WO2008112891A2 - Monoclonal antibody that recognizes a seneca valley virus (svv) cellular receptor and uses thereof - Google Patents

Monoclonal antibody that recognizes a seneca valley virus (svv) cellular receptor and uses thereof Download PDF

Info

Publication number
WO2008112891A2
WO2008112891A2 PCT/US2008/056852 US2008056852W WO2008112891A2 WO 2008112891 A2 WO2008112891 A2 WO 2008112891A2 US 2008056852 W US2008056852 W US 2008056852W WO 2008112891 A2 WO2008112891 A2 WO 2008112891A2
Authority
WO
WIPO (PCT)
Prior art keywords
svv
cell
antibody
monoclonal antibody
mmab
Prior art date
Application number
PCT/US2008/056852
Other languages
French (fr)
Other versions
WO2008112891A3 (en
Inventor
Paul Hallenbeck
Seshidhar Reddy Police
Laura M. Hales
Kevin Burroughs
Original Assignee
Neotropix, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Neotropix, Inc. filed Critical Neotropix, Inc.
Publication of WO2008112891A2 publication Critical patent/WO2008112891A2/en
Publication of WO2008112891A3 publication Critical patent/WO2008112891A3/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1009—Picornaviridae, e.g. hepatitis A virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • Seneca Valley virus is a picornavirus that can selectively kill some types of tumors in humans but is not cytotoxic to normal human cells (see International Application No. PCT/US2004/031504 and U.S. Serial No. 60/506,182, both of which are hereby incorporated by reference in their entireties, including SVV nucleotide and amino acid sequences obtained from the SVV isolate ATCC Patent Deposit Number PTA-5343).
  • 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).
  • the invention relates in part to compositions and methods stemming from the understanding that SVV entry into cells is a receptor (or co-receptor)-mediated process, and that such a receptor may confer cell-type tropism of SVV.
  • the invention provides discovery that CXCR4 is a cellular receptor for SVV, and that at least some cells normally non-permissive for SVV infection can be made permissive by expressing or overexpressing CXCR4.
  • CXCR4 is expressed on a wide-variety of cell-types, different cell-types express different CXCR4 isoforms, different sets of CXCR4 isoforms, and/or different protein levels of particular CXCR4 isoforms.
  • cell-type tropism of SVV may result from (1) expression of a particular CXCR4 isoform(s), and/or (2) overexpression of a particular CXCR4 isoform(s).
  • the invention further provides the discovery that a monoclonal antibody raised against cell surface proteins expressed on an SVV permissive cell can reduce SVV yield in a dose dependent manner.
  • the antigen to which the monoclonal antibody binds represents a candidate for an SVV cellular receptor.
  • the invention provides SVV receptors which may confer cell-type tropism of SVV.
  • the SVV receptors, as well as the monoclonal antibody, provided by the invention can be used, for example, as biomarkers for prescreening and stratification of patients.
  • the invention provides a hybridoma cell line, designated Anti-H446 MMAb 8G10 and having ATCC Accession No. , that produces a monoclonal antibody of the invention was deposited with the Patent Depository of the American Type Culture Collection (ATCC),
  • the invention provides a hybridoma cell line, designated Anti-H82 MMAb 17El 1 and having ATCC Accession No. , that produces a monoclonal antibody of the invention was deposited with the Patent Depository of the American Type Culture Collection (ATCC),
  • the invention provides monoclonal antibodies.
  • a monoclonal antibody is produced by the hybridoma cell designated Anti-H446 MMAb 8G10 deposited as ATCC
  • a monoclonal antibody is produced by the hybridoma cell designated Anti-H82 MMAb 17El 1 deposited as ATCC Accession No. .
  • a monoclonal antibody is provided, which specifically binds and forms a complex with an antigen located on the surface of a tumor cell and thereby inhibits SVV entry into the cell, the antigen being an antigen to which a monoclonal antibody produced by a hybridoma cell designated Anti-H446 MMAb 8G10 deposited as ATCC Accession No. specifically binds.
  • a monoclonal antibody which specifically binds and forms a complex with an antigen located on the surface of a tumor cell and thereby inhibits SVV entry into the cell, the antigen being an antigen to which a monoclonal antibody produced by a hybridoma cell Anti-H82 MMAb 17El 1 deposited as ATCC Accession No. specifically binds.
  • an antibody fragment is provided consisting essentially of an antigen-binding domain of the monoclonal antibody of any one the disclosed monoclonal antibodies.
  • the antibody is an chimeric antibody.
  • the antibody is a humanized monoclonal antibody.
