Patent Grant
7638318
This disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.
All patent applications, published patent applications, issued and granted patents, texts, and literature references cited in this specification are hereby incorporated herein by reference in their entirety to more fully describe the state of the art to which the present invention pertains.
Virotherapy holds great promise for treating cancer. Oncolytic viruses, which aim to specifically infect and kill cancer cells, whether native and/or engineered, may be more efficacious and less toxic than alternative treatments, such as chemotherapy and radiation. In addition, oncolytic virus therapy that uses replication competent viruses is the only therapy known that can amplify the therapeutic at the pharmacologically desired site.
A key aspect of cancer therapy is to achieve a high rate of killing of cancer cells versus normal cells. Accomplishing this goal has been difficult for many reasons, including the wide array of cell types involved, the systemic dissemination of cancer cells due to metastases, and the narrow biological differences between normal and cancer cells. While progress has been made, much still needs to be done to improve upon current cancer therapies.
In the past, surgeons have tried to remove tumors surgically without substantially harming the patient. Even complete removal of a primary tumor does not ensure survival since earlier metastases to unknown sites in the body are left undetected. There is also some research suggesting that surgical intervention may enhance the growth of distant metastases due to removal of tumor cells producing angiogenesis inhibitors. Finally, in many cases, the tumor grows back at the original site after surgical removal. Radiation aims to selectively destroy the most rapidly proliferating cells at the expense of the others. However, tumor cells can escape radiation therapy either by becoming resistant or by being in a non-dividing state during treatment. In addition, radiation is not always selective in that many normal cells are actively dividing and killed by the treatment (cells in bone marrow, gastrointestinal cells, hair follicles, etc.).
Like radiation, chemotherapy is not completely selective and thus destroys many normal cells, and does not kill all tumor cells due to drug resistance and/or division state of the cell. Thus, chemotherapy and radiation therapies exploit a small differential sensitivity that exists between normal and cancer cells, giving them a narrow therapeutic index. A small therapeutic index is clearly an undesirable property of any modality to treat cancer. Therefore, novel cancer therapeutic approaches overcoming these limitations are desired.
One such novel approach is oncolytic virus therapy. Initially, replication-defective viruses carrying cytotoxic transgenes were utilized in attempts to treat cancer. However, they were found to be inefficient in transduction of tumors, inadequate spread within the tumor mass and not adequately selective toward cancers. To overcome this limitation, viruses were either modified to replicate selectively in tumor cells or viruses were discovered to have natural tumor-selective properties. These oncolytic viruses thus had the properties to replicate, spread, and kill tumor cells selectively through a tumor mass by locally injecting the virus or by systemically delivering the virus (
Despite the early promise of this newly defined class of anti-cancer therapeutics, several limitations remain that may limit their use as a cancer therapeutic. Therefore, there is an ongoing need for novel oncolytic viruses that can be utilized for cancer therapy.
A novel RNA picornavirus has been discovered (hereafter referred to as Seneca Valley virus (“SVV”)) whose native properties include the ability to selectively kill some types of tumors. As demonstrated below in the examples, SVV selectively kills tumor lines with neurotropic properties, in most cases with a greater than 10,000 fold difference in the amount of virus necessary to kill 50% of tumor cells versus normal cells (i.e., the EC50 value). This result also translates in vivo, where tumor explants in mice are selectively eliminated. Further, in vivo results indicate that SVV is not toxic to normal cells, in that up to 1×1014 vp/kg (vector or virus particles per kilogram) systemically administered causes no mortality and no visible clinical symptoms in immune deficient or immune competent mice.
SVV elicits efficacy at doses as low as 1×108 vp/kg; therefore, a very high therapeutic index of >100,000 is achieved. Efficacy is very robust in that 100% of large pre-established tumors in mice can be completely eradicated (see Example 11). This efficacy may be mediated with a single systemic injection of SVV without any adjunct therapy. Furthermore, SVV injected mice show neither clinical symptoms nor recurrence of tumors for at least 200 days following injection. SVV can also be purified to high titer and can be produced at >200,000 virus particles per cell in permissive cell lines. SVV-based viral therapy therefore shows considerable promise as a safe, effective and new line of treatment for selected types of cancers. Further, SVV has a small and easily manipulatable genome, simple and fast lifecycle, and a well-understood biology of replication, and thus is amenable to modification. These properties, at least in part, allow for methods that generate modified SVVs that have new cell or tissue specific tropisms, such that SVV-based therapy can be directed to new tumor types resistant to infection by the original SVV isolate.
Accordingly, the present invention provides an isolated nucleic acid comprising a nucleic acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 168, or a contiguous portion of any one of these sequences that is at least 50 nucleotides in length, or 95% identical to a contiguous portion of any one of these sequences that is at least least 10, 15 or 20 nucleotides in length. The isolated nucleic acids of the invention can be RNA or DNA.
For all aspects of the invention, an isolated nucleic acid can comprise a nucleic acid sequence having at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a contiguous portion of any one of the SVV nucleic acid SEQ ID NO sequences herein, wherein the contiguous portion is at least about 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1250, 1500, 2000 or 2500 nucleotides in length, for example. The SVV nucleic acid SEQ ID NO sequences include, for example, SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 168.
For all aspects of the invention, an isolated protein or peptide can comprise an amino acid sequence having at least about 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a contiguous portion of any one of the SVV amino acid SEQ ID NO sequences herein, wherein the contiguous portion is at least at least about 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, or 350 amino acids in length, for example. The SVV amino acid SEQ ID NO sequences include, for example, SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 169.
In another aspect, the invention provides an isolated nucleic acid comprising a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:168, or to a contiguous portion of SEQ ID NO:168 that is at least 20, 50, 100, or 200 nucleotides in length. The isolated nucleic acids can comprise specific portions of SEQ ID NO:168, including but not limited to: the 5′ untranslated region (UTR) of SVV spanning nucleotides 1-666 of SEQ ID NO:168; the coding sequence for the SVV polyprotein spanning nucleotides 667-7209 of SEQ ID NO:168; the coding sequence for the leader peptide of SVV spanning nucleotides 667-903 of SEQ ID NO:168; the coding sequence for the SVV VP4 protein spanning nucleotides 904-1116 of SEQ ID NO:168; the coding sequence for the SVV VP2 protein spanning nucleotides 1117-1968 of SEQ ID NO:168; the coding sequence for the SVV VP3 protein spanning nucleotides 1969-2685 of SEQ ID NO:168; the coding sequence for the SVV VP1 protein spanning nucleotides 2686-3477 of SEQ ID NO:168; the coding sequence for the SVV 2A protein spanning nucleotides 3478-3504 of SEQ ID NO:168; the coding sequence for the SVV 2B protein spanning nucleotides 3505-3888 of SEQ ID NO:168; the coding sequence for the SVV 2C protein spanning nucleotides 3889-4854 of SEQ ID NO:168; the coding sequence for the SVV 3A protein spanning nucleotides 4855-5124 of SEQ ID NO:168; the coding sequence for the SVV 3B protein spanning nucleotides 5125-5190 of SEQ ID NO:168; the coding sequence for the SVV 3C protein spanning nucleotides 5191-5823 of SEQ ID NO:168; the coding sequence for the SVV 3D protein spanning nucleotides 5824-7209 of SEQ ID NO:168; and the 3′UTR of SVV spanning nucleotides 7210-7280 of SEQ ID NO:168.
In one aspect, the invention provides methods for using the SVV 2A, SVV leader peptide, or other SVV proteins or peptide portions thereof, to shut off host cell protein translation. In one aspect, such SVV proteins can be used to shut off host cell protein translation by interfering or inhibiting with the cap binding protein complex in the host cell.
In another aspect, the invention provides methods for using SVV 2A or other SVV proteins or peptide portions thereof in order to cleave a peptide or protein.
In other aspects, the invention provides an isolated nucleic acid that hybridizes under conditions of high, moderate stringency or low stringency to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 168, or to a contiguous portion of any one of these sequences that is at least 50 nucleotides in length.
In another aspect, the invention provides a vector comprising a nucleic acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NOS 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 168, or to a contiguous portion of any one of these sequences that is at least 50 nucleotides in length. Vector compositions can also comprise the nucleic acid regions of SEQ ID NO:168 that code for SVV proteins.
The present invention also provides an isolated polypeptide encoded by a nucleic acid having at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to a nucleic acid sequence comprising SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or to a contiguous portion of any one of these sequences that is at least 50 nucleotides in length. The invention also provides an isolated polypeptide encoded by a nucleic acid having at least 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleic acid region of SEQ ID NO:168 that encodes a SVV protein.
In one aspect, the invention provides an isolated polypeptide comprising an amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 169, or to a contiguous portion of any one of these sequences that is at least 10 amino acids in length.
In another aspect, the invention provides an isolated polypeptide comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to a contiguous portion of SEQ ID NO:169 that is at least 9, 10, 15, 20 or 50 amino acids in length. Exemplary contiguous portions of SEQ ID NO:169, include but are not limited to, regions that comprise a SVV protein, such as: the leader peptide spanning residues 1-79; VP4 spanning residues 80-150; VP2 spanning residues 151-434; VP3 spanning residues 435-673; VP1 spanning residues 674-937; 2A spanning residues 938-946; 2B spanning residues 947-1074; 2C spanning residues 1075-1396; 3A spanning residues 1397-1486; 3B spanning residues 1487-1508; 3C spanning residues 1509-1719; and 3D spanning residues 1720-2181.
In another aspect, the invention provides an isolated antibody which specifically binds a polypeptide comprising an amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 169, or to a contiguous portion of any one of these sequences that is at least 9, 10, 15, or 20 amino acids in length. The isolated antibody can be generated such that it binds to any protein epitope or antigen of SEQ ID NOS:2 or 169. Further, the antibody can be a polyclonal antibody, a monoclonal antibody or a chimeric antibody.
In one aspect, the invention provides an isolated SVV or derivative or relative thereof, having a genomic sequence comprising a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO:1 or SEQ ID NO:168.
In another asepct, the invention provides an isolated virus having all the identifying characteristics and nucleic acid sequence of American Type Culture Collection (ATCC) Patent Deposit number PTA-5343. Some of the viruses of the present invention are directed to the PTA-5343 isolate, variants, homologues, relatives, derivatives and mutants of the PTA-5343 isolate, and variants, homologues, derivatives and mutants of other viruses that are modified in respect to sequences of SVV (both wild-type and mutant) that are determined to be responsible for its oncolytic properties.
The present invention further provides an isolated SVV comprising the following characteristics: a single stranded RNA genome (positive (+) sense strand) of ˜7.5 or of ˜7.3 kilobases (kb); a diameter of ˜27 nanometers (nm); a capsid comprising at least 3 proteins that have approximate molecular weights of about 31 kDa, 36 kDa and 27 kDa; a buoyant density of approximately 1.34 g/mL on cesium chloride (CsCl) gradients; and replication competence in tumor cells. In this aspect, the 31 kDa capsid protein (VP1) can comprise an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO:8 or residues 674-937 of SEQ ID NO:169; the 36 kDa capsid protein (VP2) can comprise an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO:4 or residues 151-434 of SEQ ID NO:169; and the 27 kDa capsid protein (VP3) can comprise an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO:6 or residues 435-673 of SEQ ID NO:169.
In another aspect, the invention provides an isolated SVV derivative or relative comprising the following characteristics: replication competence in tumor cells, tumor-cell tropism, and lack of cytolysis in normal cells. An SVV relative includes SVV-like picornaviruses, including viruses from the following USDA isolates: MN 88-36695, NC 88-23626, IA 89-47552, NJ 90-10324, IL 92-48963, CA 131395; LA 1278; IL 66289; IL 94-9356; MN/GA 99-29256; MN 99197; and SC 363649. If an SVV-like picornavirus does not naturally have the characteristics of replication competence in tumor cells, tumor-cell tropism, and lack of cytolysis in non-tumor cells, then the SVV-like picornavirus can be mutated such that these characteristics are obtained. Such mutations can be designed by comparing the sequence of the SVV-like picornavirus to SVV, and making mutations into the SVV-like picornavirus such that its amino acid sequence is identical or substantially identical (in a particular region) to SVV. In another aspect, the virus is replication competent in tumor cell types having neuroendocrine properties.
In other aspects, the present invention provides: a pharmaceutical composition comprising an effective amount of a virus of the invention and a pharmaceutically acceptable carrier; a cell comprising a virus of the invention; a viral lysate containing antigens of a virus of the invention; and an isolated and purified viral antigen obtained from a virus of the invention.
In yet another aspect, the invention provides a method of purifying a virus of the invention, comprising: infecting a cell with the virus; harvesting cell lysate; subjecting cell lysate to at least one round of gradient centrifugation; and isolating the virus from the gradient.
In another aspect, the invention provides a method for treating cancer comprising administering an effective amount of a virus or derivative thereof, so as to treat the cancer, wherein the virus has a genomic sequence that comprises a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 168, or to a portion of SEQ ID NO:1 or SEQ ID NO:168. In one aspect, the invention provides a method for treating cancer a method for treating cancer comprising administering an effective amount of a virus or derivative thereof, so as to treat the cancer, wherein the virus has a genomic sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1. The virus that has a genomic sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1 can be, for example, a SVV mutant, a SVV-like picornavirus, or a cardiovirus. The SVV-like picornavirus can be, for example, a virus from one of the following isolates MN 88-36695, NC 88-23626, IA 89-47752, NJ 90-10324, IL 9248963, CA 131395; LA 1278; IL 66289; IL 94-9356; MN/GA 99-29256; MN 99197; and SC 363649. The SVV-like picornaviruses can be wild-type or mutant.
In another aspect, the invention provides a method for treating cancer comprising adminstering an effective amount of a virus comprising a capsid encoding region that comprises a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOS:3, 5, 7, nucleotides 904-3477 of SEQ ID NO:169, or to a contiguous portion thereof that is at least 75, 100, 200, or 500 nucleotides in length. The invention also provides a method for treating cancer comprising administering an effective amount of a virus comprising a capsid that comprises an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:4, 6, 8, residues 80-937 of SEQ ID NO:169, or a contiguous portion thereof that is at least 25, 50, or 100 amino acids in length.
In one aspect, the present invention provides a method for inhibiting cancer progression comprising contacting a cancer cell with a virus or derivative thereof, wherein the virus or derivative thereof specifically binds to the cancerous cell, wherein the virus has a genomic sequence that comprises a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 168.
In another aspect, the invention provides a method for inhibiting cancer progression comprising contacting a cancer cell with a virus or derivative thereof, wherein the virus or derivative thereof specifically infects the cancerous cell, wherein the virus has a genomic sequence that comprises a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 168, or to a contiguous portion of SEQ ID NO:168 that is at least 50, 100, 200, or 500 nucleotides in length.
In another aspect, the present invention provides a method for killing cancer cells comprising contacting a cancer cell with an effective amount of a virus or derivative thereof, wherein the virus has a genomic sequence that comprises a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 168.
In another aspect, the present invention provides a method for killing cancer cells comprising contacting a cancer cell with an effective amount of a virus or derivative thereof, wherein the virus has a genomic sequence that comprises a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:168, or to a contiguous portion of SEQ ID NO:168 that is at least 50, 100, 200 or 500 nucleotides in length.
In these methods directed to cancer, the virus can be a picornavirus. The picornavirus can be a cardiovirus, erbovirus, aphthovirus, kobuvirus, hepatovirus, parechovirus, teschovirus, enterovirus, rhinovirus, SVV, or an SVV-like picornavirus. The cardiovirus can be selected from the group consisting of: vilyuisk human encephalomyelitis virus, Theiler's murine encephalomyelitis virus, and encephalomyocarditis virus. The SVV can be a virus having the ATCC deposit number PTA-5343 or a virus comprising a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1 or SEQ ID NO:168, or to a contiguous portion thereof that is at least 50, 100, 200, or 500 nucleotides in length. The SVV-like picornavirus can be a virus comprising a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:168, or to a contiguous portion thereof that is at least 50, 100, 200, or 500 nucleotides in length. The SVV-like picornavirus can be, for example, a virus from one of the following isolates MN 88-36695, NC 88-23626, IA 89-47752, NJ 90-10324, IL 92-48963, CA 131395; LA 1278; IL 66289; IL 94-9356; MN/GA 99-29256; MN 99197; and SC 363649. The SVV-like picornaviruses can be wild-type or mutant.
The present invention also provides a method of killing an abnormally proliferative cell comprising contacting the cell with a virus of the invention. In one aspect, the abnormally proliferative cell is a tumor cell. In various aspects of this method, the tumor cell is selected from the group consisting of: human small cell lung cancer, human retinoblastoma, human neuroblastoma, human medulloblastoma, mouse neuroblastoma, Wilms' tumor, and human non-small cell lung cancer.
The present invention also provides a method of treating a neoplastic condition in a subject comprising administering to the subject an effective amount of a virus of the invention to the mammal. In one aspect, the neoplastic condition is a neuroendocrine cancer. In another aspect, the subject is a mammal. In another aspect, the mammal is a human.
The present invention also provides a method of producing a virus of the invention, comprising: culturing cells infected with the virus under conditions that allow for replication of the virus and recovering the virus from the cells or the supernatant. In one aspect of this method, the cells are PER.C6 cells. In another aspect of this method, the cells are H446 cells. In the various aspects of this method, the cells may produce over 200,000 virus particles per cell.
In another aspect, the present invention provides a method for detecting a virus of the invention, comprising: isolating RNA from test material suspected to contain the virus of the invention; labeling RNA corresponding to at least 15 contiguous nucleotides of SEQ ID NO:1 or SEQ ID NO:168; probing the test material with the labeled RNA; and detecting the binding of the labeled RNA with the RNA isolated from the test material, wherein binding indicates the presence of the virus. In another aspect, the present invention provides a nucleic acid probe comprising a nucleotide sequence corresponding to at least 15 contiguous nucleotides of SEQ ID NO:1 or SEQ ID NO:168, or its complement.
The present invention also provides a method for making an oncolytic virus, the method comprising: (a) comparing a SVV genomic sequence with a test virus genomic sequence; (b) identifying at least a first amino acid difference between a polypeptide encoded by the SVV genomic sequence and a polypeptide encoded by the test virus genomic sequence; (c) mutating the test virus genomic sequence such that the polypeptide encoded by the test virus genomic sequence has at least one less amino acid difference to the polypeptide encoded by the SVV genomic sequence; (d) transfecting the mutated test virus genomic sequence into a tumor cell; and (e) determining whether the tumor cell is lytically infected by the mutated test virus genomic sequence. In one aspect, the amino acid(s) mutated in the test virus are amino acids in a structural region, such as in the capsid encoding region. In another aspect, the amino acids mutated in the test virus are amino acids in a non-structural region.
