subject is identified as a strong or intermediate responder to the oncolytic virus cancer therapy regimen, administering at least a second dose of the oncolytic virus cancer therapy regimen. A method comprising:

26. 26. The method of claim 25, wherein administration of the first dose of the oncolytic viral cancer therapy regimen is intravenous.

27. 26. The method of claim 25, wherein administration of the first dose of the oncolytic viral cancer therapy regimen is intratumoral administration.

28. 28. The method of any one of claims 25 to 27, wherein the first dose of the oncolytic viral cancer therapy regimen is a non-therapeutic and non-toxic dose of the oncolytic viral cancer therapy regimen.

29. 29. The method of any one of claims 25 to 28, wherein administration of the second dose of the oncolytic viral cancer therapy regimen is intravenous or intratumoral.

30. 30. The method of any one of claims 25 to 29, wherein the oncolytic viral cancer therapy regimen comprises a nucleic acid encoding a sodium iodide symporter (NIS).

31. The method of any one of claims 25 to 30, wherein the oncolytic virus is an RNA virus.

32. The method of any one of claims 25 to 31, wherein the oncolytic virus is vesicular stomatitis virus (VSV).

33. The method of claim 32, wherein the VSV has the construct N-P-M-IFNβ-G-NIS-L.

34. 34. The method of any one of claims 25-33, further comprising administering to the subject one or more additional immuno-oncology therapeutic agents if the subject is identified as an intermediate responder to the oncolytic virus cancer therapy regimen.

35. 35. The method of any one of claims 25-34, further comprising administering to the subject a Janus kinase inhibitor (JAK inhibitor) if the subject is identified as a strong responder to the oncolytic virus cancer therapy regimen.

36. 36. The method of claim 35, wherein the JAK inhibitor is ruxolitinib.

37. 37. The method of any one of claims 25-36, wherein the level of IFNβ is assessed between about 0.5-45 days after administration of the first dose of the oncolytic virus cancer therapy regimen.

38. 38. The method of any one of claims 25-37, wherein the level of IFNbeta is assessed between about 0.5 and 3 days after administration of the first dose of the oncolytic virus cancer therapy regimen.

39. 39. The method of any one of claims 25-38, wherein the second dose of the oncolytic viral cancer therapy regimen is administered within about 1-10 days after administration of the first dose of the oncolytic viral cancer therapy regimen.

40. 40. The method of claim 39, wherein the circulating levels of IFNβ are assessed within about 12-24 hours after administration of the first dose of the oncolytic virus cancer therapy regimen.

41. The method of any one of claims 25 to 40, wherein circulating levels of IFNβ are assessed by an immunological assay.

42. The method of any one of claims 25 to 41, wherein the cancer is a solid tumor or a hematological malignancy.

43. 43. The method of claim 42, wherein the solid tumor is head and neck cancer, colon cancer, rectal cancer, pancreatic cancer, bladder cancer, breast cancer, hepatocellular carcinoma, lung cancer, medulloblastoma, or atypical teratoid / rhabdoid tumor.

44. 43. The method of claim 42, wherein the hematological malignancy is leukemia, lymphoma, or myeloma.

45. 44. The method of any one of claims 25 to 43, wherein the second administration of the oncolytic viral cancer therapy regimen is by intratumoral injection.

46. 46. ​​The method of claim 45, wherein the second intratumoral injection is administered to the subject based on the number of viral particles per unit volume of the tumor.

47. 47. The method of claim 46, wherein the second intratumoral injection is administered to the subject within a standard dosage range.

48. The method of any one of claims 25 to 44, wherein the therapeutic dose of the oncolytic virus is administered intravenously.

Images