  • the antibody is a murine monoclonal antibody.
  • the antibody is a human monoclonal antibody.
  • the antibody, or an antigen-binding fragment thereof comprises a detectable label.
  • the antibody, or an antigen-binding fragment thereof is linked to a moiety capable of producing a detectable signal.
  • a method for determining whether SVV will enter a tumor cell comprising: (a) contacting a tumor cell with a monoclonal antibody, or an antigen- binding fragment thereof, produced by (i) a hybridoma cell designated Anti-H446 MMAb
  • the tumor cell is contained within a tumor or a tumor sample.
  • the method is carried out in vitro.
  • the method is carried out in vivo.
  • the determining comprises direct detection of the monoclonal antibody.
  • the determining comprises indirect detection of the monoclonal antibody.
  • the determining comprises detecting a chromogenic signal, a fluorescent signal, a luminogenic signal or a radioactive signal.
  • a method for determining whether a subject with a tumor will respond to SVV treatment comprising: (a) contacting the tumor with a monoclonal antibody, or an antigen-binding fragment thereof, produced by (i) a hybridoma cell designated Anti-
  • the contacting is in vitro.
  • a method for delivering a compound to a tumor cell in a subject comprising (a) linking the compound to an antibody produced by (i) a hybridoma cell designated Anti-H446 MMAb 8G10 deposited as ATCC Accession No. , or (ii) a hybridoma cell designated Anti-H82 MMAb 17El 1 deposited as ATCC Accession No. , or a fragment thereof; and (b) administering the antibody to the subject.
  • the linking is via a covalent bond.
  • a kit for determining whether SVV will enter a tumor cell, the kit comprising: (a) a monoclonal antibody, or an antigen-binding fragment thereof, produced by (i) a hybridoma cell designated Anti-H446 MMAb 8G10 deposited as ATCC Accession No. , or (ii) a hybridoma cell designated Anti-H82 MMAb 17El 1 deposited as ATCC
  • kits further comprises at least one positive control cell sample to which the monoclonal antibody or fragment thereof binds.
  • the kit further comprises instructions for preparing a tumor sample.
  • the monoclonal antibody or the fragment thereof is linked to a detectable signal or a moiety capable of producing a detectable signal.
  • a method for identifying stem cells comprising: (a) contacting a cell with the monoclonal antibody, or an antigen-binding fragment thereof, produced by (i) a hybridoma cell designated Anti-H446 MMAb 8G10 deposited as ATCC
  • An antigen is provided that is specifically bound by the monoclonal antibody, or an antigen-binding fragment thereof, produced by (i) a hybridoma cell designated Anti-H446
  • MMAb 8G10 deposited as ATCC Accession No. , or (ii) a hybridoma cell designated
  • a pharmaceutical composition comprising a disclosed monoclonal antibody, and a pharmaceutically acceptable carrier.
  • a method for treating cancer in a subject comprising administering to a subject an effective amount of a monoclonal antibody which specifically binds and forms a complex with an antigen located on the surface of a tumor cell and thereby inhibits SVV entry into the cell, the antigen being an antigen to which a monoclonal antibody produced by (i) a hybridoma cell designated Anti-H446 MMAb 8G10 deposited as ATCC
  • the antibody is a monoclonal antibody produced by (i) a hybridoma cell designated Anti-H446 MMAb 8G10 deposited as ATCC Accession No. , or (ii) a hybridoma cell designated Anti-H82 MMAb
  • the antibody is a chimeric antibody.
  • the antibody is a humanized monoclonal antibody.
  • the antibody comprises a human constant region and a heavy and light chain variable region, wherein the heavy and light chain variable region comprises heavy and light chain framework regions and heavy and light chain complementarity determining regions (CDRs), at least a portion of the heavy and light chain framework regions being derived from a human antibody, and the CDRs comprising heavy- chain CDRs light-chain CDRs derived from a monoclonal antibody produced by (i) a hybridoma cell designated Anti-H446 MMAb 8G10 deposited as ATCC Accession No. , or (ii) a hybridoma cell designated Anti-H82 MMAb 17El 1 deposited as ATCC Accession No. .
  • CDRs complementarity determining regions
  • Figures IA - IH The genomic sequence (SEQ ID NO:1) and encoded polyprotein sequence (SEQ ID NO:2) of SVV. Specific features of the SVV genomic sequence, such as specific coding regions for proteins cleaved from the polyprotein sequence are described herein.
  • the SVV genome sequence was obtained from the SVV isolate that has been deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard., Manassas, Virginia, 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.