In one aspect of the method for making an oncolytic virus, the SVV genomic sequence is obtained from the isolated SVV having the ATCC deposit number PTA-5343 or from a virus comprising a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 168, or a contiguous portion thereof. In one aspect, the SVV genomic sequence is obtained from the isolated SVV having the ATCC deposit number PTA-5343 or from a virus comprising a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 168, or a contiguous portion thereof that is at least 50, 100, 200, or 500 nucleotides in length. In another aspect of this method, the step of mutating the test virus genomic sequence comprises mutating a cDNA having the test virus genomic sequence. In another aspect of this method, the step of transfecting the mutated test virus genomic sequence comprises transfecting RNA, wherein the RNA is generated from the cDNA having the mutated test virus genomic sequence.
In another aspect of the method for making an oncolytic virus, the test virus is a picornavirus. The test picornavirus can be a teschovirus, enterovirus, rhinovirus, cardiovirus, erbovirus, apthovirus, kobuvirus, hepatovirus, parechovirus or teschovirus. In another aspect, the test virus is a cardiovirus. In another aspect, the test virus is a SVV-like picornavirus. The SVV-like picornavirus can be, for example, a virus from one of the following isolates: MN 88-36695, NC 88-23626, IA 89-47552, NJ 90-10324, IL 92-48963, CA 131395; LA 1278; IL 66289; IL 94-9356; MN/GA 99-29256; MN 99197; and SC 363649. In another aspect, the amino acid differences identified in the methods for making an oncolytic virus are between a SVV capsid protein and a test virus capsid protein sequence. In another aspect for making an oncolytic virus, the test virus genomic sequence is selected from the group consisting of: Vilyuisk human encephalomyelitis virus, Theiler's murine encephalomyelitis virus, and encephalomyocarditis virus. In another aspect, the test virus genomic sequence is selected from an encephalomyocarditis virus. In yet another aspect, the encephalomyocarditis virus, the SVV-like picornavirus, or any other test virus can be selected from an isolate having a nucleic acid sequence comprising at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SVV of ATCC deposit number PTA-5343 or SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 168, or a contiguous portion thereof that is at least 50, 100, 200, or 500 nucleotides in length.
In another aspect of the method for making an oncolytic cardiovirus, the amino acid difference between the test virus and SVV is in a capsid protein region of SVV, wherein the amino acid difference is aligned within SVV SEQ ID NO:4, 6, 8, residues 80-937 of SEQ ID NO:169, residues 80-150 of SEQ ID NO:169, residues 151-434 of SEQ ID NO:169, residues 435-673 of SEQ ID NO:169, or residues 674-937 of SEQ ID NO:169.
The present invention also provides a method for making a mutant virus having an altered cell-type tropism, the method comprising: (a) creating a library of viral mutants comprising a plurality of nucleic acid sequences; (b) transfecting the library of viral mutants into a permissive cell, such that a plurality of mutant viruses is produced; (c) isolating the plurality of mutant viruses; (d) incubating a non-permissive cell with the isolated plurality of mutant viruses; and (e) recovering a mutant virus that was produced in the non-permissive cell, thereby making a mutant virus having an altered tropism. In one aspect, this method further comprises the steps of: (f) incubating the recovered mutant virus in the non-permissive cell; and (g) recovering a mutant virus that that was produced in the non-permissive cell. In another aspect, the method further comprises iteratively repeating steps (f) and (g). In another aspect, the library of viral mutants is created from a parental sequence comprising SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 168, or a contiguous portion thereof.
In one aspect of the method for making a mutant virus having an altered cell-type tropism, the incubating is conducted in a multi-well high-throughput platform wherein the platform comprises a different non-permissive cell-type in each well. In this aspect, the method can further comprise screening the platform to identify which wells contain a mutant virus that kills the cells. In another aspect, the screening is conducted by analyzing light absorbance in each well.
In another aspect of the method for making a mutant virus having an altered cell-type tropism, the non-permissive cell is a tumor cell.
In another aspect of the method for making a mutant virus having an altered cell-type tropism, the step of creating the library of viral mutants comprises: (i) providing a polynucleotide having a sequence identical to a portion of a genomic sequence of a virus; (ii) mutating the polynucleotide in order to generate a plurality of different mutant polynucleotide sequences; and (iii) ligating the plurality of mutated polynucleotides into a vector having the genomic sequence of the virus except for the portion of the genomic sequence of the virus that the polynucleotide in step (i) contains, thereby creating the library of viral mutants. In one aspect, the genomic sequence of a virus is from a picornavirus. In another aspect, the genomic sequence of a virus comprises a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 168, or a contiguous portion thereof. In another aspect, the genomic sequence of a virus comprises a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 168, or a contiguous portion thereof that is at least 50, 100, 200, or 500 nucleotides in length. In one aspect, the virus that comprises a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a contiguous portion of SEQ ID NO:168 that is at least 50, 100, 200, or 500 nucleotides in length is a SVV-like picornavirus. In another aspect, in the step of creating the library of viral mutants, the mutating of step (ii) is conducted by random insertion of nucleotides into the polynucleotide. In one aspect, the random insertion of nucleotides is conducted by trinucleotide-mutagenesis (TRIM). In another asepct, at least a portion of the nucleotides inserted into the polynucleotide encodes an epitope tag. In another aspect, in the step of creating the library of viral mutants, the mutating of step (ii) is conducted in a capsid encoding region of the polynucleotide.
The present invention also provides a method for making a mutant virus having an altered cell-type tropism, the method comprising: (a) creating a library of mutant polynucleotide sequences of a virus, wherein the creating comprises: providing a polynucleotide encoding a capsid region of the virus; mutating the polynucleotide in order to generate a plurality of different mutant capsid-encoding polynucleotide sequences; and ligating the plurality of mutated capsid-encoding polynucleotides into a vector having the genomic sequence of the virus except for the capsid-encoding region, thereby creating the library of mutant polynucleotide sequences of the virus; (b) transfecting the library of mutant polynucleotide sequences into a permissive cell, such that a plurality of mutant viruses is produced; (c) isolating the plurality of mutant viruses; (d) incubating a non-permissive cell with the isolated plurality of mutant viruses; and (e) recovering a mutant virus that that was produced in the non-permissive cell, thereby making a mutant virus having an altered tropism. In one aspect, the method further comprises the steps of: (f) incubating the recovered mutant virus in the non-permissive cell; and (g) recovering a mutant virus that that was produced in the non-permissive cell. In another aspect, the method further comprises iteratively repeating steps (f) and (g). In another aspect, the mutating is conducted by random insertion of nucleotides into the capsid-encoding polynucleotide. In another aspect, at least a portion of the nucleotides randomly inserted into the capsid-encoding polynucleotide encodes an epitope tag. In another aspect, the random insertion of nucleotides is conducted by TRIM. In another aspect, the plurality of different mutant capsid-encoding polynucleotide sequences comprises greater than 108 or 109 different capsid-encoding polynucleotide sequences. The library of mutant polynucleotide sequences can be from, for example, a cardiovirus or an SVV-like picornavirus.
In one aspect, a method for making a mutant SVV having an altered cell-type tropism comprises: (a) creating a cDNA library of SVV mutants; (b) generating SVV RNA from the cDNA library of SVV mutants; (c) transfecting the SVV RNA into a permissive cell, such that a plurality of mutant SVV is produced; (d) isolating the plurality of mutant SVV; (e) incubating a non-permissive tumor cell with the isolated plurality of mutant SVV; and (f) recovering a mutant SVV that lytically infects the non-permissive tumor cell, thereby making a mutant SVV having an altered tropism. In another aspect, the method further comprises the steps of: (g) incubating the recovered mutant SVV in the non-permissive cell; and (h) recovering a mutant SVV that lytically infects the non-permissive tumor cell. In another apsect, the method further comprises iteratively repeating steps (g) and (h). In one aspect, the incubating is conducted in a multi-well high-throughput platform wherein the platform comprises a different non-permissive tumor cell-type in each well. In another aspect, the method further comprises screening the platform to identify which wells contain a mutant SVV that lytically infects the cells. In another aspect, the screening is conducted by analyzing light absorbance in each well. In one aspect, the cDNA library of SVV mutants comprises a plurality of mutant SVV capsid polynucleotide sequences. In another aspect, the plurality of mutant SVV capsid polynucleotide sequences is generated by random insertion of nucleotides. In another aspect, at least a portion of the sequence of the nucleotides randomly inserted encodes an epitope tag. In another aspect, the random insertion of nucleotides is conducted by TRIM. In another aspect, the cDNA library of SVV mutants is generated from a SVV of ATCC deposit number PTA-5343. In another aspect, the cDNA library of SVV mutants is generated from a SVV comprising a sequence having at least 99%, 95%, 90%, 85%, 80%, 75%, 70%, or 65% sequence identity to SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 168, or to a contiguous portion thereof that is at least 50, 100, 200, or 500 nucleotides in length. In one aspect, the cDNA library of SVV mutants is generated from an SVV comprising a sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:168, or to a contiguous portion thereof that is at least 50, 100, 200, or 500 nucleotides in length. In another aspect, the non-permissive tumor cell is a tumor cell-line or a tumor cell-type isolated from a patient.
The present invention also provides a method for making a mutant virus having a tumor cell-type tropism in vivo, the method comprising: (a) creating a library of viral mutants comprising a plurality of nucleic acid sequences; (b) transfecting the library of viral mutants into a permissive cell, such that a plurality of mutant viruses is produced; (c) isolating the plurality of mutant viruses; (d) administering the isolated plurality of mutant viruses to a mammal with a tumor, wherein the mammal is not a natural host of the unmutated form of the mutant virus; and (e) recovering a virus that replicated in the tumor, thereby making a mutant virus having a tumor cell-type tropism in vivo. In one aspect, the step of creating a library of viral mutants comprises: providing a polynucleotide encoding a capsid region of a virus; mutating the polynucleotide in order to generate a plurality of different mutant capsid-encoding polynucleotide sequences; and ligating the plurality of mutated capsid-encoding polynucleotides into a vector having the genomic sequence of the virus except for the capsid-encoding region, thereby creating the library of viral mutants. In another aspect, the virus recovered in step (e) lytically infects cells of the tumor. In another aspect for a method for making a mutant virus having a tumor cell-type tropism in vivo, the tumor is a xenograft, a syngeneic tumor, an orthotopic tumor or a transgenic tumor. In another aspect, the mammal is a mouse.
For all the methods of the present invention, the virus can be a picornavirus. The picornavirus can be a cardiovirus, erbovirus, aphthovirus, kobuvirus, hepatovirus, parechovirus, teschovirus, entrovirus, rhinovirus, or a virus belonging to the genus to which SVV belongs. The virus can be a cardiovirus. The virus can be an SVV-like picornavirus. The virus can be SVV. The SVV can be a SVV having the ATCC Patent Deposit No. PTA-5343 or a SVV comprising a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO:1, 3, 6, 7, 9, 11, 13, 15, 17, 19, 21, 168, or a contiguous portion thereof. Further, the cardiovirus can be selected from the group consisting of: vilyuisk human encephalomyelitis virus, Theiler's murine encephalomyelitis virus, and encephalomyocarditis virus. In one aspect, the SVV-like picornavirus is selected from the group of isolates consisting of: MN 88-36695, NC 88-23626, IA 89-47552, NJ 90-10324, IL 92-48963, CA 131395; LA 1278; IL 66289; IL 94-9356; MN/GA 99-29256; MN 99197; and SC 363649. In another aspect, the present invention encompasses any virus that is selected from an isolate having at least 99%, 95%, 90%, 85%, 80%, 75%, 70%, or 65% sequence identity to SVV of ATCC deposit number PTA-5343 or SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 168, or a contiguous portion thereof or is otherwise considered related to SVV to by sequence homology.
In another aspect, the present invention encompasses any virus having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence identity to SVV of ATCC deposit number PTA-5343, to SEQ ID NO:168, or to a contiguous portion of SEQ ID NOS: 1 or 168 that is at least 100, 200, 300, 400, 500, 750, 1000, 1500, or 2000 nucleotides in length.
The present invention also provides an oncolytic virus made by any of the methods for making a mutant virus disclosed herein. In one aspect, the present invention provides a method for treating a patient with an oncolytic virus, the method comprising: (a) inactivating an oncolytic virus made by any of the methods for making a mutant virus disclosed herein, such that the oncolytic virus is non-infectious and the tropism of the oncolytic virus is unaffected; and (b) administering the irradiated oncolytic virus to a patient afflicted with a tumor. In another aspect, the method for treating a patient further comprises attaching a toxin to the inactivated oncolytic virus.
In another aspect, the present invention provides a method for treating a patient with a tumor with SVV, the method comprising: (a) inactivating a SVV such that the virus is non-infectious and the tropism is unaffected; and (b) administering the inactivated SVV in a patient afflicted with a tumor. In another aspect, the method for treating a patient with a tumor with SVV further comprises attaching a toxin to the inactivated SVV.
In another aspect, the present invention provides a SVV composition comprising an inactivated SVV or attenuated SVV. In another aspect, the present invention provides a SVV comprising an epitope tag incorporated in the capsid region.
The present invention also provides a method for treating a patient with a tumor with SVV, the method comprising: (a) creating a mutant SVV comprising an epitope tag encoded in the capsid; (b) attaching a toxin to the epitope tag; and (c) administering the mutant SVV with the attached toxin to a patient afflicted with a tumor. In one aspect, the creating comprises: inserting an oligonucleotide encoding an epitope tag into a capsid-encoding region polynucleotide of SVV. In one aspect, the mutant SVV does not have an altered cell-type tropism. In another aspect, the method further comprises inactivating the mutant SVV such that the mutant SVV is not infectious or cannot replicate.
The present invention also provides a method for detecting a tumor cell in a sample comprising: (a) isolating a tumor sample from a patient; (b) incubating the tumor sample with an epitope-tagged SVV; and (c) screening the tumor sample for bound SVV by detecting the epitope tag.
In one aspect, the invention provides a method for detecting a tumor cell in vivo comprising: (a) administering to a patient an inactivated epitope-tagged SVV, wherein a label is conjugated to the epitope-tag; and (b) detecting the label in the patient. In the methods for detecting a tumor cell of the present invention, the SVV can be a mutant SVV generated by the methods disclosed herein.
In one aspect, the invention provides a method for detecting a tumor cell in a sample comprising: (a) isolating a cell sample from a subject; (b) incubating the cell sample with SVV (or an SVV-like picornavirus); (c) incubating the cell sample from step (b) with an antibody specific to SVV (or an antibody specific to an SVV-like picornavirus); and (d) screening the cell sample for bound antibody, wherein bound antibody indicates that the sample contains a tumor cell.
In one aspect, the invention provides a method for determining whether a subject is candidate for SVV therapy, the method comprising: (a) isolating a cell from the subject; (b) incubating the cell with SVV; (c) incubating the sample from step (b) with an anti-SVV antibody; and (d) detecting for the presence of the anti-SVV antibody on or in the cell, wherein a positive detection indicates that the subject is a candidate for SVV therapy.
Screening a cell sample for bound antibody or detecting for the presence of an anti-SVV antibody can be conducted by adding a secondary antibody that can bind to the constant regions or non-epitope binding regions of the anti-SVV antibody, wherein the secondary antibody is conjugated or labeled with a detectable marker. The detectable marker can be, for example, a fluorophore such as fluorescein. When a secondary antibody is labeled with a detectable marker, the detectable marker can be detected, for example, by fluorescent microscopy. The cell from the subject can be from a tissue biopsy from the subject. The tissue biopsy can be from a tumor in the subject or from a region in the subject that is suspected to contain tumor cells. SVV directly labeled with flurophore can also be used in identification of tumor cells.
Further, the methods for treating neoplastic conditions, for detecting neoplastic conditions and for producing SVV, apply to wild-type SVV, mutant (including modified or variant) SVV, relatives of SVV, SVV-like picornaviruses, and other tumor-specific viruses of the invention.
The viruses of the present invention, and the compositions thereof, can be used in the manufacture of a medicament for treating the diseases mentioned herein. Further, the viruses and composition thereof of the invention can be used for the treatment of the diseases mentioned herein. Thus, in one aspect of the present invention, the present invention provides the use of SVV (or mutants, derivatives, relatives, and compositions thereof) for the treatment of cancer or in the manufacture of a medicament for treating cancer.
SVV and SVV-like viruses for gene therapy: Replication defective SVV expressing gene(s) of interest can be used to deliver genes to correct genetic disoders. SVV and SVV-like viruses can also be used as delivery vehicle for siRNA to prevent any specific gene expression. Replication defective viruses can be grown in complementing cell lines and/or in the presence of a helper virus to provide for missing functions in the recombinant virus.
IRES of picornaviruses known to play a role in expression of genes in a tissue specific manner. IRES of SVV and SVV-like viruses can be used to replace IRES of other picornaviruses. This strategy can be used to generate viruses with altered tissue tropism. In one aspect, the invention provides an IRES of SVV or an IRES from an SVV-related virus for the purpose of expressing two genes from a single promoter in a tissue specific manner.
Self-cleavage properties of 2A protease of SVV can be used to express more than one gene in equal amounts using single promoter and transcription termination signal sequences. In one aspect, the invention provides a self-cleaving 2A peptide of SVV or of an SVV-related virus for the purpose of expressing of two or more proteins in equal amounts under the control of single promoter and a single poly(A) signal. In another aspect, the invention provides the use of an SVV or an SVV-related virus 3C protease to cleave polypeptides for production of proteins from a eukaryotic cell. In another aspect, the invention provides for the use of an SVV or an SVV-like virus leader peptide to cause shut off of cell protein synthesis in tumor cells or another cell type of interest.
Virus like particles of SVV can be generated and used as vaccines and identify a particular cell type in a mixed population of cells.
The following material has been deposited with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Va., 20110-2209, U.S.A., under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. All restrictions on the availability of the deposited material will be irrevocably removed upon the granting of a patent. Material: Seneca Valley Virus (SVV). ATCC Patent Deposit Number: PTA-5343. Date of Deposit: Jul. 25, 2003.
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The terms “virus,” “viral particle,” “virus particle,” and “virion” are used interchangeably.
The terms “vector particle” and “viral vector particle” are interchangeable and are to be understood broadly—for example—as meaning infectious viral particles that are formed when, e.g., a viral vector of the invention is transduced or transfected into an appropriate cell or cell line for the generation of infectious particles.
