Oncolytic poliovirus for human tumors

Abstract

To provide an oncolytic virus anti-tumor therapy.SOLUTION: According to the present invention, there is provided a method for treating a human harboring a solid tumor that expresses NECL5 (nectin-like protein 5). The method comprises the steps of: administering directly to the tumor in the human a chimeric poliovirus construct comprising a Sabin type I strain of poliovirus with a human rhinovirus 2 (HRV2) internal ribosome entry site (IRES) in the poliovirus' 5' untranslated region between the poliovirus' cloverleaf and the poliovirus' open reading frame.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention was made with funding provided by the U.S. Government, which retains certain rights pursuant to grants from the National Institutes of Health R01 CA87537, P50 NS20023, R01 CA124756, and R01 CA140510.

[0002] Technical field of the invention The present invention relates to the field of antitumor therapy, in particular to antitumor therapy with oncolytic viruses. [Background technology]

[0003] Background of the Invention PVS-RIPO is a recombinant oncolytic poliovirus (PV). PVS-RIPO consists of a live-attenuated human rhinovirus type 1 (Sabin) PV vaccine containing a foreign internal ribosome entry site (IRES) from human rhinovirus type 2 (HRV2). The IRES is a cis-acting genetic element located within the 5' untranslated region of the PV genome that mediates viral replication. 7 Mediates G-cap-independent translation.

[0004] Oncolytic therapy with PVS-RIPO has been reported in tissue culture assays (6, 7, 10, 15-17) and animal tumor models, but not in human clinical trials. Efficacy cannot be predicted due to differences between tissue culture, animal models, and humans. Furthermore, because the virus preparations used in preclinical studies are often impure, any activity cannot be attributed to the agent under investigation.

[0005] The art has not provided examples of oncolytic viral agents whose biological activity in tumor models accurately predicted efficacy in patients. This is because oncolytic viral therapy is the result of a complex triangular relationship between (a) infected malignant cells, (b) the non-malignant tumor microenvironment, and (c) the host immune system. This complex and intricate system has not been reproduced in any animal model.

[0006] There is a continuing need in the art for the identification and development of effective anti-cancer treatments for humans, particularly for brain tumor patients. Summary of the Invention

[0007] One aspect of the present invention provides a method for treating a human with a solid tumor that expresses NECL5 (CD155, HVED, Nec1-5, PVS, TAGE4, ​​nectin-like 5; nectin-like protein 5). A chimeric poliovirus construct is administered directly to the tumor in the human. The chimeric poliovirus comprises a Sabin I strain poliovirus that contains a human rhinovirus 2 (HRV2) internal ribosome entry site (IRES) in the 5' untranslated region of the poliovirus between the cloverleaf region and the open reading frame of the poliovirus.

[0008] These and other aspects which will become apparent to those skilled in the art upon reading this specification provide the art with methods of treating tumors, including brain tumors. [The present invention 1001] 1. A method of treating a human having a solid tumor that expresses NECL5 (nectin-like protein 5), comprising the steps of: A process of directly administering to the tumor in the human a chimeric poliovirus construct comprising a Sabin type I strain poliovirus, wherein the poliovirus has a human rhinovirus 2 (HRV2) internal ribosome entry site (IRES) in the 5' untranslated region of the poliovirus between the cloverleaf region of the poliovirus and the open reading frame of the poliovirus. [The present invention 1002] 1001. The method of claim 1001, wherein convection-enhanced delivery is used to administer said chimeric poliovirus construct. [The present invention 1003] 1001. The method of claim 1001, wherein said solid tumor is glioblastoma. [The present invention 1004] 1001. The method of claim 1001, wherein said solid tumor is a prostate tumor. [The present invention 1005] 1001. The method of claim 1001, wherein said administration is intracerebral administration. [The present invention 1006] 1001. The method of claim 1001, wherein said solid tumor is a medulloblastoma. [The present invention 1007] 1001. The method of claim 1001, wherein said solid tumor is a breast tumor. [The present invention 1008] 1001. The method of claim 1001, wherein said solid tumor is a lung tumor. [The present invention 1009] 1001. The method of claim 1001, wherein said solid tumor is a colorectal tumor. [The present invention 1010] 1001. The method of claim 1001, wherein said administering is intracerebral injection with convection-enhanced delivery. [The present invention 1011] The method of claim 1010, wherein said administering is stereotactically guided. [The present invention 1012] 1001. The method of claim 1001, wherein the human is an adult. [The present invention 1013] 1001. The method of claim 1001, wherein said human is a child. [The present invention 1014] 1013. The method of claim 1012, wherein concurrent or sequential chemotherapy is administered to said human. [The present invention 1015] The method of claim 1013, wherein concurrent or sequential radiation therapy is administered to said human. [The present invention 1016] 1001. The method of claim 1001, wherein said solid tumor is surgically resected before or after administration of the non-pathogenic oncolytic poliovirus. [The present invention 1017] 1002. The method of claim 1001, wherein said solid tumor is tested for expression of NECL5 prior to said administering step. [The present invention 1018] 1. A method of treating a human having a solid tumor that expresses NECL5 (nectin-like protein 5), comprising the steps of: testing the solid tumor to confirm that it expresses NECL5; Administering a chimeric poliovirus construct comprising a Sabin type I strain poliovirus, wherein the poliovirus has a human rhinovirus 2 (HRV2) internal ribosome entry site (IRES) in the 5' untranslated region of the poliovirus between the cloverleaf domain of the poliovirus and the open reading frame of the poliovirus, directly to the tumor in the human, wherein the administration is by intracerebral injection via stereotactically guided, convection-enhanced delivery. [Brief explanation of the drawings]