  • ATCC American Type Culture Collection
  • Figure 4 An analysis of the epidemiology of SVV.
  • SVV is a unique virus, phylogenetically similar to Cardioviruses, but classified in a separate tree.
  • Figure 5 The protein products made from proteolytic processing of a picornavirus polyprotein.
  • Figure 6 A screening assay that tests the ability of cDNAs to confer SVV permissiveness on cell lines originally non-permissive for SVV infection.
  • cDNAs can be transfected en masse to non- permissive cells in culture prior to aliquoting the cells onto a multi-well plate.
  • Figure 7 Results of an expression cloning experiment to determine whether the expression of a particular cellular receptor in the non-permissive cell-line Hep3b confers SVV permissiveness to the cells. As shown in the Figure, only CXCR4 conferred permissiveness to the same degree as the positive control (the permissive cell line PER.C6). The SVV cellular receptor candidates were identified based upon the results of differential gene expression profiles of non-permissive and permissive cell lines.
  • Figures 8A - 8B The anti-CXCR4 antibody 12G5 can inhibit SVV infection.
  • Figure 9 The amino acid sequence of human CXCR4 (SEQ ID NO:3; GenBank Accession No. P61073). Residues 1-39, 100-110, 176-200 and 262-285 are extracellular domain residues (i.e., the approximate residues for the four extracellular domains). Potential N-linked glycosylation sites at least include positions 11-13 and 176-178. At least residues 2-9, 7, 11, 12, 14, 15, 18, 20, 21, 176, 187 and 268 may play a role in SDF-I ⁇ binding.
  • Figures 1OA - 1OB Serum collected from mice following a second injection ( Figure 10A) or a fourth injection ( Figure 10B) of NCI-H446 cells was screened with a virus blocking assay to determine the presence of antibodies specific for cell surface proteins as described in Example 3. Mice were identified as numbers 2-1 through 2-14. Serum was added to the cells at a dilution of 1 :4 or 1 :8. Gray boxes represent wells in which a cytopathic effect (CPE) was observed. White boxes represent wells in which no CPE was observed.
  • CPE cytopathic effect
  • Figure 11 Generation of hybridoma clones and screening for virus entry blocking monoclonal antibodies. Mice were immunized with H446 cells or membrane extracts of H82, and spleenocytes from the mice were used for fusion with myeloma cells to generate hybridomas which were selected in the presence of HAT medium. Hybridomas secreting antibody which blocked viral infection were selected and subjected to single cell cloning.
  • Figures 12A - 12B Supernatants of selected hybridoma clones were tested in duplicate in a virus blocking assay as described in Example 3. For each positive hybridoma clone, ten different single cell clones were tested in a virus blocking assay for the presence of monoclonal antibodies that block virus entry into NCI-H446 cells. Dark blue wells indicate that viral replication was blocked, and indicate the presence in the supernatant of monoclonal antibodies that block SVV entry into NCI-H446 cells.
  • Figures 13A - 13B Supernatant from hybridoma clone 8G10 was incubated with NCI-H446 cells, then SVV-GFP (200 PPC) was added to the cells to determine if the monoclonal antibody in the supernatant blocked virus entry into the cells. Cells were analyzed by fluorescence microscopy.
  • Figure 13A shows virus uptake in NCI-H446 cells in the absence of monoclonal antibody.
  • Figure 13B shows a significant reduction in virus uptake in the presence of 8G10 monoclonal antibody.
  • Figures 14A - 14D Monoclonal antibody purified from the supernatant of hybridoma clone 8G10 was incubated with PER.C6 cells, then SVV-GFP (200 PPC) was added to the cells to determine if the mAb blocked SVV-GFP entry into the cells. Fluorescence microscopy images ( Figures 14A - 14B) show a reduction in SVV-GFP uptake in the presence of 8G10 mAb. Fig. 14C and Fig. 14D.
  • Figures 15A - 15C Proteins were octyl D-glucoside extracted from membranes of NCI-H446 and PER.C6 cells.
  • Figure 15A shows an SDS-PAGE analysis of the extracted membrane proteins (50 ⁇ g/lane). The SDS-PAGE gel was stained with Coomassie blue.
  • Figure 15B is a Western blot analysis of the membrane proteins (20 ⁇ g/lane) with 8G10 mAb (IgM isotype; 1 :10 dilution).
  • Figure 15C NCI-H446 membrane proteins (25 ⁇ g/lane) were analyzed by Western blot with monoclonal antibodies (IgM isotype, 1 :10 dilution) produced by several different hybridoma clones.