The terms “derivative,” “mutant,” “variant” and “modified” are used interchangeably to generally indicate that a derivative, mutant, variant or modified virus can have a nucleic acid or amino acid sequence difference in respect to a template viral nucleic acid or amino acid sequence. For example, a SVV derivative, mutant, variant or modified SVV may refer to a SVV that has a nucleic acid or amino acid sequence difference with respect to the wild-type SVV nucleic acid or amino acid sequence of ATCC Patent Deposit Number PTA-5343.
An “SVV-like picornavirus” as used herein can have at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SVV at the nucleotide level (see SEQ ID NO:168,
As used herein, the terms “cancer,” “cancer cells,” “neoplastic cells,” “neoplasia,” “tumor,” and “tumor cells,” are used interchangeably, and refer to cells that exhibit relatively autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation. Neoplastic cells can be malignant or benign. According to the present invention, one type of preferred tumor cells are those with neurotropic properties.
The terms “identical” or percent “identity” in the context of two or more nucleic acid or protein sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm such as Protein-Protein BLAST (Protein-Protein BLAST of GenBank databases (Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990) “Basic local alignment search tool.” J. Mol. Biol. 215:403-410)) or by visual inspection. The BLAST algortihm is described in Altschul et al., J. Mol. Biol., 215:403-410 (1990), and publicly available BLAST software is provided through the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nhn.nih.gov/).
For example, as used herein, the term “at least 90% identical to” refers to percent identities from 90 to 100 relative to the reference polypeptides (or polynucleotides). Identity at a level of 90% or more is indicative of the fact that, assuming for exemplification purposes a test and reference polypeptide length of 100 amino acids are compared, no more than 10% (i.e., 10 out of 100) amino acids in the test polypeptide differs from that of the reference polypeptide. Similar comparisons can be made between a test and reference polynucleotide. Such differences can be represented as point mutations randomly distributed over the entire length of an amino acid sequence or they can be clustered in one or more locations of varying length up to the maximum allowable, e.g., 10 out of 100 amino acid differences (90% identity). Differences are defined as nucleic acid or amino acid substitutions, insertions or deletions. At the level of identities above about 85-90%, the result should be independent of the program and gap paramaters set; such high levels of identity can be assessed readily, often without relying on software.
The concepts of “high stringency,” “intermediate stringency” or “low stringency” refer to nucleic acid hybridization conditions. High stringency conditions refers to conditions that require a greater identity between a target's nucleic acid sequence and a probe's nucleic acid sequence in order for annealing or hybridization to occur between the target and the probe. Low stringency conditions refer to conditions that require a lower identity between a target's nucleic acid sequence and a probe's nucleic acid sequence in order for annealing or hybridization to occur between the target and the probe. Stringency conditions can be controlled by the salt concentration of the buffer or by the temperature at which the hybridization is carried out, where higher salt concentrations result in less stringent conditions and where higher temperatures result in more stringent conditions. Although stringency conditions will vary based on the length and nucleic acid content of the sequences undergoing hybridization, representative conditions of high, intermediate and low stringency are described in the following exemplary conditions. A commonly used hybridization buffer is SSC (sodium chloride sodium citrate) with a 20× stock concentration corresponding to 0.3 M trisodium citrate and 3 M NaCl. For high stringency conditions, the working concentration of SSC can be 0.1×-0.5× (1.5-7.5 mM trisodium citrate, 15-75 mM NaCl) with the hybridization temperature set at 65° C. Intermediate conditions typically utilize a 0.5×-2×SSC concentration (7.5-30 mM trisodium citrate, 75-300 mM NaCl) at a temperature of 55-62° C. Hybridizations conducted under low stringency conditions can use a 2×-5×SSC concentration (30-75 mM trisodium citrate, 300-750 mM NaCl) at a temperature of 50-55° C. Note that these conditions are merely exemplary and are not to be considered limitations.
Seneca Valley Virus (SVV):
SVV is a novel, heretofore undiscovered RNA virus, and with respect to previously characterized picornaviruses, SVV is most closely related to members from the genus Cardiovirus in the family Picornaviridae (see International Application No. PCT/US2004/031594). The results of sequence analyses between SVV and other cardioviruses are discussed in PCT/US2004/031594, which is hereby incorporated by reference in its entirety. Since the time of the sequence analysis of SVV described in PCT/US2004/031594, the Picornavirus Study Group has initiated discussion as to whether SVV will be a member of a new genus.
From initial sequence comparisons to known picornaviruses (see International Application No. PCT/US2004/031504), there were two phylogenetic classification options: (1) to include SVV as a new species in the genus Cardiovirus; or (2) assign SVV to a new genus. At that time and for the International application, SVV was designated to be a novel member of the genus Cardiovirus. After further analyses however, it has been found that several characteristics of SVV differ with that of cardioviruses. For example, some cardiovirus genomes contain an extended internal poly(C) tract in their 5′ UTRs. SVV does not contain a poly(C) tract. From the additional 5′ sequence information, the Internal Ribosome Entry Sequence (IRES) of SVV has been mapped and compared to other picornaviruses, and it has been determined that the SVV IRES is Type IV, whereas cardiovirus IRES's are Type II. The cardioviruses have a long (150 amino acid (aa)) 2A protease while SVV has a short (9 aa) 2A protease. The size of this protein as well as others (Leader peptide, 3A) differs significantly between SVV and cardioviruses. From the study of other picornaviruses, it is know that these proteins are likely involved in host cell interactions including tropism and virulence. Lastly, it is now thought that the overall sequences differ too much in a number of genome regions and SVV should therefore be considered to form a new genus. Additionally, multiple unique picornaviruses have been discovered at the USDA that are more similar to SVV than SVV is to other cardioviruses. Therefore, it has been decided by the Executive Committee of the International Committee for the Taxonomy of Viruses (ICVT) based on recommendations made by the Picornavirus Study Group that SVV will make up a new species of picornavirus, named Seneca Valley virus. However, currently, SVV and these unique USDA picornaviruses (herein referred to as being members of the group of SVV-like picornaviruses) are currently unassigned to any genus.
Several of the SVV-like picornaviruses discovered at the USDA are about 95-98% identical to SVV at the nucleotide level (for example, see
SVV, like cardioviruses, can be distinguished from other picornaviruses by special features of their genome organization, common pathological properites, and the dissociability of their virions at pHs between 5 and 7 in 0.1 M NaCl (Scraba, D. et al., “Cardioviruses (Picornaviridae),” in Encyclopedia of Virology, 2nd Edition, R. G. Webseter and A. Granoff, Editors, 1999). The genome of SVV consists of one single-stranded positive (+) sense strand RNA molecule having a size of 7,310 nucleotides including a poly(A) tail of 30 nucleotides in length (see
The invention provides the isolated SVV virus (ATCC Patent Deposit number PTA-5343) and the complete genomic content of SVV therefrom. At first, the largest SVV genomic fragment that was sequenced is an isolated SVV nucleic acid, derived from the PTA-5343 isolate, that comprises the majority of the SVV genomic sequence, and is listed in
The invention provides isolated (or purified) portions of SEQ ID NO:1, including SEQ ID NOS:3, 5, 7, 9, 11, 13, 15, 17, 19 and 21, and isolated portions of SEQ ID NO:168, including the 5′UTR region (1-666), coding region for the leader peptide (667-903), coding region for the VP4 protein (904-1116), coding region for the VP2 protein (1117-1968), coding region for the VP3 protein (1969-2685), coding region for the VP1 protein (2686-3474), coding region for the coding region for the 2A protein (3478-3504), coding region for the 2B protein (3505-3888), coding region for the 2C protein (3889-4854), coding region for the 3A protein (4855-5124), coding region for the 3B protein (5125-5190), coding region for the 3C protein (5191-5823), coding region for the 3D protein (5824-7209), and the 3′UTR region including the poly(A) tail (7210-7310). The invention also provides isolated nucleic acids that are portions of the specified portions listed above. The invention also provides mutants or derivatives of such isolated portions. The isolated portions of SEQ ID NOS:1 and 168 can be subcloned into expression vectors such that polypeptides encoded by these portions can be isolated. Further encompassed by the invention are isolated nucleic acids that can hybridize to SEQ ID NO:1 or SEQ ID NO:168, or any portion thereof, under high, moderate or low stringency conditions. The following table lists the nucleotides of SEQ ID NO:168 that encode the SVV proteins. The invention provides isolated (or purified) SVV proteins or portions thereof. The table also lists the amino acid sequences of the SVV proteins with respect to the polyprotein sequence listed in SEQ ID NO:169.
The invention provides an isolated SVV leader sequence peptide with the amino acid sequence of residues 1-79 of SEQ ID NO:169, which is encoded by nucleotides 667-903 of SEQ ID NO:168.
The invention provides an isolated SVV VP4 (1A) protein with the amino acid sequence of residues 80-150 of SEQ ID NO:169, which is encoded by nucleotides 904-1116 of SEQ ID NO:168.
The invention provides an isolated SVV VP2 (1B) protein with the amino acid sequence of residues 151-434 of SEQ ID NO:169, which is encoded by nucleotides 1117-1968 of SEQ ID NO:168. The invention also provides an isolated partial SVV VP2 (1B) protein with the amino acid sequence of SEQ ID NO:4, as listed in
The invention provides an isolated SVV VP3 (1C) protein with the amino acid sequence of residues 435-673 of SEQ ID NO:169, which is encoded by nucleotides 1969-2685 of SEQ ID NO:168. The invention also provides an isolated SVV VP3 (1C) protein with the amino acid sequence of SEQ ID NO:6, as listed in
The invention provides an isolated SVV VP1 (1D) protein with the amino acid sequence of residues 674-937 of SEQ ID NO:169, which is encoded by nucleotides 2686-3477 of SEQ ID NO:168. The invention also provides an isolated SVV VP1 (1D) protein with the amino acid sequence of SEQ ID NO:8, as listed in
The invention provides an isolated SVV 2A protein with the amino acid sequence of residues 938-946 of SEQ ID NO:169, which is encoded by nucleotides 3478-3504 of SEQ ID NO:168. The invention also provides an isolated SVV 2A protein with the amino acid sequence of SEQ ID NO:10, as listed in
The invention provides an isolated SVV 2B protein with the amino acid sequence of residues 947-1074 of SEQ ID NO:169, which is encoded by nucleotides 3505-3888 of SEQ ID NO:168. The present invention also provides an isolated SVV 2B protein with the amino acid sequence of SEQ ID NO:12, as listed in
The invention provides an isolated SVV 2C protein with the amino acid sequence of residues 1075-1396 of SEQ ID NO:169, which is encoded by nucleotides 3889-4854 of SEQ ID NO:168. The invention also provides an isolated SVV 2C protein with the amino acid sequence of SEQ ID NO:14, as listed in
The invention provides an isolated SVV 3A protein with the amino acid sequence of residues 1397-1486 of SEQ ID NO:169, which is encoded by nucleotides 4855-5124 of SEQ ID NO:168. The invention also provides an isolated SVV 3A protein with the amino acid sequence of SEQ ID NO:16, as listed in
The invention provides an isolated SVV 3B (VPg) protein with the amino acid sequence of residues 1487-1508 of SEQ ID NO:169, which is encoded by nucleotides 5125-5190 of SEQ ID NO:168. The invention also provides an isolated SVV 3B protein with the amino acid sequence of SEQ ID NO:18, as listed in
The invention provides an isolated SVV 3C (“pro” or “protease”) protein with the amino acid sequence of residues 1509-1719 of SEQ ID NO:169, which is encoded by nucleotides 5191-5823 of SEQ ID NO:168. The invention also provides an isolated SVV 3C protein with the amino acid sequence of SEQ ID NO:20, as listed in
The invention provides an isolated SVV 3D (“pol” or “polymerase”) protein with the amino acid sequence of residues 1720-2181 of SEQ ID NO:169, which is encoded by nucleotides 5824-7209 of SEQ ID NO:168. The invention also provides an isolated SVV 3D protein with the amino acid sequence of SEQ ID NO:22, as listed in
The nucleic acids of the present invention include both RNA and DNA forms, and implicitly, the complementary sequences of the provided listings.
Thus, the isolated SVV nucleic acid depicted by SEQ ID NO:168 has a length of 7,310 nucleotides that encodes a polyprotein with the amino acid sequence depicted by SEQ ID NO:169. The isolated SVV nucleic acid depicted by SEQ ID NO:1 has a length of 5,752 nucleotides that encodes a polypeptide with the amino acid sequence depicted by SEQ ID NO:2. The SVV genomic sequence is translated as a single polyprotein that is cleaved into various downstream “translation products.” The present invention encompasses all nucleic acid fragments of SEQ ID NO:168 and SEQ ID NO:1, and all polypeptides encoded by such fragments.
The full-length SVV polyprotein amino acid sequence is depicted by SEQ ID NO:169 and is encoded by nucleotides 667-7209 of SEQ ID NO:168. The majority of the full-length SVV polyprotein amino acid sequence is encoded by nucleotides 1-5673 of SEQ ID NO:1. The polyprotein is cleaved into three precursor proteins, P1, P2 and P3 (see
In certain embodiments, the invention provides isolated nucleic acids that comprise: (i) the coding sequence of 1ABCD or the capsid region (nucleotides 904-3477 of SEQ ID NO:168); (ii) the coding sequence of 1ABC (nucleotides 904-2685 of SEQ ID NO:168); (iii) the coding sequence of VP0 (nucleotides 904-1968 of SEQ ID NO:168); (iv) the coding sequence of 2ABC (nucleotides 3478-4854 of SEQ ID NO:168; nucleotides 1924-3315 of SEQ ID NO:1); (v) the coding sequence of 2BC (nucleotides 3505-4854 of SEQ ID NO:168; nucleotides 1966-3315 of SEQ ID NO:1); (iii) the coding sequence of 3ABCD (nucleotides 4855-7209 of SEQ ID NO:168; nucleotides 3316-5673 of SEQ ID NO:1); (iv) the coding sequence of 3AB (nucleotides 4855-5190 of SEQ ID NO:168; nucleotides 3316-3651 of SEQ ID NO:1); and (v) the coding sequence of 3CD (nucleotides 5191-7209 of SEQ ID NO:168; nucleotides 3652-5673 of SEQ ID NO:1). The invention also provides isolated proteins or peptides encoded by the coding sequences described above, including fragments thereof.
The basic capsid structure of picornaviruses consists of a densely packed icosahedral arrangement of 60 protomers, each consisting of 4 polypeptides, VP1, VP2, VP3 and VP4, all of which are derived from the cleavage of the original protomer, VP0. The SVV virus particle is about 27 nm in diameter (see
The kinetics of picornavirus replication is rapid, the cycle being completed in about 5-10 hours (typically by about 8 hours) (see
The 5′ UTR contains the IRES. Normally, eukaryotic translation is initiated when ribosomes bind to the 5′ methylated cap and then scans along the mRNA to find the first AUG initiation codon. The IRES overcomes this process and allows Picornavirus RNA's to continue to be translated after degradation of CBC. In one embodiment, the invention provides for an isolated nucleic acid comprising the SVV IRES, wherein the IRES is contained within the 5′UTR. In one embodiment, the SVV IRES can be from nucleotides 300-366 of SEQ ID NO:168. The 5′UTR of SVV is present at nucleotides 1-666 of SEQ ID NO:168.
The virus polyprotein is initially cleaved by the 2A protease into polyproteins P1, P2 and P3 (see
The (+) strand RNA genome is believed to be packaged into preformed capsids, although the molecular interactions between the genome and the capsid are not clear. Empty capsids are common in all picornavirus infections. The capsid is assembled by cleavage of the P1 polyprotein precursor into a protomer consisting of VP0, VP3, and VP1, which join together enclosing the genome. Maturation and infectivity of the virus particle relies on an internal autocatalytic cleavage of VP0 into VP2 and VP4. Release of newly formed virus particles occurs when the cell lyses.
The present invention also provides an isolated virus having all the identifying characteristics and nucleic acid sequence of ATCC Patent Deposit number PTA-5343. Viruses of the present invention can be directed to the PTA-5343 isolate, variants, homologues, derivatives and mutants of the PTA-5343 isolate, and variants, homologues, derivatives and mutants of other picornaviruses that are modified in respect to sequences of SVV (both wild-type as disclosed herein and mutant) that are determined to be responsible for its oncolytic properties.
The present invention further provides antibodies that are specific against: the isolated SVV having the ATTC Patent Deposit number PTA-5343, and epitopes from the isolated SVV proteins having the amino acid sequences SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 169 (including entire polyprotein, VP4, VP2, VP3, VP1, 2A, 2B, 2C, 3A, 3B, 3C, 3D, and portions thereof; see Table A supra for amino acids in SEQ ID NO:169 that make up these proteins). The invention also includes antibodies that are specific against epitopes from the proteins that are encoded by fragments or portions of SEQ ID NO:1 or SEQ ID NO:168.
Comparative analyses of the RNA sequences from a variety of cardiovirus isolates have shown >45% nucleotide identity between genomes. Cardioviruses can be subclassified into the EMC-like viruses (“EMCV”—such as, Mengo, B, R; and also MM, ME, Columbia-SK), the Theiler's-like viruses (“TMEV”—such as, BeAn, DA and GD VII strains), and the Vilyuisk viruses.
In analyzing the SVV sequence to other viruses, it appears that SVV is a cardiovirus (see Example 4 and Figures referenced therein). If EMCV and TMEV are taken as the standard cardioviruses, SVV is clearly not a typical cardiovirus. However, even these two viruses have their differences, notably in the 5′ UTR (Pevear et al., 1987, J. Virol., 61: 1507-1516). Phylogenetically SVV clusters with EMCV and TMEV in much of its polyprotein (P1, 2C, 3Cpro and 3Dpol regions; see
SVV is phylogenetically similar to cardioviruses, but it has now been determined to be in a separate tree (see
Methods for Treating Cancer:
The present invention provides methods for cancer therapy using viruses modified in view of the oncolytic properties of SVV, including picornaviruses (including SVV-like picornaviruses), derivatives, variants, mutants or homologues thereof. The present invention shows that wild-type SVV (i.e., ATTC Patent Deposit number PTA-5343) has the ability to selectively kill some types of tumors. For example, SVV can selectively kill tumor cells that have neurotropic or neuroendocrine properties, including small cell lung cancer (SCLC) and neuroblastomas. Other examples of neuroendocrine tumors that are contemplated to be treated by the viruses of the present invention include, but are not limited to: adrenal pheochromocytomas, gastrinomas (causing Zollinger-Ellison syndrome), glucagonomas, insulinomas, medullary carcinomas (including medullary thyroid carcinoma), multiple endocrine neoplasia syndromes, pancreatic endocrine tumors, paragangliomas, VIPomas (vasoactive intestinal polypeptide tumor), islet cell tumors, and pheochromocytoma.