[0009] [Figure 1] Figures 1A-1B (formerly Figure 8). Intratumoral injection of PVS-RIPO induces gradual tumor regression. Figure 1A: Tumor volume after treatment with mock (□) or PVS-RIPO (■). Figure 1B: Mean virus recovery from tumors over the indicated time periods. [Figure 2] Figure 2 (formerly Figure 12). MRI on 4 / 16 / 2012. Axial post-contrast T1-weighted MRI showing disease progression. [Figure 3] Figure 3 (formerly Figure 13). MRI on May 9, 2012. Axial post-contrast T1-weighted MRI obtained before PVS-RIPO injection. [Figure 4] Figure 4 (formerly Figure 14). MRI from 5 / 11 / 2012. Axial, post-contrast, T1-weighted MRI showing the distribution of Gd-DTPA contrast and (presumably) PVS-RIPO in the brain. [Figure 5] Figure 5 (formerly Figure 15). MRI on 6 / 6 / 2012. Axial post-contrast T1-weighted MRI showing stable disease. [Figure 6] Figure 6 (formerly Figure 16). MRI on 7 / 9 / 2012. Axial post-contrast T1-weighted MRI revealed concern for disease progression. [Figure 7]Figure 7 (formerly Figure 17). 18-FDG PET scan from 7 / 11 / 2012. Results suggest a lack of hypermetabolic activity in the area of ​​concern on the MRI. DETAILED DESCRIPTION OF THE INVENTION

[0010] Detailed Description of the Invention The present inventors have developed a viral construct for use in humans. Previously, laboratory-grade preparations of the viral construct have been tested in cell culture and animal models. However, these tests are insufficient to attribute any effects to the viral construct itself, rather than to other components in the crude laboratory-grade preparation. Furthermore, as is well known in the art, cell culture and animal models are not predictive of efficacy in humans.

[0011] Because poliovirus is a potential pathogen, particular precautions must be taken to avoid introducing disease-causing agents into subjects. Good manufacturing practices and purification have been used to produce preparations that are sufficiently pure to be introduced into humans in studies.

[0012] Any technique for administering the preparation directly to the tumor may be used. Direct administration does not rely on blood vasculature to reach the tumor. The formulation may be painted onto the tumor surface, injected into the tumor, instilled into or at the tumor site during surgery, injected into the tumor via a catheter, etc. One particular technique that may be used is convection-enhanced delivery.