  • Figures 16A - 16C Figure 16A, Immunoprecipitation of proteins recognized by 17El 1. Immunoprecipitation of H446 proteins using the monoclonal antibody 17El 1. The arrow points to the specific protein immunoprecipitated by 17El 1.
  • Figure 16B Immunoprecipitation of proteins recognized by 8G10. Immunoprecipitation of S 35 labeled H446 proteins using the monoclonal antibody 8G10. Decay accelerating factor was used as positive control in immunoprecipitation reactions.
  • Figure 16C Two-dimensional gel electrophoresis and Western blot analysis.
  • Figure 17 Analysis of concentrated 8G10 mAb produced by the cell line flask system. Samples from the first cell line flask (lanes 1 (1 ⁇ .1 sample), 2 (2 ⁇ l sample) and 3 (3 ⁇ l sample)) and the second cell line flask (lanes 4 (1 ⁇ l sample), 5 (2 ⁇ l sample) and 6 (4 ⁇ l sample)) were compared to an IgM isotype control (lanes 7 (1 ⁇ l control), 8 (2 ⁇ l control) and 9 (4 ⁇ l control)).
  • Figures 18A - 18L To determine the cellular location of the 8G10 antigen, SVV permissive cells (NCI-H446 and PER.C6) and SVV non-permissive cells (NCI-H460 and Hep3B) were analyzed by immunofluorescence staining as described in Example 6. Cells were analyzed under non-permeabilizing ( Figures 18A - 18D) or permeabilizing ( Figures 18E - 18H) conditions. Cell nuclei are stained with DAPI (blue) and the red fluorescence indicates the location of the bound 8G10 mAb. Cells were also stained with a control IgM isotype antibody. The cells were visualized under confocal microscopy.
  • FIG. 8G10 shows distinct surface staining of permissive cells, but not non-permissive cells.
  • Figures 181 - 18L Immunofluorescence for detection of protein recognized by 17El 1. Cells were stained with 17El 1 as well as a control IgM isotype antibody (data not shown). 17El 1 gave surface staining as well as cytoplasmic staining of permissive cells, but not non-permissive cells.
  • Figure 19 Results of a dose-response study to determine SVV-OOl -mediated killing of NCI-H446 cells in the absence (triangles) and presence (squares) of 8G10 mAb. See Example 5 for details.
  • Figures 2OA - 2OB 8G10 treatment lowers yields of SVV-OOl .
  • the cells were treated with 8G10 at 25, 75 ⁇ g/mL or isotype control antibody (75 ⁇ g/mL) and incubated at room temperature for one 40 minutes.
  • the cells were then infected with SVV-OOl at 1 virus particle/cell and incubated at 37°C for one hour.
  • the cells were washed three times and further incubated for 14 hours.
  • the virus yields were assayed on PER.C6 cells and shown as total yields (TCID50/mL- Figure 20A) and percent of IgM isotype control (Figure 20B).
  • Figures 21A - 21B Figure 21A, 8G10 treatment lowers yields of SVV-OOl.
  • H446 cells were treated with 8G10 at varying amounts and infected with 1 or 10 virus particles/cell. The cells were washed three times and further incubated for 14 hours. The virus yields were assayed on PER.C6 cells and shown as percent of IgM isotype control.
  • Figure 21B Virus yield assays in the presence or absence of 17El 1 and 8G10. Shown is a representative competition experiment with 8G10 and 17El 1 blocking viral production on H446 cells.
  • Figure 22 Immunohistochemistry staining of xenografts of SVV permissive (H446) and non-permissive (H460) cells shows that permissive cells are infected by SVV and express 8G10 antigen and integrin ⁇ 4, while non-permissive cells are not infected by SVV and do not express detectable levels of 8G10 antigen or integrin ⁇ 4.
  • Figure 23 Immunohistochemistry analysis of cell markers. All markers co-stain the same cells on an invasive edge of a xenograft tumor from a permissive cell line. Ki-67 is expressed in proliferating cells. NCAM; neuronal cell adhesion molecule.
  • Figure 24 Results of immunohistochemistry analysis conducted with the 8G10 mAb on human SCLC tissue microarrays.
  • the 8G10 mAb strongly stained SCLC tissue (+++; upper panels), with no staining of the control tissue (-; lower panels).
  • Figure 25 Schematic of a proposed mechanism of receptor-mediated binding of SVV to permissive cells.
  • Figures 26 A - 26B Figure 26 A depicts the viral titer of SVV as detected by RT- PCR in the serum of four patients who received an intravenous infusion of SVV.
  • Figure 26B depicts the titer of anti-SVV neutralizing antibodies generated in each patient.