In one embodiment, the invention provides methods for treating or reducing neuroendocrine tumors by administering to a subject SVV or an SVV-like picornavirus, where the neuroendocrine tumor expresses (or overexpresses) one or more neuroendocrine tumor markers, including but not limited to, NTR (Neurotensin receptor), ATOH (, GL11, Myc, GRP receptors, GRP, Neuronal enolase (neuron specific enolase (NSE)), carcinoembryonic antigen (CEA), chromoganin A, NCAM, IgF2, BCL-2, sonic hedgehog pathway, and a chemokine receptor.
Also encompassed in the present invention are the four types of neuroendocrine lung tumors. The most serious type, small cell lung cancer (SCLC), is among the most rapidly growing and spreading of all cancers. Large cell neuroendocrine carcinoma is a rare cancer that, with the exception of the size of the cells forming the cancer, is very similar to SCLC in its prognosis and in how patients are treated. Carcinoid tumors, also known as carcinoids, comprise the other 2 types of lung neuroendocrine cancer. These two types are typical carcinoid and atypical carcinoid.
Not being bound by theory, the ability of SVV to specifically kill tumor cells may include, but is not limited to: selective replication, cell protein synthesis shut-off, apoptosis, lysis via tumor-selective cell entry, tumor-selective translation, tumor-selective proteolysis, tumor-selective RNA replication, and combinations thereof.
SVV has many advantageous characteristics over other oncolytic viruses, including modified adenoviruses, for example: (i) SVV has a very high selectivity for cancers with neural properties, including SCLC, Wilms' tumor, retinoblastoma, and neuroblastoma—for example, SVV shows a greater than 10,000-fold selectivity toward neuroendocrine tumor cells; (ii) SVV has been shown to have a 1,000 fold better cell-killing specificity than chemotherapy treatments; (iii) SVV exhibits no overt toxicity in mice following systemic administration with as high as 1014 viral particles per kilogram; (iv) the efficacy of SVV is very robust in that 100% of large pre-established tumors can be eradicated in mice, with no recurrence of tumor growth; (v) SVV can be purified to high titer and can be produced at more than 200,000 particles per cell in permissive cell lines; (vi) SVV has a small size (the SVV virus particle is less than 30 nm in diameter) enabling better penetration and spread in tumors than other oncolytic viruses, (vii) SVV replicates quickly (less than 12 hours) and (viii) no modification of SVV is necessary for its use as a specific anti-tumor agent.
Further, initial studies (see Example 6) indicate some additional factors that make SVV an advantageous tool for oncolytic viral therapy: (i) human serum samples do not contain neutralizing antibodies directed against SVV; (ii) SVV is not inhibited by complement; and (iii) SVV does not produce hemagglutination of human erythrocytes. All of these factors contribute to the fact that SVV exhibits a longer circulation time in vivo than other oncolytic viruses (for example, see Example 7).
The present invention provides methods for selectively killing a neoplastic cell in a cell population that comprises contacting an effective amount of SVV with said cell population under conditions where the virus can transduce the neoplastic cells in the cell population, replicate and kill the neoplastic cells. Besides methods where SVV kills tumor cells in vivo, the present methods encompass embodiments where the tumors can be: (1) cultured in vitro when infected by SVV; (2) cultured in the presence of non-tumor cells; and (3) the cells are mammalian (both tumor and non-tumor cells), including where the cells are human cells. The in vitro culturing of cells and infection by SVV can have various applications. For example, in vitro infection be used as a method to produce large amounts of SVV, as method for determining or detecting whether neoplastic cells are present in a cell population, or as a method for screening whether a mutant SVV can specifically target and kill various tumor cell or tissue types.
The present invention further provides an ex vivo method of treating cancer wherein cells are isolated from a human cancer patient, cultured in vitro, infected with a SVV which selectively kills the cancer cells, and the non-tumor cells are introduced back to the patient. Alternatively, cells isolated form a patient can be infected with SVV and immediately introduced back to the patient as a method for administering SVV to a patient. In one embodiment, the cancer cells are of a hematopoietic origin. Optionally, the patient may receive treatment (e.g., chemotherapy or radiation) to destroy the patient's tumor cell in vivo before the cultured cells are introduced back to the patient. In one embodiment, the treatment may be used to destroy the patient's bone marrow cells.
Polymer coated SVV can be used to target the SVV to any specific cell type. This coating strategy can also be used to overcome antibodies to SVV.
SVV possesses potent antitumor activity against tumor cell-types with neural characteristics. SVV does not exhibit cytolytic activity against tested normal human. Further SVV is not cytotoxic to primary human hepatocytes. Table 1 below summarizes initial studies that have been conducted to determine the in vitro cytolytic potency of SVV against selected tumor cell types.
Table 1-A below provides a list of cell lines that are permissive are non-permissive to SVV infection. The Table shows the cytolytic potency and selectivity of SVV.
Table 1-A lists the results of SVV permissivity experiments on 165 primary cells and cell lines, representing 22 tissues from 8 different species. The results indicate that virtually all adult normal are nonpermissive for SVV. Thirteen primary adult human cell cultures tested were nonpermissive. Of the twelve bovine, ovine, porcine and primate normal cell cultures tested, only three cell cultures were permissive, which were porcine smooth muscle cells. This result is consistent with the hypothesis that the natural host for SVV may be pigs. Besides the porcine smooth muscle cells, only neuroendocrine cancer cell lines or most fetal lines were permissive.
Murine studies (see Examples) show that SVV can specifically kill tumors with great efficacy and specificity in vivo. These in vivo studies show that SVV has a number of advantages over other oncolytic viruses. For example, one important factor affecting the ability of an oncolytic tumor virus to eradicate established tumors is viral penetration. In studies with adenoviral vectors, Ad5 based vectors had no effect on SCLC tumor development in athymic mice. Based on immunohistochemical results, adenovirus did not appear to penetrate the established tumors. In contrast, SVV was able to eliminate H446 SCLC tumors in athymic nude mice following a single systemic administration. SVV has a small size (<30 nm in diameter) enabling better penetration and spread in tumor tissue than other viruses, and thus, the small size of SVV may contribute to its ability to successfully penetrate and eradicate established tumors.
Additional in vivo tests demonstrate the efficacy of a single intravenous dose of SVV in murine tumor models using athymic nude mice and immunocompetent mice. The tumor models tested were: (1) H446 (human SCLC); (2) Y79 (human retinoblastoma); (3) H69AR (human multi-drug resistant SCLC); (4) H1299 (human NSCLC); and (5) N1E-115 (murine neuroblastoma). The results of these tests are shown in
Chemoresistance is a major issue facing any patient that receives chemotherapy as a facet of cancer therapy. Patients that become chemoresistant often, if not always, have a much poorer prognosis and may be left with no alternative therapy. It is well known that one of the major causes of chemoresistance is the expression, over expression, or increased activity of a family of proteins called Multiple Drug Resistant proteins (MRPs). Applicants have found that a sensitivity of certain tumor cells for SVV is also correlated with the chemoresistant state of cancer cells and MRP expression. H69 is a chemosensitive (adriamycin) cell line that is resistant to SVV in vitro, whereas H69AR is a chemoresistant cell line that overexpresses MRPs and is sensitive to SVV (see Table 1). Evidence indicates that overexpression of MRPs, including MDR, correlates with sensitivity of cells to SVV killing. Thus, in one embodiment, the present invention provides a method for treating cancer wherein SVV kills cells overexpressing an MRP.
The invention also provides methods for treating diseases that are a result of abnormal cells, such as abnormally proliferative cells. The method comprises contacting said abnormal cells with SVV in a manner that results in the destruction of a portion or all of the abnormal cells. SVV can be used to treat a variety of diseases that are a result of abnormal cells. Examples of these diseases include, but are not limited to, cancers wherein the tumor cells display neuroendocrine features and neurofibromatosis.
Neuroendocrine tumors can be identified by a variety of methods. For example, neuroendocrine tumors produce and secrete a multitude of peptide hormones and amines. Some of these substances cause a specific clinical syndrome: carcinoid, Zollinger-Ellison, hyperglycemic, glucagonoma and WDHA syndrome. Specific markers for these syndromes are basal and/or stimulated levels of urinary 5-HIAA, serum or plasma gastrin, insulin, glucagon and vasoactive intestinal polypeptide, respectively. Some carcinoid tumors and about one third of endocrine pancreatic tumors do not present any clinical symptoms and are called ‘nonfunctioning’ tumors. Therefore, general tumor markers such as chromogranin A, pancreatic polypeptide, serum neuron-specific enolase and subunits of glycoprotein hormones have been used for screening purposes in patients without distinct clinical hormone-related symptoms. Among these general tumor markers chromogranin A, although its precise function is not yet established, has been shown to be a very sensitive and specific serum marker for various types of neuroendocrine tumors. This is because it may also be elevated in many cases of less well-differentiated tumors of neuroendocrine origin that do not secrete known hormones. At the moment, chromogranin A is considered the best general neuroendocrine serum or plasma marker available both for diagnosis and therapeutic evaluation and is increased in 50-100% of patients with various neuroendocrine tumors. Chromogranin A serum or plasma levels reflect tumor load, and it may be an independent marker of prognosis in patients with midgut carcinoids.
The invention also provides a pharmaceutical composition comprising SVV and a pharmaceutically acceptable carrier. Such compositions, which can comprise an effective amount of SVV in a pharmaceutically acceptable carrier, are suitable for local or systemic administration to individuals in unit dosage forms, sterile parenteral solutions or suspensions, sterile non-parenteral solutions or oral solutions or suspensions, oil in water or water in oil emulsions, and the like. Formulations for parenteral and non-parenteral drug delivery are known in the art. Compositions also include lyophilized and/or reconstituted forms of SVV. Acceptable pharmaceutical carriers are, for example, saline solution, protamine sulfate (Elkins-Sinn, Inc., Cherry Hill, N.J.), water, aqueous buffers, such as phosphate buffers and Tris buffers, or Polybrene (Sigma Chemical, St. Louis, Mo.) and phosphate-buffered saline and sucrose. The selection of a suitable pharmaceutical carrier is deemed to be apparent to those skilled in the art from the teachings contained herein. These solutions are sterile and generally free particulate matter other than SVV. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. Excipients that enhance infection of cells by SVV may be included.
SVV is administered to a host or subject in an amount that is effective to inhibit, prevent or destroy the growth of the tumor cells through replication of the virus in the tumor cells. Methods that utilize SVV for cancer therapy include systemic, regional or local delivery of the virus at safe, developable, and tolerable doses to elicit therapeutically useful destruction of tumor cells. Even following systemic administration, the therapeutic index for SVV is at least 10, preferably at least 100 or more preferably at least 1000. In general, SVV is administered in an amount of between 1×108 and 1×1014 vp/kg. The exact dosage to be administered is dependent upon a variety of factors including the age, weight, and sex of the patient, and the size and severity of the tumor being treated. The viruses may be administered one or more times, which may be dependent upon the immune response potential of the host. Single or multiple administrations of the compositions can be carried out with dose levels and pattern being selected by the treating physician. If necessary, the immune response may be diminished by employing a variety of immunosuppressants, so as to permit repetitive administration and/or enhance replication by reducing the immune response to the viruses. Anti-neoplastic viral therapy of the present invention may be combined with other anti-neoplastic protocols. Delivery can be achieved in a variety of ways, employing liposomes, direct injection, catheters, topical application, inhalation, etc. Further, a DNA copy of the SVV genomic RNA, or portions thereof, can also be a method of delivery, where the DNA is subsequently transcribed by cells to produce SVV virus particles or particular SVV polypeptides.
A therapeutically effective dose refers to that amount of the virus that results in amelioration of symptoms or a prolongation of survival in a patient. Toxicity and therapeutic efficacy of viruses can be determined by standard procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population of animals or cells; for viruses, the dose is in units of vp/kg) and the ED50 (the dose—vp/kg—therapeutically effective in 50% of the population of animals or cells) or the EC50 (the effective concentration—vp/cell (see Table 1 for example)—in 50% of the population of animals or cells). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50 or EC50. Viruses which exhibit high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of viruses lies preferably within a range of circulating concentrations that include the ED50 or EC50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
In yet another aspect, a method for treating a host organism having a neoplastic condition is provided, comprising administering a therapeutically effective amount of a viral composition of the invention to said host organism. In one embodiment, the neoplastic tissue is abnormally proliferating, and the neoplastic tissue can be malignant tumor tissue. Preferably, the virus is distributed throughout the tissue or tumor mass due to its capacity for selective replication in the tumor tissue. Neoplastic conditions potentially amenable to treatment with the methods of the invention include those with neurotropic properties.
Methods for Producing the Viruses of the Present Invention:
Methods for producing the present viruses to very high titers and yields are additional aspects of the invention. As stated, SVV can be purified to high titer and can be produced at more than 200,000 particles per cell in permissive cell lines. Cells that are capable of producing high quantities of viruses include, but are not limited to, PER.C6 (Fallaux et al., Human Gene Therapy, 9:1909-1917, 1998), H446 (ATCC# HTB-171) and the other cell lines listed in Table 1 where the EC50 value is less than 10.
For example, the cultivation of picornaviruses can be conducted as follows. The virus of interest is plaque purified and a well-isolated plaque is picked and amplified in a permissive cell line, such as PER.C6. A crude virus lysate (CVL) from the infected cells can be made by multiple cycles of freeze and thaw, and used to infect large numbers of permissive cells. The permissive cells can be grown in various tissue culture flasks, for example, 50×150 cm2 flasks using various media, such as Dulbecco's modified Eagle medium (DMEM, Invitrogen, Carlsbad, Calif.)) containing 10% fetal bovine serum (Biowhitaker, Walkersvile, Md.) and 10 mM magnesium chloride (Sigma, St Louis, Mo.). The infected cells can be harvested between 12 and 48 hours after infection or when complete cytopathic effects (CPE) are noticed, and are collected by centrifugation at 1500 rpm for 10 minutes at 4° C. The cell pellet is resuspended in the cell culture supernatant and is subjected to multiple cycles of freeze and thaw. The resulting CVL is clarified by centrifugation at 1500 rpm for 10 minutes at 4° C. Virus can be purified by gradient centrifugation. For example, two rounds of CsCl gradients can suffice for SVV purification: a one-step gradient (density of CsCl 1.24 g/ml and 1.4 g/ml) followed by one continuous gradient centrifugation (density of CsCl 1.33 g/ml). The purified virus concentration is determined spectrophotometrically, assuming 1A260=9.5×1012 particles (Scraba D. G., and Palmenberg, A. C. 1999. Cardioviruses (Picornaviridae). In: Encyclopedia of Virology, Second edition, R. G. Webster and A Granoff Eds). Infectivity titers of purified virus are also determined by a standard plaque and/or tissue culture infective dose 50 (TCID50) assay using PER.C6 or any other suitable cell type. The yield of SVV from PER.C6 cells are greater than 200,000 particles per cell with particles to PFU ratio of about 100. The yields of SVV from other permissive cells (H446-ATCC# HTB-171) may be at least this high or higher. SVV can also be purified by column chromatography.
In addition, several steps in a commercially attractive large scale Good Manufacturing Processes (GMP) are applicable to the purification of SVV. The invention also contemplates methods for purifying SVV that are based on methods for purifying adenoviruses. These methods include isolating SVV based on its density, since SVV has a very similar density to adenovirus and can be co-purified with adenovirus.
Methods for Detecting and Studying Tumors:
The present invention provides methods for detecting tumor or neoplastic cells in a patient using the viruses of the present invention. Cellular samples can be obtained from a patient and screened by incubating the sample with an epitope-tagged SVV (or other tumor-specific viruses provided by the invention, i.e., tumor-specific mutant cardioviruses), and then screening the sample for bound SVV by detecting the epitope tag. Alternatively, the sample can be screened by detecting whether the SVV causes any cellular lysis. If SVV does cause cellular lysis, or if SVV can bind specifically to cells in the sample, this would indicate the possibility that the sample contains neoplastic or tumor cells known to be capable of being bound and/or infected by SVV.
Additionally, SVV can be used in a method for detecting a tumor cell in vivo. In such a method, epitope-tagged SVV can first be inactivated in a manner where SVV can still bind to tumor cells specifically but cannot replicate. Tumor cells that have bound SVV can be detected by assaying for the epitope tag. Detection of the epitope tag can be accomplished by antibodies that specifically bind the epitope, where the antibodies are either labeled (for example, fluorescently) or where the antibodies can then be detected by labeled secondary antibodies.
The present methods of detection encompass detecting any type of tumor or neoplastic cell that is specifically targeted by any virus of the present invention. Specific tumor types include, for example, neuroendocrine-type tumors, such as retinoblastomas, SCLC, neuroblastomas glioblastomas and medulloblastomas.
The present invention also provides the use of SVV as a tool to study tumor cells. SVV selectively destroys some tumor cell types, and has very little, if any, toxic effects on non-tumor cells. Because of these characteristics, SVV can be used to study tumors and possibly discover a new tumor specific gene and/or pathway. In other words, there is some characteristic of the tumor cells that allows replication of SVV, wherein normal cells do not exhibit said characteristic. Upon identification of a new tumor specific gene and/or pathway, therapeutic antibodies or small molecules can then be designed or screened to determine whether these reagents are anti-tumor agents.
The present invention also provides a method for identifying all types of cancers that respond to SVV. In one embodiment, the method for identifying SVV-responsive cells comprises obtaining cells, contacting said cells with SVV and detecting cell killing or detecting viral replication. Cell killing can be detected using various methods known to one skilled in the art (e.g., MTS assay, see High-Throughput section herein). Methods of detecting virus replication are also known to one skilled in the art (e.g., observance of CPE, plaque assay, DNA quantification methods, FACS to detect quantity of virus in the tumor cells, RT-PCR assays to detect viral RNA, etc.). In one embodiment, the cells are cancer cells. Examples of cancer cells include, but are not limited to, established tumor cell lines and tumor cells obtained from a mammal. In one embodiment, the mammal is a human. In a further embodiment, the cells are cancer cells obtained from a human cancer patient.