[0013] Any human tumor can be treated, including both pediatric and adult tumors. The tumor can be located in any organ, such as the brain, prostate, breast, lung, colon, and rectum. Various types of tumors can be treated, including glioblastoma, medulloblastoma, cell carcinoma, adenocarcinoma, etc. Other examples of tumors include adrenocortical carcinoma, anal carcinoma, appendix carcinoma, grade I (anaplastic) astrocytoma, grade II astrocytoma, grade III astrocytoma, grade IV astrocytoma, atypical teratoid / rhabdomyosarcoma-like tumor of the central nervous system, basal cell carcinoma, bladder carcinoma, breast sarcoma, bronchial carcinoma, bronchioloalveolar carcinoma, cervical carcinoma, craniopharyngioma, endometrial carcinoma, endometrial carcinoma, ependymoblastoma, ependymoblastoma, ependymoblastoma, ependymoblastoma, endothelial carcin ... tumor, esophageal cancer, nasal neuroblastoma, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct carcinoma, fibrous histiocytoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid, gastrointestinal stromal tumor, gestational trophoblastic tumor, gestational trophoblastic tumor, glioma, head and neck cancer, hepatocellular carcinoma, anal bile duct carcinoma, hypopharyngeal cancer, intraocular melanoma, pancreatic islet cell tumor, Kaposi's sarcoma, Langerhans cell histiocytosis, large cell undifferentiated tumor Lung cancer, laryngeal cancer, lip cancer, lung adenocarcinoma, malignant fibrous histiocytoma, medulloepithelioma, melanoma, Merkel cell carcinoma, mesothelioma, endocrine neoplasms, nasal cavity cancer, nasopharyngeal carcinoma, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian clear cell carcinoma, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, papilloma, paranasal sinus cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pineal parenchymal tumor, pineoblastoma, pituitary tumor, pleuropulmonary blastoma, kidney These include: cell carcinoma, respiratory tract cancer including mutations in chromosome 15, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous non-small cell lung cancer, squamous cervical cancer, supratentorial primitive neuroectodermal tumor, supratentorial primitive neuroectodermal tumor, testicular tumor, pharyngeal cancer, thymic carcinoma, thymoma, goiter, renal pelvis cancer, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Wilms' tumor.

[0014] Optionally, patients may be stratified based on NECL5 expression, which can be analyzed at the RNA or protein level using, for example, probes, primers, or antibodies. NECL5 expression may guide the decision to treat or not treat with a chimeric poliovirus of the invention. NECL5 expression may also be used to guide the aggressiveness of treatment, including the dose, frequency, and duration of treatment.

[0015] In addition to delivery of the chimeric poliovirus construct, the treatment regimen may include surgical removal of the tumor, surgical reduction of the tumor, chemotherapy, biological therapy, or radiation therapy. These treatments are the standard of care in many disease states, and patients are not denied these standard of care. The chimeric poliovirus may be administered before, during, or after the standard of care. The chimeric poliovirus may also be administered after failure of the standard of care.

[0016] Applicants have found that clinical pharmaceutical preparations of chimeric polioviruses have remarkable genetic stability and homogeneity. This is particularly advantageous because polioviruses are known to be highly variable both in culture and in natural bioaccumulation. Any suitable assay for genetic stability and homogeneity can be used. One assay for stability involves testing for inability to grow at 39.5°C. Another assay involves population sequencing. Yet another assay involves testing for primate neurovirulence.

[0017] While applicants do not wish to be bound by a particular mechanism of action, multiple mechanisms are believed to contribute to its efficacy. These include lysis of cancer cells, recruitment of immune cells, and specificity for cancer cells. Additionally, the virus is neuroattenuated.

[0018] The above disclosure generally describes the present invention. All references disclosed herein are expressly incorporated by reference. A more complete understanding can be obtained by reference to the following specific examples, which are provided for illustrative purposes only and are not intended to limit the scope of the invention. [Example]

[0019] Example 1 Animal tumor models.An IND efficacy study of PVS-RIPO was conducted in an HTB-15 GBM xenograft model in athymic mice. PVS-RIPO (from a clinical lot) was administered at the maximum FDA-approved starting dose, "adjusted for mice" [the FDA-approved maximum starting dose (10e8 TCID) was adjusted to account for the smaller tumor size in mice (6.7 x 10e6 TCID)]. Delivery mimicked the intended clinical route, i.e., slow intratumoral injection. Under these conditions, PVS-RIPO induced complete tumor regression in all animals after 15 days (Figure 8A). Although virus was recovered from treated tumors by day 10, the amount was at most modest, suggesting that direct viral tumor cell killing alone does not explain the efficacy of this treatment (Figure 8B).

[0020] Evidence from animal tumor models suggests that intratumoral inoculation of PVS-RIPO directly induces virus-induced tumor cell killing and elicits a strong host immune response against the infected / killed tumor (3, 7, 10). The response to virus injection is characterized by a strong, localized response that leads to immune infiltration of the tumor. Ultimately, a slow tissue response to PVS-RIPO injection leads to the disappearance of the tumor mass, which is replaced by a scar.

[0021] Example 2 Clinical trials. IND No. 14,735, "Dose-Finding and Safety Study of PVSRIPO for Recurrent Glioblastoma," was approved by the FDA on June 19, 2011, and approved by the IRB on October 27, 2011. A Phase I / II clinical trial in patients with recurrent glioblastoma (GBM) (NCT01491893) is currently enrolling patients.