The method for identifying SVV-responsive cancer cells may be used to discover tumor cell lines or tumor tissues that are permissive for SVV replication. Also, by determining the characteristics of permissive tumor cells, one may be able to identify characteristics of tumor cells that cause the cells to be selectively killed by SVV. The discovery of these characteristics could lead to new targets for cancer drugs. Also, the methods for identifying SVV responsive cancer cells could be used as a screen for human cancer patients who would benefit from treatment with SVV.
For example, antibodies against SVV or an SVV-like picornavirus (polyclonal, monoclonal, etc.) can be used in a viral binding assay to pre-screen patients prior to SVV or SVV-like picornavirus therapy. The pre-screening can be conducted generally as follows: (1) cells from a patient are isolated, the cells can be from a tumor biopsy for example, (2) the cells are stained with anti-SVV or anti-SVV-like picornavirus antibodies, (3) a secondary antibody conjugated with a marker (such as fluoroscein or some other detectable dye or fluorophore) that is specific to the anti-SVV or anti-SVV-like picornavirus antibodies is added (for example, if the antibodies were raised in a rabbit, then the secondary antibody would be specific for rabbit immunoglobulins), and (4) detection for the marker is conducted—for example, fluorescence microscopy can be conducted where the marker is fluorescein. (Step 3 is optional if the anti-SVV or anti-SVV-like picornavirus antibodies are directly conjugated, i.e, where the antibodies are monoclonal. If the antibodies are polyclonal, indirect immunofluorescence—use of a secondary antibody—is suggested.) If the patient's tumor cells are permissive for SVV or SVV-like picornavirus infection, then the patient is a candidate for SVV or SVV-like picornavirus therapy. In a virus binding assay, the patient's tumor cells can be determined to be permissive for SVV if the cells are positive for antibody staining. For example,
In pre-screening patients with a viral binding assay, the cell sample from the patient can also be a tissue section of a tissue suspected to contain tumor cells. The tissue section can then be prepared into sections and incubated with SVV prior to histochemistry with anti-SVV or anti-SVV-like picornavirus antibodies.
The invention also provides methods of detecting SVV. In one embodiment, the detection assay is based on antibodies specific to SVV polypeptide epitopes. In another embodiment, the detection assay is based on the hybridization of nucleic acids. In one embodiment, RNA is isolated from SVV, labeled (e.g., radioactive, chemiluminsecence; fluorescence, etc.) to make a probe. RNA is then isolated from test material, bound to nitrocellulose (or a similar or functionally equivalent substrate), probed with the labeled SVV RNA, and the amount of bound probe detected. Also, the RNA of the virus may be directly or indirectly sequenced and a PCR assay developed based on the sequences. In one embodiment, the PCR assay is a real time PCR assay.
Methods for Making Viruses with Altered Tropism:
The present invention provides methods for constructing SVV mutants (or variants or derivatives) where these mutants have an altered cell-type tropism. SVV-like picornaviruses may also be mutated in order to provide a particular cell-type tropism. Specifically, SVV and SVV-like picornavirus mutants are selected for their ability to specifically bind and/or kill tumor or neoplastic cells that are known to be resistant to wild-type SVV or wild-type SVV-like picornavirus binding.
The native or wild-type SVV has a simple genome and structure that allow the modification of the native virus to target other cancer indications. These new derivatives have an expanded tropism toward non-neural cancers and still maintain the high therapeutic index found in the native SVV. One possible means of targeting is the inclusion of tissue-specific peptides or ligands onto the virus.
To select cancer-targeting viral candidates, the present invention provides methods to construct and screen an oncolytic virus library with a genetic insertion that encodes a random peptide sequence in the capsid region of the native SVV. A random peptide library with a diversity of 108 is believed to be sufficient and should yield peptides that specifically direct the virus to tumor tissue.
Various studies have shown that tumor cells exhibit different characteristics from normal cells such as: (1) tumor cells have more permeable cell membranes; (2) tumors have specific stromal cell types such as angiogenic endothelial cells which have previously been shown to express cell type specific receptors; and (3) tumor cells differentially express certain receptors, antigens and extracellular matrix proteins (Arap, W. et al., Nat. Med., 2002, 8(2): 121-127; Kolonin, M. et al., Curr. Opin. Chem. Biol., 2001, 5(3): 308-313; St. Croix, B. et al., Science, 2000, 289(5482): 1997-1202). These studies demonstrated that tumor and normal tissues are distinct at the molecular level and targeted drug delivery and treatment of cancer is feasible. Specifically, several peptides selected by homing to blood vessels in mouse models have been used for targeted delivery of cytotoxic drugs (Arap, W. et al., Science, 1998, 279(5349): 377-380), pro-apoptotic peptides (Ellerby, H. M. et al., Nat. Med., 1999, 17(8): 768-774), metalloprotease inhibitor (Koivunen, E. et al., Nat. Biotechnol, 1999, 17(8): 768-774), cytokine (Curnis, F. et al., Nat. Biotechnol., 2000, 18(11): 1185-1190), fluorophores (Hong. F. D. and Clayman, G. L., Cancer Res., 2000, 60(23): 6551-6556) and genes (Trepel, M. et al., Hum. Gene Ther., 2000, 11(14): 1971-1981). The tumor-targeting peptides have proven to increase the efficacy and lower the toxicity of the parental drugs.
A library of SVV derivatives can be generated by the insertion of a random peptide sequence into the capsid region of the virus. As shown in
The oligonucleotides that are inserted into the capsid encoding region can be either random oligonucleotides, non-random oligonucleotides (i.e., the sequence of the oligonucleotide is pre-determined), or semi-random (i.e., a portion of the oligonucleotide is pre-determined and a portion has a random sequence). The non-random aspect of the contemplated oligonucleotides can comprise an epitope-encoding region. Contemplated epitopes include, but are not limited to, c-myc—a 10 amino acid segment of the human protooncogene myc (EQKLISEEDL (SEQ ID NO: 35); HA—haemoglutinin protein from human influenza hemagglutinin protein (YPYDVPDYA (SEQ ID NO: 36)); and His6 (SEQ ID NO:116)—a sequence encoding for six consecutive histidines.
The library of mutant capsid polynucleotides (for example, “pSVV capsid library” in
The peptide encoded by the oligonucleotide inserted into the capsid encoding region can serve as a targeting moiety for specific viral infection. The viruses that target a specific type of cancer would selectively infect only those cancer cells that have a receptor to the peptide, replicate in those cells, kill those cells, and spread to only those same types of cells. This methodology enables the identification of novel tumor-targeting peptides and ligands, tumor-selective receptors, therapeutic SVV derivatives and other virus derivatives, including picornavirus derivatives.
In vitro and in vivo screening of SVV mutant libraries have several advantages over other technologies such as peptide bead libraries and phage display. Unlike these other technologies, the desirable candidate here, i.e. an SVV derivative that selectively binds to a cancer cell, will replicate in situ. This replication-based library approach has numerous advantages over prior methods of discovering new cell binding moieties, such as phage display. First, the screening of a SVV library is based on replication. Only the desired viral derivatives can replicate in the target tissue, in this case certain cancer cells. The screening/selection process will yield very specific viral candidates that have both the targeting peptide moiety and may be a cancer therapeutic itself. On the contrary, phage display screens will only result in binding events and yields only the targeting peptide candidates. Thus, SVV library screening offers a much faster and selective approach. Second, during in vitro or in vivo phage display screening, phages recovered from the target cells have to be amplified in bacteria, while SVV derivatives can be directly recovered and purified from infected cells (or from the culture supernatant of lytically infected cells). Third, SVV has a smaller genome that renders easier manipulability; thus it is possible to randomly insert the genetic information into the capsid region to ensure an optimized insertion. Therefore, construction and screening of the SVV library has a high possibility to produce highly effective viral derivatives. These derivatives are designed and screened to specifically infect cancers with non-neural properties.
The insertion of oligonucleotides into the capsid encoding region will result in the generation of some defective mutants. Mutants may be defective in the sense that the insertion of an oligonucleotide sequence can result in a stop codon, such that the viral polyprotein will not be produced. Also, mutants may be defective in the sense that the insertion of an oligonucleotide sequence may result in the alteration of the capsid structure such that capsid can no longer be assembled. To decrease the probability that the insertion of oligonucleotide sequences may result in stop codon or untenable capsid structure, random oligonucleotides can be designed such that they do not encode for stop codons or for certain amino acids using methods such as TRIM.
To determine whether there is an optimal insertion point in the capsid region for oligonucleotides, one can generate an RGD-SVV library (see Example 16). The polynucleotide encoding the SVV capsid is randomly cut, for example, with CviJI. The randomly linearized capsid polynucleotides are then ligated to oligonucleotides encoding at least the RGD amino acid sequence (arginine-glycine-aspartic acid). These RGD-capsid sequences are then ligated into SVV full-length sequence vectors that are missing the capsid sequence. RGD-SVV derivatives viruses are produced and tested for their ability to infect and replicate in certain integrin-expressing cell lines (as the RGD peptide has been shown to target entities to integrin receptors). The RGD-SVV derivatives that are successful in infecting the integrin-expressing cell lines are then analyzed to determine whether there is a predominant insertion site for the RGD oligonucleotide. This site can then be used for site-directed insertion of random, non-random or semi-random oligonucleotides.
Further, in comparing portions of the capsid encoding region between SVV and other picornaviruses (see
Inactivated SVV as a Tumor-Specific Therapeutic:
Since SVV and SVV-capsid derivatives can target specific tumor cell-types and/or tissues, the SVV particle itself can be used as a delivery vehicle for therapeutics. In such a method, the need for the oncolytic abilities of SVV becomes optional, as the delivered therapeutic can kill the targeted tumor cell.
For example, the wild-type SVV can be inactivated such that the virus no longer lyses infected cells, but where the virus can still specifically bind and enter targeted tumor cell-types. There are many standard methods known in the art to inactivate the replicative functions of viruses. For example, whole virus vaccines are inactivated by formalin or β-propiolactone such that the viruses cannot replicate. The wild-type SVV may itself contain peptides that cause the apoptosis of cells. Alternatively, SVV can be irradiated. However, irradiated viruses should first be tested to ensure that they are still capable of specifically targeting tumor cells, as certain irradiation conditions may cause protein, and thus capsid, alterations. Further, mutant SVVs can be generated where the packaging signal sequence is deleted. These SVV mutants are able to specifically bind and enter target cells, but replicated SVV genomic RNA will not be packaged and assembled into capsids. However, this method may prove to be useful as initial entry of these mutant SVVs will cause host-protein synthesis shut-off such that tumor-cell death is still achieved.
Derivative SVVs having mutant capsids can also be inactivated and used to kill cancer cells. Derivative SVVs with oligonucleotides encoding epitope tags inserted into the capsid region can be used as vehicles to deliver toxins to tumor cells. As described herein, derivative SVVs can be randomly mutagenized and screened for tumor-specific tropisms. Toxins can be attached to the epitope tags, such that the virus delivers the toxin to tumor cells. Alternatively, therapeutic antibodies that specifically bind to the epitope tag can be used, such that the virus delivers the therapeutic antibody to the tumor cell.
High-Throughput Screening:
The present invention encompasses high-throughput methods for screening viruses that have the ability to specifically infect different cell-lines. The specificity of infection can be detected by assaying for cytopathic effects. For example, a number of different tumor cell-lines can be grown in different wells of a multi-well plate that is amenable for high-throughput screening, for example a 384 well-plate. To each well, a sample of virus is added to test whether the cells are killed by virus-mediated lysis. From those wells that show cytopathic effects, the media is collected such that any viruses in the media can be amplified by infecting permissive cell lines in flasks or large tissue culture plates. The viruses are grown such that the RNA can be isolated and the sequence analyzed to determine sequence mutations that may be responsible for providing a tumor cell-type specific tropism for a virus.
Various colorimetric and fluorometric methods can quickly assay cytopathic effects, including fluorescent-dye based assays, ATP-based assays, MTS assays and LDH assays. Fluorescent-dye based assays can include nucleic acid stains to detect dead-cell populations, as cell-impermeant nucleic acid stains can specifically detect dead-cell populations. If it is desired to simultaneously detect both live-cell and dead-cell populations, nucleic acid stains can be used in combination With intracellular esterase substrates, membrane-permeant nucleic acid stains, membrane potential-sensitive probes, organelle probes or other cell-permeant indicators to detect the live-cell population. For example, Invitrogen (Carlsbad, Calif.) offers various SYTOX™ nucleic acid stains that only penetrate cells with compromised plasma membranes. Ethidium bromide and propidium iodide can also be used to detect dead or dying cells. These stains are high-affinity nucleic acid stains that can be detected by any light-absorbance reader
For example, lysis can be based on the measurement of lactate dehydrogenase (LDH) activity released from the cytosol of damaged cells into the supernatant. To detect the presence of LDH in cell culture supernatants, a substrate mixture can be added such that LDH will reduce the tetrazolium salt INT to formazan by a coupled enzymatic reaction. The formazan dye can then be detected by a light-absorbance reader. Alternatively, an MTS assay [3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] using phenzine methosulfate (PMS) as the electron coupling reagent can also be used to detect cytotoxicity. Promega (Madison, Wis.) offers a CellTiter 96® AQueous One Solution Cell Proliferation Assay kit where the solution reagent is added directly to culture wells, incubated for 1-4 hours and then absorbance is recorded at 490 μm. The quantity of formazan product as measured by the amount of 490 nm absorbance is directly proportional to the number of living cells in culture.
There are numerous high-throughput devices for reading light-absorbance. For example, SpectraMax Plus 384 Absorbance Platereader (Molecular Devices) can detect wavelengths from 190-1000 nm in 1 μm increments. The device can read 96-well microplates in 5 seconds and 384-well microplates in 16 seconds for ultra fast sample throughput.
Virus replication can also be assayed as an indication of successful infection, and such detection methods can be used in a high-throughput manner. For example, real-time RT-PCR methods can be used to detect the presence of virus transcripts in cell-culture supernatants. Upon reverse-transcription of viral RNA into cDNA, the cDNA can be amplified and detected by PCR with the use of double-stranded DNA-binding dyes (for example, SYBR® Green, Qiagen GmbH, Germany). The amount of PCR product can then be directly measured using a fluorimeter.
Viruses from the wells showing cytopathic effects are grown up and tested in further in vitro (re-testing of tumor and normal cell lines) and in vivo models (testing whether the virus can kill explanted tumors in mice).
Antibodies:
The present invention is also directed to antibodies that specifically bind to the viruses of the present invention, including the proteins of the viruses. Antibodies of the present invention include naturally occurring as well as non-naturally occurring antibodies, including, for example, single chain antibodies, chimeric antibodies, bifunctional antibodies and humanized antibodies, as well as antigen-binding fragments thereof. Such non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly or can be obtained, for example, by screening combinatorial libraries consisting of variable heavy chains and variable light chains (Huse et al., Science 246:1275-1281, 1989). These and other methods of making, for example, chimeric, humanized, CDR-grafted, single chain, and bifunctional antibodies are well known to those skilled in the art (Winter and Harris, Immunol. Today 14:243-246, 1993; Ward et al., Nature 341:544-546, 1989; Harlow and Lane, Antibodies: A laboratory manual, Cold Spring Harbor Laboratory Press, 1988); Hilyard et al., Protein Engineering: A practical approach, IRL Press 1992; Borrabeck, Antibody Engineering, 2d ed., Oxford University Press 1995). Antibodies of the invention include intact molecules as well as fragments thereof, such as Fab, F(ab′)2, and Fv which are capable of binding to an epitopic determinant present in a polypeptide of the present invention.
Where a peptide portion of a SVV polypeptide of the invention (i.e., any peptide fragment from SEQ ID NO:2 or SEQ ID NO:169) or peptide portion of another viral polypeptide of the invention used as an immunogen for antibody generation is non-immunogenic, it can be made immunogenic by coupling the hapten to a carrier molecule such as bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH), or by expressing the peptide portion as a fusion protein. Various other carrier molecules and methods for coupling a hapten to a carrier molecule are well known in the art (for example, by Harlow and Lane, supra, 1988). Methods for raising polyclonal antibodies, for example, in a rabbit, goat, mouse or other mammal, are well known in the art (see, for example, Green et al, “Production of Polyclonal Antisera,” in Immunochemical Protocols, Manson, ed., Humana Press 1992, pages 1-5; Coligan et al., “Production of Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters,” in Curr. Protocols Immunol. (1992), section 2.4.1).
Monoclonal antibodies also can be obtained using methods that are well known and routine in the art (Kohler and Milstein, Nature 256:495, 1975; Coligan et al., supra, 1992, sections 2.5.1-2.6.7; Harlow and Lane, supra, 1988). For example, spleen cells from a mouse immunized with a virus, viral polypeptide or fragment thereof, can be fused to an appropriate myeloma cell line to produce hybridoma cells. Cloned hybridoma cell lines can be screened using, for example, labeled SVV polypeptide to identify clones that secrete monoclonal antibodies having the appropriate specificity, and hybridomas expressing antibodies having a desirable specificity and affinity can be isolated and utilized as a continuous source of the antibodies. Polyclonal antibodies similarly can be isolated, for example, from serum of an immunized animal. Such antibodies, in addition to being useful for performing a method of the invention, also are useful, for example, for preparing standardized kits. A recombinant phage that expresses, for example, a single chain antibody also provides an antibody that can used for preparing standardized kits. Monoclonal antibodies, for example, can be isolated and purified from hybridoma cultures by a variety of well established techniques, including, for example, affinity chromatography with Protein-A SEPHAROSE gel, size exclusion chromatography, and ion exchange chromatography (Barnes et al., in Meth. Mol. Biol. 10:79-104, Humana Press 1992); Coligan et al., supra, 1992, see sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3).
An antigen-binding fragment of an antibody can be prepared by proteolytic hydrolysis of a particular antibody, or by expression of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab′)2. This fragment can be further cleaved using a thiol-reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab′ fragments and an Fc fragment directly (see, for example, Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647; Nisonhoff et al., Arch. Biochem. Biophys. 89:230. 1960; Porter, Biochem. J. 73:119, 1959; Edelman et al., Meth. Enzymol., 1:422 (Academic Press 1967); Coligan et al., supra, 1992, see sections 2.8.1-2.8.10 and 2.10.1-2.10.4).
Another example of an antigen binding fragment of an antibody is a peptide coding for a single complementarity determining region (CDR). CDR peptides can be obtained by constructing polynucleotides encoding the CDR of an antibody of interest. Such polynucleotides can be prepared, for example, using the polymerase chain reaction to synthesize a variable region encoded by RNA obtained from antibody-producing cells (for example, Larrick et al., Methods: A Companion to Methods in Enzymology 2:106, 1991).
The antibodies of the invention are suited for use, for example, in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier. In addition, the antibodies in these immunoassays can be detectably labeled in various ways. Examples of types of immunoassays which can utilize antibodies of the invention are competitive and non-competitive immunoassays in either a direct or indirect format. Examples of such immunoassays are the radioimmunoassay (RIA) and the sandwich (immunometric) assay. Detection of the antigens using the antibodies of the invention can be done utilizing immunoassays which are run in either the forward, reverse, or simultaneous modes, including immunohistochemical assays on physiological samples. Those of skill in the art will know, or can readily discern, other immunoassay formats without undue experimentation.
There are many different labels and methods of labeling antibodies known to those of ordinary skill in the art. Examples of the types of labels which can be used in the present invention include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, phosphorescent compounds, and bioluminescent compounds. Those of ordinary skill in the art will know of other suitable labels for binding to the antibody, or alternatively to the antigen, or will be able to ascertain such, using routine experimentation.
As various changes can be made in the above methods and compositions without departing from the scope and spirit of the invention as described, it is intended that all subject matter contained in the above description, shown in the accompanying drawings, or defined in the appended claims be interpreted as illustrative, and not in a limiting sense.
The examples described below are provided to illustrate the present invention and are not included for the purpose of limiting the invention.
Cultivation of SVV in PER.C6 cells: SVV is plaque purified once and a well isolated plaque is picked and amplified in PER.C6 cells (Fallaux et al., 1998). A crude virus lysate (CVL) from SVV infected PER.C6 cells is made by three cycles of freeze and thaw and used to infect PER.C6 cells. PER.C6 cells are grown in 50×150 cm2 T.C. flasks using Dulbecco's modified Eagle medium (DMEM, Invitrogen, Carlsbad, Calif., USA)) containing 10% fetal bovine serum (Biowhitaker, Walkersvile, Md., USA) and 10 mM magnesium chloride (Sigma, St Louis, Mo., USA). The infected cells harvested 30 hr after infection when complete CPE is noticed and are collected by centrifugation at 1500 rpm for 10 minutes at 4° C. The cell pellet is resuspended in the cell culture supernatant (30 ml) and is subjected to three cycles of freeze and thaw. The resulting CVL is clarified by centrifugation at 1500 rpm for 10 minutes at 4° C. Virus is purified by two rounds of CsCl gradients: a one-step gradient (density of CsCl 1.24 g/ml and 1.4 g/ml) followed by one continuous gradient centrifugation (density of CsCl 1.33 g/ml). The purified virus concentration is determined spectrophotometrically, assuming 1A260=9.5×1012 particles (Scraba D. G., and Palmenberg, A. C. 1999. Cardioviruses (Picornaviridae). In: Encyclopedia of Virology, Second edition, R. G. Webster and A Granoff Eds). Titers of purified virus are also determined by a standard plaque assay using PER.C6 cells. The yield of SVV from PER.C6 cells are greater than 200, 000 particles per cell with particles to PFU ratio of about 100. The yields of SVV from other permissive cells (H446-ATCC# HTB-171) may be at least this high or higher.
SVV is mounted onto formvar carbon-coated grids using the direct application method, stained with uranyl acetate, and examined in a transmission electron microscope. Representative micrographs of the virus are taken at high magnification. For the transmission electron microscope, ultra-thin sections of SVV-infected PER.C6 cells are cut from the embedded blocks, and the resulting sections are examined in the transmission electron microscope.
The purified SVV particles are spherical and about 27 nm in diameter, appearing singly or in small aggregates on the grid. A representative picture of SVV is shown in
RNA Isolation: SVV genomic RNA was extracted using guanidium thiocyanate and a phenol extraction method using Trizol (Invitrogen). Isolation was performed according to the supplier's recommendations. Briefly, 250 μl of the purified SVV was mixed with 3 volumes TRIZOL and 240 μl of chloroform. The aqueous phase containing RNA was precipitated with 600 μl isopropanol. The RNA pellet was washed twice with 70% ethanol, dried and dissolved in DEPC-treated water. The quantity of RNA extracted was estimated by optical density measurements at 260 μm. An aliquot of RNA was resolved through a 1.25% denaturing agarose gel (Cambrex Bio Sciences Rockland Inc., Rockland, Me. USA) and the band was visualized by ethidium bromide staining and photographed (
cDNA synthesis: cDNA of the SVV genome was synthesized by RT-PCR. Synthesis of cDNA was performed under standard conditions using 1 μg of RNA, AMV reverse transcriptase, and random 14-mer oligonucleotide or oligo-dT. Fragments of the cDNA were amplified, cloned into plasmids and the clones are sequenced
Part I: SVV SEQ ID NO:1
The nucleotide sequence of SVV SEQ ID NO:1 was analyzed to determine its evolutionary relationship to other viruses. The translated product (SEQ ID NO:2) for this ORF was picornavirus-like and reached from the middle of VP2 to the termination codon at the end of the 3D polymerase and was 1890 amino acids in length (
Preliminary comparisons (not shown) of three partial genome segments of SVV had revealed that SVV was most closely related members of the genus Cardiovirus (family Picornaviridae). Therefore an alignment of the polyprotein sequences of SVV, encephalomyocarditis virus (EMCV; species Encephalomyocarditis virus, Theiler's murine encephalomyelitis virus (TMEV; species Theilovirus), Vilyuisk human encephalomyelitis virus (VHEV; species Theilovirus) and a rat TMEV-like agent (TLV; species Theilovirus) was constructed (
In picornaviruses, most polyprotein cleavages are carried out by one or more virus-encoded proteases, although in cardio-, aphtho-, erbo- and teschoviruses the cleavage between P1-2A and 2B is carried out by a poorly understood cis-acting mechanism related to the 2A sequence itself and critically involving the sequence “NPG/P”, where “/” represents the break between the 2A and 2B polypeptides (Donnelly et al., 1997, J. Gen. Virol. 78: 13-21). One of the parechoviruses, Ljungan virus, has this sequence (NPGP) present upstream of a typical parechovirus 2A and is either an additional 2A or is the C-terminal end of the P1 capsid region. In all nine currently recognised picornavirus genera, 3Cpro carries out all but the cis-acting self-cleaving reactions (i.e. 2A cleaves at its N-terminus in entero- and rhinoviruses and L cleaves at its C-terminus in aphthoviruses and erboviruses). The post-assembly cleavage of the capsid polypeptide VP0 to VP4 and VP2 is not carried out by 3Cpro, but by an unknown mechanism which may involve the virus RNA. The VP0 cleavage does not occur in parechoviruses and kobuviruses. The normal cardiovirus 3Cpro cleavage site has either a glutamine (Q) or glutamate (E) at the −1 position and glycine (G), serine (S), adenine (A) or asparagine (N) at the +1 position (Table 2). The cleavages of the SVV polyprotein conform to this pattern except for the VP3/VP1 site which is histidine (H)/serine (S) (Table 2); however, H/S is probably present as the cleavage site between 3A and 3BVPg in at least one strain of equine rhinitis A virus (ERAV; genus Aphthovirus) (Wutz et al., 1996, J. Gen. Virol. 77:1719-1730).
Primary cleavages (P1/P2 and P2/P3) of SVV: These primary cleavage events are predicted to occur in a similar fashion to cardio-, aphtho-, erbo- and teschoviruses, involving separation of P1-2A from 2B by a novel mechanism involving the sequence NPG/P (SEQ ID NO:111) and a traditional cleavage event by 3Cpro between 2BC and P3 (Table 2).
P1 cleavages: Cleavages within the SVV P1 capsid coding region were relatively easy to predict by alignment with sequence with EMCV and TMEV (Table 2).
P2 cleavages: The 2C protein is involved in RNA synthesis. The 2C polypeptide of SVV contains NTP-binding motifs GxxGxGKS/T (SEQ ID NO:112) (domain A) and hyhyhyxxD (in which hy is any hydrophobic residue; domain B) present in putative helicases and all picornavirus 2Cs (
P3 cleavages: Prediction of the P3 cleavage sites was also relatively straightforward. Little is known about the function of the 3A polypeptide. However, all picornavirus 3A proteins contain a putative transmembrane alpha-helix. Primary sequence identity is low in this protein between SVV and cardioviruses (See
The genome-linked polypeptide, VPg, which is encoded by the 3B region, shares few amino acids in common with the other cardioviruses, however, the third residue is a tyrosine, consistent with its linkage to the 5′ end of the virus genome (Rothberg et al., 1978). See
The three-dimensional structure of four picornavirus 3C cysteine proteases have been solved and the active-site residues identified (HAV, Allaire et al., 1994, Nature, 369: 72-76; Bergmann et al., 1997, J. Virol., 71: 2436-2448; PV-1, Mosimann et al., 1997, J. Mol. Biol., 273: 1032-1047; HRV-14, Matthews et al., 1994, Cell, 77: 761-771; and HRV-2, Matthews et al., 1999, Proc. Natl. Acad. Sci. USA, 96: 11000-11007). The cysteine bolded in
The 3D polypeptide is the major component of the RNA-dependent RNA polymerase and SVV contains motifs conserved in picorna-like virus RNA-dependent RNA polymerases, i.e. KDEL/IR (SEQ ID NO:113), PSG, YGDD (SEQ ID NO:114) and FLKR (SEQ ID NO:115) (
Myristoylation of the N-terminus of P1: In most picornaviruses the P1 precursor polypeptide is covalently bound by its N-terminal glycine residue (when present the N-terminal methionine is removed) to a molecule of myristic acid via an amide linkage (Chow et al., 1987, Nature, 327: 482-486). Consequently the cleavage products VP0 and VP4 which contain the P1 N-terminus are also myristoylated. This myristoylation is carried out by myristoyl transferase which recognises an eight amino acid signal beginning with glycine. In picornaviruses, a five residue consensus sequence motif, G-x-x-x-T/S, has been identified (Palmenberg, 1989, In Molecular Aspects of picornavirus Infection and Detection, pp. 211-241, Ed. Semler & Ehrenfeld, Washington D.C., Amer. Soc. for Micro.). Parechoviruses (Human parechovirus and Ljungan virus) as well as not having a maturation cleavage of VP0 are apparently not myristoylated, however, there appears to be some type of molecule blocking the N-terminus of VP0 for these viruses.
Comparisons of the Individual SVV Polypeptides with the Public Sequence Databases
Each of the SVV polypeptides (SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22) were compared to the public protein sequence databases using the FASTA online program at the European Bioinformatics Institute (EBI; http://www.ebi.ac.uk/). The results (best matches) of these comparisons are shown in Table 3. The capsid polypeptides (VP2, VP3 and VP1) taken as a whole, along with 2C, 3Cpro and 3Dpol are most closely related to members of the cardiovirus genus, however, the short predicted 2A sequence is closer to that of Ljungan virus (genus Parechovirus). A more detailed comparison of the SVV 2A nucleotide sequence with similar sequences is shown in
Ureaplasma urealyticum
Chlorobium tepidum TLS*
The significance of the matches of SVV 2B with Ureaplasma urealyticum multiple banded antigen or 3A with Chlorobium tepidum endolase 2 is not clear, however, these relationships maybe worthy of further investigation.
Phylogenetic Comparison of SVV Polypeptides with Other Picornaviruses
Those SVV polypeptides which could be aligned with the cardioviruses (VP2, VP3, VP1, 2C, 3C and 3D) were compared with the same proteins of representative members of each of the picornavirus species (Table 4). The programs BioEdit v5.0.9 (Hall, 1999, Nucl. Acids. Symp. Ser., 41: 95-98) and Clustal X v1.83 (Thompson et al., 1997, Nucl. Acids Res., 25:4876-4882) were used to make the alignments and to construct distance matrices and unrooted Neighbor-joining trees according to the algorithm of Saitou and Nei (Satiou and Nei, 1987, Mol. Biol. Evol., 4: 406-425). Confidence limits on branches were accessed by bootstrap resampling (1000 pseudo-replicates). The trees were drawn using TreeView 1.6.6 (Page, 1996) (
Enterovirus
Poliovirus
Human enterovirus A
Human enterovirus B
Human enterovirus C
Human enterovirus D
Simian enterovirus A
Bovine enterovirus
Porcine enterovirus B
Porcine enterovirus A
Rhinovirus
Human rhinovirus A
Human rhinovirus B
Cardiovirus
Encephalomyocarditis virus
Theilovirus
Aphthovirus
Foot-and-mouth disease virus
Equine rhinitis A virus
Hepatovirus
Hepatitis A virus
Parechovirus
Human parechovirus
Ljungan virus
Kobuvirus
Aichi virus
Bovine kobuvirus
Erbovirus
Equine rhinitis B virus
Teschovirus
Porcine teschovirus
The trees of the individual capsid proteins (
Seneca Valley Virus as a Member of the Cardiovirus Genus
Clearly the 3Dpol of SVV is related to the cardioviruses, almost as closely as EMCV and TMEV are to each other (
Seneca Valley virus is clearly not a typical cardiovirus if EMCV and TMEV are taken as the standard. However, even these two viruses have their differences, notably in the 5′ UTR (Pevear et al., 1987, J. Gen. Virol., 61: 1507-1516). However, phylogenetically SVV clusters with EMCV and TMEV in much of its polyprotein (P1, 2C, 3Cpro and 3Dpol regions). Ultimately, the taxonomic position of SVV within the Picornaviridae will be decided by the Executive Committee (EC) of the International Committee for the Taxonomy of Viruses (ICTV) following recommendations by the Picornaviridae Study Group and supporting published material. There are two options: i) include SVV as a new species in the cardiovirus genus; or ii) assign SVV to a new genus.
Part II: SVV SEQ ID NO:168
The full-length genome of SVV (
The features of the SVV full-length genome with respect to its untranslated and coding regions are listed at Table A supra. The features of the full-length SVV in comparison to EMCV and TMEV-GDVII are listed in the table below.
The cleavage sites of SVV (based on full-length sequence, see also bolded amino acids between at protein boundaries in
Multiple unique viruses were discovered at the USDA that are more similar to SVV than SVV is to other cardioviruses. These USDA virus isolates, herein considered to be members of the group called “SVV-like picornaviruses,” are: MN 88-36695, NC 88-23626, IA 89-47552, NJ 90-10324, IL 92-48963, CA 131395; LA 1278; IL 66289; IL 94-9356; MN/GA 99-29256; MN 99197; and SC 363649. These SVV-like picornaviruses and SVV are considered to comprise a new picornavirus genus.
Each of these SVV-like picornaviruses are unique, and are about 95%-98% identical to SVV at the nucleotide level (see
Part III: Serum Studies
Pigs are a permissive host for the USDA virus isolates identified above. The isolate MN 88-36695 was inoculated into a gnobiotic pig and antisera generated (GP102). The antisera binds to all of the other USDA isolates listed above and to SVV. The antisera does not react with 24 common porcine virus pathogens indicating its specificity. Porcine sera was also tested for neutralizing antibodies to 1278 (Plum Island virus). Sera were collected in the US and 8/29 sera were positive with titers ranging from 1:57 to 1:36,500.
To test whether the pig is the natural source for SVV, serum samples from various animals were obtained and tested for their ability to act as neutralizing antibodies against SVV infection of permissive cells. The Serum Neutralization Assay is conducted as follows: (1) Dilute various serums 1:2 and 1:4; (2) Mix with 100 TCID50 of virus (SVV; but any virus can be tested to determine whether a serum can neutralize its infection); (3) Incubate at 37° C. for 1 hour; (4) Add to 1×104 PER.C6 cells (or other permissive cell type); (5) Incubate at 37° C. for 3 days; and (6) Measure CPE using MTS assay. The neutraliziation titer is defined as the highest dilution of sera that neutralizes SVV (or other virus in question) at 100%.
The serum neutralization results showed that there is a minimal or no presence of neutralizing antibodies in human and primate populations. In one experiment, 0/22 human sera contained neutralizing antibodies to SVV. In another experiment, only 1/28 human sera contained neutralizing antibodies. In a third experiment, 0/50 human sera from Amish farmers were neutralizing. In another experiment, 0/52 primate sera from four species were neutralizing.
The serum neutralization results showed that there is a prevalence of neutralizing antibodies in farm animal populations. In one experiment, 27/71 porcine sera from farms were neutralizing. In another experiment, 4/30 porcine sera from a disease-free farm were neutralizing. In another experiment, 10/50 bovine sera were neutralizing. In yet another experiment, 5/35 wild mouse sera were neutralizing. Because antibodies cross-reactive to SVV and/or SVV-like picornaviruses have been found in pigs, cows, and mice, these data indicate that SVV and/or SVV-like picornaviruses may be prevalent in a wide-variety of non-primate animals.
A crude viral lysate of MN 88-36695 was tested to assess its cytotoxicity ability on two cell lines permissive (NCI-H446; HEK293) for SVV and on two cell lines non-permissive (NCI-H460 and S8) for SVV. The cytotoxicity profile for MN 88-36695 was identical to SVV: the TCID50 for NCI-H446 was 1.6×10−6; the TCID50 for HEK293 was 1.3×10−2; and NCI-H460 and S8 were non-permissive for MN 88-36695. This data indicates that SVV-like picornaviruses have the potential to be used in the present methods directed to cancer therapy. In one embodiment, the invention provides for the use of the MN 88-36695 SVV-like picornavirus in any of the methods directed to cancer therapy, diagnosis, or screening.
Antisera to MN 88-36694 and SVV were tested in serum neutralization assays on each virus. Anti-SVV mouse serum was able to neutralize infection by both MN 88-36695 and SVV (neutralization titers on infection were 1:640 for MN 88-36695 and 1:1000 for SVV). Anti-MN 88-36695 gnobiotic pig serum was able to neutralize infection by both MN 88-36695 and SVV (neutralization titers on infection were 1:5120 for MN 88-36695 and 1:100 for SVV).
These data indicate that SVV is genetically and serologically linked to the porcine USDA virus isolates.
Purified SVV is subjected to electrophoresis using NuPAGE pre-cast Bis-Tris polyacrylamide mini-gel electrophoresis system (Novex, San Diego, Calif., USA). One half of the gel is visualized by silver stain while the other half is used to prepare samples for amino acid sequencing of the N-termini of the capsid proteins. Prior to transfer of proteins to membrane, the gel is soaked in 10 mM CAPS buffer, pH 11, for 1 hour, and a PVDF membrane (Amersham) is wetted in methanol. Proteins are transferred to the PVDF membrane. After transfer, proteins are visualized by staining with Amido black for approximately 1 minute, and bands of interest are excised with a scalpel and air dried. The proteins can be subjected to automated N-terminal sequence determination by Edman degradation using a pulsed phase sequencer.