[0022] Two human subjects have been treated with PVS-RIPO to date under IRB-approved protocols. Preliminary findings from the first subject are described in Example 3.

[0023] Example 3 Preliminary findings in the first human subject.The patient was a 21-year-old female nursing student diagnosed with GBM (WHO grade IV) of the right frontal lobe. She was first diagnosed in June 2011 at age 20, following a history of severe headaches and unsuccessful treatment for a suspected sinus infection. Brain imaging was obtained on June 17, 2011, and revealed a large mass measuring approximately 5 x 6 cm in the right frontal lobe. The patient underwent partial resection of the right frontal lobe mass on June 22, 2011, which was pathologically confirmed as GBM (WHO grade IV). Given the patient's young age, excellent performance status, and partial tumor resection, six weeks of radiation therapy and daily oral administration of 75 mg / m 2 It was decided to aggressively treat the patient with a combination of Temodar chemotherapy and bevacizumab (an anti-angiogenic drug) every two weeks. The patient completed six weeks of treatment on September 18, 2011. On October 3, 2011, the patient began adjuvant treatment with Temodar chemotherapy for five days each month in addition to bevacizumab 10 mg / kg every two weeks.

[0024] The patient reported to the clinic on April 16, 2012, after experiencing her first generalized seizure while sleeping. At this time, the patient had completed six months of Temodar and bevacizumab. Because the patient was completing her degree to become a pediatric oncology nurse, she attributed the seizures to increased stress at school, despite the GBM diagnosis and ongoing chemotherapy treatment. A brain MRI obtained that day showed tumor recurrence with new nodular shadows along the medial aspect of the resection cavity (Figure 12).

[0025] The patient was offered multiple treatment options but elected to proceed with the PVS-RIPO clinical trial. After the first generalized seizure, he was prescribed Keppra but occasionally forgot to take it, and as a result of this and the discovered tumor recurrence, he experienced a second generalized seizure while sleeping on May 6, 2012. The patient returned to baseline neurological status and underwent a workup for protocol enrollment.

[0026] A follow-up MRI was obtained on May 9, 2012 (Figure 13) before the patient received a PVS-RIPO injection at the maximum FDA-approved starting dose (10e8) on May 11, 2012, using the intended clinical delivery method (convection-enhanced intratumoral delivery of 3 mL of viral suspension containing the contrast agent Gd-DTPA over 6 hours; see Example 4), but no related neurological or other complications were experienced.

[0027] An MRI obtained immediately after the completion of the injection demonstrates the distribution of the injectate (Figure 14).

[0028] Our research team followed the patient on a weekly basis and met with the patient in clinic two weeks after the infusion, at which time the patient denied any neurological symptoms, recurrent seizures, fatigue, shortness of breath, or weakness. The patient was again evaluated in clinic on June 7, 2012, and the patient's physical and neurological symptoms remained normal. A brain MRI obtained at that visit showed stable disease (Figure 15).

[0029] We met with the patient in the clinic on July 9, 2012. The patient again denied the presence of any new neurological symptoms, including no recurrence of any seizure activity since the seizure observed on May 6, 2012, prior to the PVS-RIPO infusion. The patient also reported feeling well, being satisfied with her progress in nursing school, and feeling better able to concentrate on school since the infusion. The patient was also excited about moving in with two roommates and being able to exercise regularly. The patient's brain MRI, obtained that day, showed a slight increase in the mass effect and minimal enhancement of the superior streak, raising concerns about disease progression (Figure 16).

[0030] Given the worrisome radiographic changes without clinical deterioration, we decided to obtain an 18-FDG PET scan. The 18-FDG PET scan showed low metabolic activity in the area of ​​concern on the MRI, suggestive of a necrotic process (treatment response effect; Figure 17). The PET scan on July 9 suggested the absence of viable tumor. After discussion with the patient and her mother, we decided to follow the patient clinically and radiographically.

[0031] At examinations on August 27 and October 22, the patient denied the presence of any new neurological symptoms, including the absence of any seizure activity since the seizure observed on May 6, 2012 (prior to PVS-RIPO infusion). The patient reported improvement in cognitive / memory function and motor function (movement). As of October 26, the patient was neurologically normal.

[0032] A PET scan was not performed due to a positive radiographic result on August 27. The patient was rescanned on October 22 and had a quantifiable radiographic response.

[0033] MRI / PET overlay shows absence of signal from the entire area of ​​tumor recurrence.

[0034] Example 4 Convective injection. Preoperatively, the BrainLab iPlan Flow system is used to design catheter paths based on predicted distributions using information from preoperative MRI.

[0035] The present invention provides an alternative tracer for identifying the distribution of poliovirus, 124 1 mM gadolinium is used along with I-labeled human serum albumin. This can be used in other drug infusions as well. Gadolinium and radiolabeled albumin are injected along with the drug, and various MRI sequences and PET imaging are used to quantify its distribution.

[0036] The entire volume of medication to be delivered will be preloaded into the syringe and connected to the catheter by the study pharmacist under sterile conditions in the operating room or NICU immediately prior to the start of the infusion. Due to the complexity of planning all the necessary elements for the infusion (operating room time, pharmacy time, and radiology appointments), a +1-day gap was established in the study for study medication infusion. This means that the infusion may begin the day after biopsy / catheter placement. For purposes of the protocol and timing of subsequent events, this will still be considered "day 0." At the time of virus infusion, rescue medications including epinephrine and diphenhydramine will be available, and neurological status, oxygen saturation, and cardiac rhythm will be monitored. Medication infusions will be performed in the neurosurgical intensive care unit (NSCU) so that all other emergency facilities are available. Patients will begin with induction of anesthesia for catheter placement and will be treated with prophylactic antibiotics such as nafcillin, a second-generation cephalosporin, or vancomycin.

[0037] Based on our own experience, a previously published report (19), and an IRB- and FDA-approved study (IRB#4774-03-4R0) using similar infusion techniques, patients would receive an infusion at a rate of 500 μL / h. The Medfusion 3500 infusion pump would be preprogrammed for a delivery rate of 500 μL / h. The drug (which would total 10 mL to fill the 3.3723 mL "dead space" in the infusion system) would be loaded into a 20 mL syringe in the syringe pump at the start to avoid any interruptions in the infusion. The total volume of inoculum delivered to the patient would be 3 mL. It is not possible to preload the catheter itself (30 cm long, 1 mm internal diameter) with the virus suspension. Therefore, the first approximately 250 μL of the infusion would be preservative-free saline in the "dead space" of the indwelling catheter. Commensurate with this, the infusion pump would be programmed to deliver 3.250 mL. The infusion will be performed using a Medfusion 3500 (Medex, Inc., Duluth, GA) syringe infusion pump. The viral infusion process will be completed within 6.5 hours. The catheter will be removed immediately after delivery of the PVSRIPO.

[0038] The infusion catheter (PIC030) and infusion tubing (PIT400) will be supplied by Sohysa, Inc. (Crown Point, IN). The Infusion Catheter Kit is a 30-cm clear, open-ended catheter (1.0 mm ID / 2.0 mm OD) marked every 20 cm. The catheter includes a 30-cm stainless steel stylet, a capped barbed female Luer lock, and a stainless steel trocar. The Infusion Tubing Kit consists of a three-way stopcock connector with air filter, 4 m of fine-bore tubing with an antisiphon valve, a red open-hole cap, and a white Luer lock cap. The catheter products are packaged sterile, non-pyrogenic, and intended for single-use only. Infusions will be performed using a Medfusion 3500 (Medex, Inc., Duluth, GA) syringe infusion pump.

[0039] References The disclosure of each cited reference is expressly incorporated herein. TIFF0007765896000001.tif230161TIFF0007765896000002.tif237161

Claims

1. A pharmaceutical for treating melanoma tumors expressing NECL5 (nectin-like protein 5) in patients with said melanoma tumors, comprising a chimeric poliovirus construct comprising a Sabin type I poliovirus strain having a human rhinovirus 2 (HRV2) internal ribosome entry site (IRES) in the 5' untranslated region of the poliovirus between the cloverleaf domain of the Sabin type I poliovirus strain and the open reading frame of the poliovirus, characterized in that the chimeric poliovirus construct is used to be administered directly to the melanoma tumor.

2. The pharmaceutical composition of claim 1, wherein the patient is an adult.

3. The pharmaceutical composition of claim 1, wherein the patient is a child.

4. The medicament according to any one of claims 1 to 3, characterized in that it is used in combination with simultaneous or sequential chemotherapy.

5. 5. The medicament according to any one of claims 1 to 4, characterized in that the medicament is used in combination with surgical resection of a melanoma tumor before or after administration of the chimeric poliovirus construct.

6. The method of any one of claims 1 to 5, wherein melanoma tumors in patients are tested for expression of NECL5 prior to administration of the chimeric poliovirus construct.

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