Three major structural proteins of the purified SVV are shown in
Preexisting antibodies to particular viral vectors may limit the use of such vectors for systemic delivery applications such as for treatment of metastatic cancer, because preexisting antibodies may bind to systemically delivered vectors and neutralize them before the vectors have a chance to transduce the targeted tissue or organ. Therefore, it is desirable to ensure that humans do not carry neutralization antibodies to viral vectors selected for systemic delivery. To determine whether human sera samples contain SVV-specific neutralizing antibodies, neutralization assays are carried out using randomly collected human sera samples.
Tissue culture infective dose 50: One day before the experiment, 180 μl of PER.C6 cell suspension containing 1×104 cells are plated in 96-well tissue culture dish. The crude virus lysate (CVL) of SVV is diluted in log steps from 10−0 to 10−11 in DMEM medium (Dulbecco's Modified Eagle's Medium) and 20 μl of each dilution is transferred to three wells of a Falcon 96-well tissue culture plate containing PER.C6 cells. The plates are incubated at 37° C. in 5% CO2 and read at 3 days for microscopic evidence of cytopathic effect (CPE), and the tissue culture infective dose 50 (TCID50) is calculated.
Neutralization assay: First, 40 μl of medium is placed in all the wells and then 40 μl of heat-inactivated serum is added to the first well and mixed by pipeting, making a 1:4 dilution used for screening purposes. 40 μl is then transferred to the next well to perform a two-fold dilution of the serum samples. 40 μl of SVV virus, containing 100 TCID50, is added to wells containing diluted serum samples. Plates are incubated at 37° C. for 1 hour. 40 μl of the mix is taken and transferred to a plate containing PER.C6 cells (1×104 cells/160 μl/well). The plates are incubated at 37° C. for 3 days. After this time, the cultures are read microscopically for CPE.
In a representative neutralization assay performed as described above, twenty-two human sera samples randomly collected from USA, Europe and Japan were examined for SVV specific neutralizing antibodies. The serum samples were serially diluted and mixed with a fixed amount of SVV containing 100 TCID50. Serum-virus mixtures were then used to infect PER.C6 cells and incubated for 24 hours. Neutralizing antibody titer was determined as the reciprocal of the highest dilution of serum able to block CPE formation. In this experiment, no dilution of serum blocked CPE formation indicating that the human serum samples did not contain SVV neutralizing antibodies.
Further SVV infection of PER.C6 was not inhibited by incubation with human blood (see Example 6), indicating that SVV infection was not inhibited by complement or by hemagglutination. As a result, SVV exhibits a longer circulation time in vivo than other oncolytic viruses, which is a significant problem with the use of oncolytic adenoviruses.
Various viral serotypes have been shown to cause in vitro hemagglutination of erythrocytes isolated from blood of various animal species. Hemagglutination or binding to erythrocytes may cause toxicity in vivo and may also affect in vivo biodistribution and the efficacy of a viral vector. Therefore, it is desirable to analyze the erythrocyte agglutination properties of a viral vector selected for systemic administration to treat metastatic cancers.
Hemagglutination assay: To determine whether SVV causes agglutination of human erythrocytes, hemagglutination assays are carried out in U-bottom 96-well plates. Purified SVV is serially diluted in 25 μl PBS (Phosphate Buffered Saline) in duplicates, and an equal volume of 1% erythrocyte suspension is added to each well. Blood samples used for isolation of erythrocytes are obtained from healthy individuals with heparin as an anticoagulant. Erythrocytes are prepared by washing the blood three times in cold PBS to remove the plasma and the white blood cells. After the last wash, erythrocytes are suspended in PBS to make a 1% (V/V) cell suspension. The virus and erythrocytes are gently mixed and the plates are incubated at room temperature for 1 hour and monitored for a hemagglutination pattern.
Whole blood inactivation assay: To rule out direct inactivation of SVV by blood components, aliquots of virus are incubated with heparinized human blood belonging to A, B, AB and O blood groups or PBS for 30 minutes or 1 hour at room temperature prior to separation of plasma, after which PER.C6 cells are infected and titers are calculated.
In representative assays performed as described above, no hemagglutination of human erythrocytes of different blood groups (A, B, AB and O) was seen at any tested dilutions of SVV. A slight increase in the virus titer is noticed when SVV is mixed with blood human samples and incubated for 30 minutes and 1 hour, indicating that the virus is not inactivated by blood components but becomes more infectious under tested conditions.
Blood circulation time: To determine the blood circulation time and the amount of the virus in the tumor, H446 tumor bearing nude mice were treated with SVV at a dose of 1×1012 vp/kg by tail vein injection. The mice were bled at 0, 1, 3, 6, 24, 48, 72 hours and 7 days (189 hours) post-injection and the plasma was separated from the blood immediately after collection, diluted in infection medium, and used to infect PER.C6 cells. The injected mice were sacrificed at 6, 24, 48, 72 hours and 7 days post-injection and the tumors were collected. The tumors were cut into small sections and suspended in one ml of medium and subjected to three cycles of freeze and thaw to release the virus from the infected cells. Serial log dilutions of supernatants were made and assayed for titer on PER.C6 cells. SVV titers were expressed as pfu/ml. The tumor sections were also subjected to H&E staining and immunohistochemistry to detect the virus capsid proteins in the tumor.
The circulating levels of virus particles in the blood were determined based on the assumption that 7.3% of mouse body weight is blood. In representative assays performed as essentially as described above, within 6 hours of virus administration, the circulating levels of SVV reduced to zero particles and SVV was not detectable at later time points (
SVV also exhibits a substantially longer resident time in the blood compared to similar doses of i.v. adenovirus. Following a single i.v. dose, SVV remains present in the blood for up to 6 hours (
In vitro cell killing activity of SVV: To determine the susceptibility of human, bovine, porcine, and mouse cells, normal and tumor cells were obtained from various sources and infected with SVV. All cell types were cultured in media and under the conditions recommended by the supplier. Primary human hepatocytes may be purchased from In Vitro Technologies (Baltimore, Md.) and cultured in Hepatocye Culture Media (HCM™, BioWhittaker/Clonetics Inc., San Diego, Calif.).
In vitro cytopathic assay: To determine which types of cells are susceptible to SVV infection, monolayers of proliferating normal cells and tumor cells were infected with serial dilutions of purified SVV. The cells were monitored for CPE and compared with uninfected cells. Three days following infection, a MTS cytotoxic assay is performed and effective concentration 50 (Ec50) values in particles per cell are calculated. See Tables 5 and 6 below and Table 1A supra.
The MTS assay was performed according to the manufacturer's instructions (CellTiter 96® AQueous Assay by Promega, Madison, Wis.). The CellTiter 96® AQueous Assay preferably uses the tetrazolium compound (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS) and an electron coupling reagent, phenazine methosulfate (PMS). Contact-inhibited normal human cells evaluated in the study include: HUVEC (human umbilical vein endothelial cells), HAEC (human aortic endothelial cells, Clonetics/BioWhittaker # CC-2535), Wi38 (normal human embryo lung fibroblasts, ATCC # CCL-75), IMR90 (human normal lung fibroblasts, ATCC CCL-186), MRC-5 (human normal lung fibroblasts, ATCC, # CCL-171) and HRCE (human renal cortical epithelial cells, Clonetics/BioWhittaker # CC-2554).
SVV does not produce CPE in any of the above contact-inhibited normal cells. No virus-induced CPE was seen in the following human tumor cell lines: Hep3B (ATCC # HB-8064), HepG2 (human hepatocellular carcinoma, ATCC # HB-8065), LNCaP (human prostate carcinoma, ATCC # CRL-10995), PC3M-2AC6, SVV620 (human colorectal adenocarcinoma, ATCC # CCL-227), SVV 839 (human kidney adenocarcinoma, ATCC # HTB-49), 5637 (human urinary bladder carcinoma, ATCC # HTB-9), DMS-114 (small cell lung cancer, ATCC # CRL-2066), DMS 153 (human small cell lung cancer, ATCC # CRL-2064), A549 (human lung carcinoma, ATCC # CCL-185), HeLa S3 (human cervical adenocarcinoma, ATCC # CCL-2.2), NCI-H460 (human large cell lung cancer, ATCC # HTB-177), KK (glioblastoma), and U-118 MG (human glioblastoma, ATCC # HTB-15). Note—the cell lines in Table 6 with EC50 values greater than 1000 are most likely not permissive for SVV replication and/or virion production; although the possibility remains that SVV can bind and enter into these cells but CPE is not observed because SVV replication cannot occur inside the cell or that replication does occur but CPE is not observed because there is some other post-entry block (i.e., no packaging of replicated SVV genomes into virions). However, considering the absence of CPE in these cell lines, these cell-lines, and potentially tumor-types thereof, are good candidates to test which cell and tumor-types are permissive or non-permissive for SVV replication. Although wild-type SVV is tumor-specific, and has been shown to target neuroendocrine tumors, including small cell lung cancer and neuroblastomas, there may be individual patients that have types of etiologies such that SVV is not permissive in their form of neuroendocrine tumor. Therefore, the invention does contemplate the generation of SVV derivatives that can kill tumor cell-types isolated from individual patients where the tumors are non-permissive to the wild-type SVV, and the tumor-types isolated from these individuals can include, for example, glioblastoma, lymphoma, small cell lung cancer, large cell lung cancer, melanoma, prostate cancer, liver carcinoma, colon cancer, kidney cancer, colon cancer, bladder cancer, rectal cancer and squamous cell lung cancer.
SVV-mediated cytotoxicity on primary human hepatocytes (In Vitro Technologies) was determined by LDH release assay (CytoTox® 96 Non-Radioactive Cytotoxicity Assay, Promega, # G1780). Primary human hepatocytes plated in collagen coated 12-well plates were infected with SVV at 1, 10 and 100 and 1000 particles per cell (ppc). After 3 hours of infection, the infection medium was replaced with 2 ml of growth medium and incubated for 3 days in a CO2 incubator. The cell associated lactate dehydrogenase (LDH) and LDH in the culture supernatant was measured separately. Percent cytotoxicity is determined as a ratio of LDH units in supernatant over maximal cellular LDH plus supernatant LDH.
The data shown in
To assess the replicative abilities of SVV, several selected contact-inhibited normal cells and actively dividing tumor cells were infected with SVV at one virus particle per cell (ppc). After 72 hours, cells and the medium were subjected to three freeze-thaw cycles and centrifuged to collect the supernatant. Serial log dilutions of supernatants were made and assayed for titer on PER.C6 cells. For each cell line, the efficiency of SVV replication was expressed as pfu/ml (
The maximum tolerated dose (MTD) is defined as the dosage immediately preceding the dose at which animals (e.g. mice) demonstrate a dose limiting toxicity (DLT) after the treatment with SVV. DLT is defined as the dose at which the animals exhibit a loss in body weight, symptoms, and mortality attributed to SVV administration during the entire duration of the study. Neutralizing antibodies to SVV were assessed at baseline, day 15, and day 21. Neutralization assays were carried as described earlier.
Escalating doses (1×108-1×1014 vp/kg) of SVV were administered intravenously into both immune deficient nude and caesarean derived-1 (CD-1) out-bred immune competent mice purchased from Harlan Sprague Dawley (Indianapolis, Ind., USA) to determine the MTD with 10 mice per dose level. The virus was well-tolerated at all tested dose levels without exhibiting any clinical symptoms and without loss in body weight (FIG. 50). Mice were bled at day 15 and 21 and the sera was monitored for the presence of SVV-specific neutralizing antibodies in neutralization assays. SVV injected CD1 mice develop neutralizing antibodies and the titers range from 1/1024 to greater than 1/4096.
Another toxicity study was conducted on the immunocompetent mouse strain (A/J). It has been demonstrated that SVV exhibits cell killing activity and replication in N1E-115 cells (see Table 1). The murine cell line N1E-115 (a neuroblastoma cell line, i.e., neuroendocrine cancer) is derived from the A/J mouse strain. Thus, a syngeneic mouse model was established where N1E-115 cells were implanted subcutaneously in A/J mice to form tumors, and the mice were then treated with SVV to investigate its efficacy and toxicity.
In the A/J study, mice were i.v. injected with SVV to determine whether A/J mice can tolerate systemic administration of SVV. Blood hematology results were obtained to look for signs of toxicity, and serum chemistry results can also be obtained. The study design is shown in Table 7 below:
The A/J were 8-10 week old females obtained from The Jackson Laboratory (Bar Harbor, Me.). SVV was prepared by storing isolated virions at −80° C. until use. SVV was prepared fresh by thawing on ice and diluting with HBSS (Hank's balanced salt solution). SVV was diluted to concentrations of 107 particles/mL for group 2, 1010 particles/mL for group 3, and 1013 particle/mL for group 4. HBSS was used as the vehicle control for group 1. All dosing solutions were kept on wet ice until dosing.
SVV was administered to animals intravenous injection via the tail vein at a dose volume of 10 mL/kg body weight. Animals were weighed on the day of dosing and dose volumes were adjusted based on body weight (i.e., a 0.0200 kg mouse gets 0.200 mL of dosing solution). Mice were monitored twice daily for morbidity and mortality. Mice were weighed twice weekly. Information relating to moribund animals and animals exhibiting any unusual symptoms (physically or behaviorally) are recorded immediately.
Post-mortem observations and measurements entail the collection of blood from all surviving animals at terminal sacrifice for standard hematology and serum chemistry (AST, ALT, BUN, CK, LDH). The following organs are to be collected at sacrifice: brain, heart, lung, kidney, liver, and gonads. Half of each organ sample is snap frozen on dry ice and the other half will be placed in formalin.
Initial blood hematology results (CBC, differential) were obtained two weeks after SVV injection and the results are summarized below in Table 8 below. Five mice were tested from each test group (see Table 7):
These results show that there are no abnormalities in blood hematology profiles obtained from mice treated with low, medium and high doses of SVV compared to blood hematology profiles obtained from untreated mice. From this study, it can be concluded that there are no measureable signs of toxicity following systemic administration of SVV, indicating that SVV is tolerated by A/J mice following i.v. injection.
Athymic female nude mice (nu/nu) aged 6-7 weeks purchased from Harlan Sprague Dawley (Indianapolis, Ind.) were used in efficacy studies. Mice were injected subcutaneously with 5×106 H446 cells into the right flank using manual restraint. Tumor sizes were measured regularly, and the volumes were calculated using the formula π/6×W×L2, where L=length and W=width of the tumor. When the tumors reach approximately 100-150 mm3, mice (n=10) were randomly divided into groups. Mice were injected with escalating doses of SVV by tail vein injections at a dose volume of 10 ml/kg. A control group of mice was injected with an equivalent volume of HBSS. Dose escalation proceeds from 1×107 to 1×1013 particles per kilogram body weight. Antitumoral efficacy was determined by measuring tumor volumes twice weekly following SVV administration. Complete response was defined as complete disappearance of xenograft; partial response as regression of the tumor volume by equal to or more than 50%; and no response as continuous growth of tumor as in the control group.
Tumors from mice treated with HBSS grew rapidly and the tumor volumes reached more than 2000 mm3 by study day 20 (
Additional experiments to test the efficacy of a single intravenous dose of SVV was conducted in murine tumor models that express neuroendocrine markers. The tumor models tested included H446 (human SCLC) (see
The results show that a single intravenous dose of SVV has efficacy in all of the murine neuroendocrine tumor models. The results also show that SVV is efficacious in the N1E-115 immunocompetent murine neuroblastoma model.
In vitro efficacy data for SVV for specific tumor cell lines is shown in Tables 1, 1A, and 5. The data shows that SVV specifically infects particular tumor cell types and does not infect normal adult cells (except for porcine normal cells), a significant advantage over any other known oncolytic virus. SVV has been shown to have 1,000 times better cell killing specificity than chemotherapy treatments (cell killing specifity values for SVV have been shown to be greater than 10,000, whereas cell killing specificity values for chemotherapy are around 10).
Specific cytotoxic activity of SVV was demonstrated in H446 human SCLC cells. Following a two-day incubation with increasing concentrations of SVV, cell viability was determined. The results are shown in
Pharmacokinetic and biodistribution study of SVV is performed in normal mice and immunocompromised athymic nude mice bearing H446 SCLC tumors. This study evaluates the biodistribution, elimination and persistence of SVV following a single intravenous administration to both normal and immunocompromised tumor-bearing mice. Groups of mice each receive a single i.v. dose of control buffer or one of three doses of SVV (108, 1010, or 1012 vp/kg) and are monitored for clinical signs. Blood samples are obtained from groups of 5 mice at 1, 6, 24 and 48 hours post dose, and at 1, 2, 4, and 12 weeks post dose. Dose levels include a known low efficacious dose and two higher dose levels to determine linearity of virus elimination. Groups of mice are sacrificed at 24 hours, and 2, 4 and 12 weeks post dose. Selected tissues, including liver, heart, lung, spleen, kidney, lymph nodes, bone marrow, brain and spinal cord tissues are aseptically collected and tested for the presence of SVV RNA using a validated RT-PCR assay.
Samples of urine and feces are obtained at sacrifice, at 24 hours, and at 2, 4 and 12 weeks post dose and are examined for the presence of infectious virus. The design of the experiments in this Example are shown in Table 9 below:
Acute i.v. toxicology studies were also performed in both normal and immunocompromised athymic nude mice bearing H446 SCLC tumors. Preliminary i.v. studies in normal and SCLC tumor bearing mice indicate safety of SVV at doses up to 1014 vp/kg. No adverse clinical signs were observed and there was no loss of body weight up to 2 weeks following a single i.v. dose of 1014 vp/kg.
The purpose of this Example is to determine if SVV is transmissible following cohabitation of noninfected normal mice with mice injected with a high concentration of SVV. Because SVV does not replicate in normal, non-tumor bearing mice, tumor bearing mice can also be injected with high concentrations of SVV and subsequently exposed to normal, healthy animals to better simulate the clinical scenario. A secondary purpose is to assess the potential transmissibility of SVV from an infected female to an uninfected pregnant DAM, and subsequently to the developing fetus.
Three groups of five naive male and female CD-1 mice are exposed to a single mouse of the same sex infected with either 108, 1010 or 1012 vp/kg, and are monitored for the presence of SVV by blood sampling.
Similarly, an SVV exposed female is co-mingled with a number of timed pregnant females, and the ability of the virus to transmit from the infected female to an uninfected pregnant female, and subsequently to the developing fetus is determined.
The safety, toxicity and toxicokinetics of SVV are also determined in non-human primates. In a dose range-finding phase, individual monkeys receive a single i.v. dose of SVV at 108 vp/kg and are closely monitored for clinical signs of infection or toxicity. If this dose is well tolerated, additional animals are treated with a higher i.v. dose until a dose of 1012 vp/kg is achieved. Subsequently, the main study consists of groups of three male and female monkeys, and each monkey is dosed once weekly for six weeks with either vehicle alone or one of three doses of SVV and monitored for signs of toxicity. An additional two monkeys per sex are dosed with the vehicle alone and with the high dose level of SVV for six weeks, and are allowed an additional four weeks recovery prior to sacrifice.
Blood samples are obtained following dosing during week 1 and week 6. Clinical pathological and hematology blood samples are obtained prior to the initial dose and prior to sacrifice. Additional blood samples are obtained following each dose for assessing the presence of neutralizing antibodies to SVV.
Surviving monkeys are euthanized and subjected to a full gross necropsy and a full tissue list is collected from the main study and recovery monkeys. Tissues from the control and high dose groups are evaluated histopathologically. Urine and fecal samples are collected following dosing on weeks 1 and 6 and are evaluated for presence of infectious SVV. The overall design of this Example is shown in Table 10 below.
With SEQ ID NO:1, only about 1.5-2 Kb of the 5′ genomic sequence of SVV remains to be sequenced, representing the nucleotide region covering the 5′ UTR, 1A (VP4) and part of 1B (VP2). To clone the 5′ end missing in SEQ ID NO:1, polymerases that function at high temperatures and reagents that can enable a polymerase to read through secondary structures were used. Additional SVV cDNAs were prepared from isolated SVV of ATCC deposit number PTA-5343. SVV particles were infected into a permissive cell line, such as PER.C6, and viruses are isolated. Viral RNA was then recovered from the virus particles such that cDNA copies are made therefrom. Individual cDNA clones were sequenced, such that selected cDNA clones are combined into one full-length clone in a plasmid having a T7 promoter upstream of the 5′ end of the SVV sequence. The full-length genomic sequence of SVV is listed in
The methodology was as follows. RNA Isolation: SVV genomic RNA was extracted using guanidium thiocyanate and a phenol extraction method using Trizol (Invitrogen). Briefly, 250 μl of the purified SVV (˜3×1012 virus particles) was mixed with 3 volumes of Trizol and 240 μl of chloroform. The aqueous phase containing RNA was precipitated with 600 μl isopropanol. The RNA pellet was washed twice with 70% ethanol, dried and dissolved in sterile DEPC-treated water. The quantity of RNA extracted can be estimated by optical density measurements at 260 nm. An aliquot of RNA can be resolved through a 1.25% denaturing agarose gel (Cambrex Bio Sciences Rockland Inc., Rockland, Me. USA) and the band visualized by ethidium bromide staining and photographed.
cDNA synthesis: cDNA of the SVV genome was synthesized by RT-PCR. Synthesis of cDNA was performed under standard conditions using 1 μg of RNA, AMV reverse transcriptase, and oligo-dT primers. Random 14-mer oligonucleotide can also be used. Fragments of the cDNA were amplified and cloned into the plasmid pGEM-3Z (Promega) and the clones were sequenced. The sequence at the 5′ end of the viral genome was cloned by RACE and the sequence determined. Sequence data was compiled to generate the complete genome sequence of SVV.
Cloning of full length genome: Three cDNA fragments representing the full-length SVV genome were amplified by three PCR reactions employing six sets of SVV-specific primers. Turbo pfu polymerase (Stratagene) was used in PCR reactions. First, a fragment representing the 5′ end of SVV genome was amplified with primers 5′SVV-A (SEQ ID NO:219) and SVV1029RT-RI (SEQ ID NO:220) and the resulting fragment was cut with ApaI and EcoRI and gel purified. The gel purified fragment was ligated to Nde-ApaT7SVV (SEQ ID NO:221), an annealed oligo duplex containing engineered Nde1 site at 5′ end, T7 core promoter sequence in the middle and first 17 nucleotides of SVV with ready to use Apa1 site at 3′ end and cloned into Nde I and Eco RI sites of pGEM-3Z (Promega) by three-way ligation to generate pNTX-03. Second, a fragment representing 3′ end of viral genome was amplified by PCR with primers SVV6056 (SEQ ID NO:222) and SVV7309NsiB (SEQ ID NO:223). The antisense primer, SVV7309NsiB was used to introduced poly(A) tail of 30 nucleotides in length and Nsi I recognition sequence at 3′ end to clone into PstI site of pGEM-3Z plasmid. The resulting PCR product was digested with BamHI and gel purified. A fragment covering the internal part of the viral genome was amplified with primers SVV911L (SEQ ID NO:224) and SVV6157R (SEQ ID NO:225). The resulting PCR product was cut with EcoRI and BamHI and gel purified. The two gel purified fragments representing the middle and 3′end of SVV genome were cloned into EcoRI and SmaI sites of pGEM-4Z by three-way ligation to generate pNTX-02. To generate full-length SVV cDNA, pNTX-02 was digested with EcoRI and NsiI and the resulting 6.3 kb fragment was gel purified cloned into EcoRI and PstI sites of pNTX-03. The resulting full-length plasmid was called pNTX-04.
The full-length plasmid pNTX-04 was further modified at both 5′ and 3′ ends to facilitate in vitro transcription and rescuing of the virus following RNA transfection into PER.C6 cells. First, a SwaI restriction enzyme site was inserted immediately downstream of the poly(A) tail to liberate the 3′ end of SVV-cDNA from the plasmid backbone prior to in vitro transcription. A PCR approach was used to insert the site utilizing a primer pair of SVV6056 (SEQ ID NO:222) and SVVSwaRev (SEQ ID NO:226) and pNTX-04 as template. The antisense primer SVV3SwaRev contained 58 nucleotides representing the 3′end of the SVV sequence and recognition sequences for SwaI and SphI restriction enzyme sites. The resulting PCR fragment was digested with BamHI and SphI and used to replace the corresponding fragment from pNTX-04 to generate pNTX-06. Second, an extra four nucleotides present between the T7 promoter transcription start site and 5′end of SVV cDNA in pNTX-06 were removed using annealed oligo duplex approach. The duplex nucleotides were engineered to contain KpnI recognition site, T7 core promoter sequence and the first 17 nucleotides of SVV with a ready to use Apa1 site at the 3′end (SEQ ID NO:227). The annealed oligos were used to replace the corresponding portion of pNTX-06 using KpnI and ApaI sites to generate pNTX-07. Finally, a two base pair deletion noticed in the polymerase encoding region of pNTX-07 was restored by replacing BamHI and SphI fragment with a corresponding fragment amplified from SVV cDNA by PCR to generate pNTX-09.
In vitro transcription: Infectivity of in vitro transcribed RNA was tested by first digesting pNTX-09 with SwaI to liberate 3′end of SVV sequence from plasmid backbone. The linearized plasmid was subjected to in vitro transcription using T7 polymerase (Promega).
Transfection of in vitro transcribed RNA into PER.C6 cells: One day prior to transfection, PER.C6 cells were plated in 6-well tissue-culture dishes. On the next day, Lipofetamine reagent (Invitrogen) was used to transfect in vitro transcribed RNA (1.5 μg) into the cells following the recommendations of the supplier. Cytopathic effect (CPE) due to virus production was noticed within 36 hour post-transfection. The transfected cells were subjected to three cycles of freeze-thaw and the viruses in lysate were further confirmed by infecting PER.C6 cells. Thus, the full-length SVV cDNA clone proved to be infectious.
As described above, the plasmid with the full-length genome of SVV can be reverse-transcribed following standard protocols. The viral RNA (100 ng) can be used to transfect any cell line known to be permissive for the native SVV, but the most efficient cell line for viral RNA transfection can be empirically determined among a variety of cell lines.
To find the optimal insertion position for the construction of SVV capsid mutants generated with random with oligonucleotides encoding random peptide sequences, a simple model system (RGD) is employed. RGD (arginine, glycine, aspartic acid) is a short peptide ligand that binds to integrins. A successful RGD-SVV derivative should contain the following characteristics: (1) the genetic insertion should not alter any of SVV's unique and desirable properties; and (2) a successful RGD derivative virus should have tropism toward αVβ5 integrin containing cells.
A SVV plasmid containing just the contiguous capsid region will be singly cleaved at random positions and a short model peptide sequence, referred to as RGD, will be inserted at each position. The virus SVV-RGD library will be constructed from this plasmid library utilizing the general technology described in
Random insertion of the cRGD oligonucleotide into the capsid region is conducted. In brief, a plasmid is constructed that just encodes the contiguous 2.1 Kb capsid region of SVV (see
The restriction enzyme CviJI has several advantages over other random cleavage methods such as sonication or shearing. First, as CviJI is a blunt ended cutter no repair is necessary. Second, CviJI has been demonstrated to cleave at random locations such that no hot spots will occur. The procedure is also simple and rapid. Briefly, the concentration of CviJI and/or time of digestion are increasingly lowered until the majority of cleaved DNA is a linearized plasmid, i.e. a single cleavage. This can be observed by standard agarose gel electrophoresis as depicted in
Another method that may be utilized to randomally cleave DNA is the endonuclease V method (Kiyazaki, K., Nucleic Acids Res., 2002, 30(24): e139). Endonuclease V nicks uracil-containing DNA at the second or third phosphodiester bond 3′ to uracil sites. This method is also expected to randomly cleave DNA, the frequency is simply determined by adjusting the concentration of dUTP in the polymerase chain reaction. Although the cleavage sites are always two or three bases downstream of a thymidine (substituted by uracil) site, this method is expected to produce much fewer hot and cold spots than other methodologies.
The randomly linearized plasmids are ligated with the cRGD oligonucleotides. The resultant pSVV capsid library is then amplified, such that a population of polynucleotides encoding the capsid region with randomly inserted cRGD regions can be purified (see
In vitro selection of the RGD-displaying SVV library. The SVV-RGD library is screened to determine which insertion site enabled an expanded tropism of SVV. The RGD-SVV library is allowed to infect αVβ5 integrin-expressing NSCLC lines (non-small cell lung cancer cell lines, i.e., A549 expressing αVβ5). Only those SVV derivatives that contain a functional and properly displayed RGD motif can infect these cells and replicate.
In vitro screening is carried out by a high throughput automation system (TECAN) that is capable of liquid handling, concurrent incubation of 20 cell lines and measurement in 384-well plates (see
Analysis of recovered RGD-SVV derivatives. A small pool of individual RGD-displaying SVV derivatives (about 10-50 different derivataives) are analyzed. The viral mixture is diluted and single viral particles are expanded for analysis. Each derivative is tested to determine whether they have gained the ability to infect αVβ5-expressing cells efficiently and specifically. The capsid region of each derivative with this property is then be sequenced to determine the site of RGD insertion. The recovered cRGD-displaying SVV derivatives should possess the following properties: (1) the original properties of the virus are still intact; and (2) the derivatives have gained the ability to infect cells that express high levels of integrins that bind to RGD. This approach aims to identify one or more sites that enable an expanded tropism with RGD insertion, such that random oligonucleotides can be inserted into these sites to generate SVV derivatives with altered tropism.
The sequenced cRGD-SVV derivatives are numbered and ranked by their binding abilities to integrin. To test the binding activity, recombinant β2 integrin is immobilized on a 96-well microtiter plate in PBS, washed twice with PBS, blocked with 3% BSA in PBS, and then incubated with a unique RGD-displaying virus. The native virus without peptide insertions is used as a negative control. After 1-5 hr of incubation, the wells are washed at least three times with PBS. Then, the viruses that are bound to the plate will be detected by anti-SVV antibodies. RGD peptide or antibodies against integrin should be able to compete with the binding of the RGD-SVV derivatives to the integrin-bound plate.
The cRGD-SVV derivatives (20) that have the strongest binding to integrin are analyzed to determine the ‘successful’ location(s) of cRGD oligonucleotide insertion. The insertion sites provide insights into the tropism of SVV. Based on the analysis of the insertion sites and other known structures, an ideal location to place a random peptide library can be determined (this method is an alternative method for generating SVV derivatives, where oligonucleotides (known sequence or random sequence) are inserted into random locations in the capsid). SVV derivatives generated with random sequence oligonucleotides are constructed in essentially the same manner as described above for the RGD-SVV library, except for two additional and novel methodologies. To avoid unwanted stop codons and deleterious amino acid insertions (e.g. cysteines or prolines) within a desired coding region, TRIM (trinucleotide-mutagenesis) technology developed by Morphosys (Munich, Germany) can be used to generate random oligonucleotides for capsid insertion. TRIM utilizes trinucleotides which only code for amino acids at the desired position (Virnekas, B. et al., Nucleic Acids Res, 1994, 22(25): 5600-5607). The random-peptide displaying SVV with a diversity of 108 is believed to be sufficient and expected to yield peptides that specifically direct the virus to targeted tumor tissues. Random-peptide displaying SVV is tested in vitro as described above, or in vivo using tumor-bearing mice.
Pigs are a permissive host for the USDA virus isolates identified above. The isolate MN 88-36695 was inoculated into a gnotobiotic pig and antisera generated (GP102). The antisera binds to all of the other USDA isolates listed above and to SVV. The antisera does not react with 24 common porcine virus pathogens indicating its specificity. Porcine sera was also tested for neutralizing antibodies to 1278 (Plum Island virus). Sera were collected in the US and 8/29 sera were positive with titers ranging from 1:57 to 1:36,500.
To test whether the pig is the natural source for SVV, serum samples from various animals were obtained and tested for their ability to act as neutralizing antibodies against SVV infection of permissive cells. The Serum Neutralization Assay is conducted as follows: (1) Dilute various serums 1:2 and 1:4 and serially in increasing dilutions if necessary; (2) Mix with 100 TCID50 of virus (SVV; but any virus can be tested to determine whether a serum can neutralize its infection); (3) Incubate at 37° C. for 1 hour; (4) Add the mixture to 1×104 PER.C6® cells (or other permissive cell type); (5) Incubate at 37° C. for 3 days; and (6) Measure CPE using a tetrazolium based dye cytotoxicity (such as MTS) assay. The neutralization titer is defined as the highest dilution of sera that neutralizes SVV (or other virus in question) at 100%.
The serum neutralization results showed that there is a minimal or no presence of neutralizing antibodies in human and primate populations. In one experiment, 0/22 human sera contained neutralizing antibodies to SVV. In another experiment, only 1/28 human sera contained neutralizing antibodies. In a third experiment, 0/50 human sera from Amish farmers were neutralizing. In another experiment, 0/52 primate sera from four species were neutralizing.
The serum neutralization results showed that there is a prevalence of SVV neutralizing antibodies in farm animal populations. In one experiment, 27/71 porcine sera from farms were neutralizing. In another experiment, 4/30 porcine sera from a disease-free farm were neutralizing. In another experiment, 10/50 bovine sera were neutralizing. In yet another experiment, 5/35 wild mouse sera were neutralizing.
Antisera to MN 88-36694 were tested in serum neutralization assays on SVV (see Example 2). Anti-MN 88-36695 gnotobiotic pig serum was able to neutralize infection by SVV (neutralization titer on infection was 1:100 for SVV). As stated above, the antisera binds to all of the other USDA isolates and to SVV, indicating that the herein disclosed USDA isolates are SVV-like picornaviruses due to their serological cross-reactivity with the gnotobiotic pig serum as measured in an indirect immunofluorescence assay.
These data indicate that SVV is genetically and serologically linked to the porcine USDA virus isolates.
The grouping of the following isolates: MN 88-36695, NC 88-23626, IA 89-47552, NJ 90-10324, IL 92-48963, CA 131395; LA 1278; IL 66289; IL 94-9356; MN/GA 99-29256; MN 99197; and SC 363649, was deduced in part from indirect immunofluorescence experiments. Antisera GP102 was raised against isolate MN 88-36695 by inoculation of the virus into a gnotobiotic pig. The antisera binds to all twelve isolates demonstrating that they are serologically related to one another.
The GP102 antisera was tested in a neutralization assay with SVV. In this assay, serial dilutions of antisera are mixed with a known quantity of SVV (100 TCID50). The mixtures are placed at 37° C. for 1 hour. An aliquot of the mixture is then added to 1×104 PER.C6® cells, or another cell line that is also permissive for SVV, and the mixtures are placed at 37° C. for 3 days. The wells are then checked for a cytopathic effect of the virus (CPE). If the serum contains neutralizing antibodies, it would neutralize the virus and inhibit the infection of the PER.C6® cells by the virus. CPE is measured quantitatively by using a tetrazolium based dye reagent that changes absorbance based on the number of live cells present. The results are expressed as the percent of viable cells of an uninfected control vs. the log dilution of serum, and are shown in
Additionally, the viral lysate of MN 88-36695 was tested in cytotoxicity assays with four different cell lines and the results are shown in Table 4. The permissivity profile is identical to that of SVV in that NCI-H446 and HEK293 are permissive for SVV, and NCI-H460 and S8 are not. Additionally, MN 88-36695, like SVV, was cytotoxic to PER.C6® cells. Further, polyclonal antisera to SVV raised in mice was used in a neutralization assay along with MN 88-36695 virus. The results are shown in
Partial genomic sequence analysis of several of the USDA isolates revealed that they are all very closely related to SVV (see
Further sequencing of parts of the P1 (
This application is a continuation-in-part of International Application No. PCT/US2004/031504 (International Publication No. WO 2005/030139), which was filed on Sep. 23, 2004, which claims priority to U.S. Ser. No. 60/506,182, which was filed on Sep. 26, 2003. This application also claims priority to U.S. Ser. No. 60/664,442, which was filed on Mar. 23, 2005 and U.S. Ser. No. 60/726,313, which was filed on Oct. 13, 2005. These applications are hereby incorporated by reference in their entirety.
| Number | Name | Date | Kind |
|---|---|---|---|
| 5708157 | Jacobs et al. | Jan 1998 | A |
| Number | Date | Country |
|---|---|---|
| WO-9908692 | Feb 1999 | WO |
| 2005030139 | Apr 2005 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20060159659 A1 | Jul 2006 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60506182 | Sep 2003 | US | |
| 60664442 | Mar 2005 | US | |
| 60726313 | Oct 2005 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2004/031504 | Sep 2004 | US |
| Child | 11335891 | US |