  • Figures 27A - 27B SVV replication is tumor-selective and continues in the presence of immune response.
  • Figure 27A depicts immunohistochemistry analysis of tissue samples obtained from one patient. The metastatic tumor cells stained positive for viral capsid, while surrounding normal tissue was not stained.
  • Figure 27B depicts the serum levels of SVV and neutralizing antibodies present in the patient of Figure 27A.
  • Figure 28 Kinetics of Seneca Valley Virus (SVV-OOl) in serum of A/J mice following intravenous injection.
  • SVV-OOl was injected intravenously at 10 9 vp/kg into female A/J mice.
  • Figures 29A - 29B Following a 10 9 vp/kg intravenous bolus dose of SVV, SVV concentration was measured in the tissues of tumor-bearing mice ( Figure 29A) and normal mice (no tumor) ( Figure 29B). SVV replication is tumor selective.
  • Figure 30 Immunohistochemistry analysis of SVV replication in tissues from a tumor-bearing mouse. Positive staining for SVV replication can be seen in the tumor sample; staining is not detected in other tissues.
  • Figures 31A - 31B Figure 31A shows the increasing titer of anti-SVV neutralizing antibodies, indicating an immune response in the mouse.
  • Figure 3 IB show the concentration of SVV in mouse tissues. SVV replicates in tumor cells despite the immune response.
  • Figures 32A - 32B Figure 32A, In vivo efficacy of Seneca Valley Virus-001 (SVV-OOl) following systemic administration.
  • SVV-OOl Seneca Valley Virus-001
  • a single intravenous injection of saline vehicle or SVV-OOl at 10 7 or 10 8 vp/kg (virus particles per kg) was given to athymic mice bearing pre-established human small-cell lung H446 xenograft tumors (n 10 per group) on study day 1 (denoted by ⁇ ). Mean tumor volumes and 95% confidence intervals are plotted as a function of time.
  • Figure 32B Body weight change in A/J mice following intravenous injection of Seneca Valley Virus (SVV-OOl).
  • Figure 33 Infectivity (IFT)-based TCID50 assay was used to monitor viral load in serum of patients with solid tumors expressing at least one neuroendocrine marker.
  • IFT Infectivity
  • Figure 34 Quantitative, real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was used to monitor viral load in serum of patients with solid tumors expressing at least one neuroendocrine marker.
  • RT-PCR real-time reverse transcriptase-polymerase chain reaction
  • Figure 35 Viral neutralization in serum of patients with solid tumors expressing at least one neuroendocrine matker.
  • Figure 36 In one exemplary patient, serum RNA is degraded at later time points, indicating lack of full-length circulating viral RNA at later time points.
  • Figure 37 Anti-SVV immunohistochemistry in patient tissue shows specific anti- SVV staining in a liver metastasis, but not in surrounding normal liver tissue or in a panel of normal organs.
  • the invention provides for the discovery that a monoclonal antibody, raised against surface proteins of an SVV permissive cell, blocks SVV infection of permissive cells.
  • the invention further provides for the discovery that the antigen of the monoclonal antibody functions as a cellular receptor (or co-receptor) for SVV.
  • the invention further provides that upon identification of SVV receptors as tumor markers (i.e., where an SVV receptor is expressed or over-expressed in a tumor cell-type as compared to a normal cell of that cell-type (the normal cell either does not express the SVV receptor or expresses the SVV receptor at a lower level than the tumor cell); or where an SVV receptor is only expressed or over-expressed in a particular stage of a tumor), therapeutic molecules can be generated to target the SVV receptor.
  • tumor markers i.e., where an SVV receptor is expressed or over-expressed in a tumor cell-type as compared to a normal cell of that cell-type (the normal cell either does not express the SVV receptor or expresses the SVV receptor at a lower level than the tumor cell); or where an SVV receptor is only expressed or over-expressed in a particular stage of a tumor.
  • SVV or an SVV-like picornavirus can bind to the SVV receptor that is expressed or overexpressed in a tumor cell-type as compared to the normal cell-type, then SVV or the SVV-like picornavirus can be used to target and kill the tumor. Additionally, if it is determined that SVV or an SVV-like picornavirus can bind to the SVV receptor that is expressed or overexpressed in a tumor cell-type as compared to the normal cell-type, then SVV or the SVV-like picornavirus can be used in diagnostic methods to detect the tumor in a subject.
  • SVV or components or derivatives thereof can be used in methods for inhibiting or decreasing HIV infection in a subject.
  • Cells that express CXCR4 and can be infected by HIV can also be examined to see whether these cells express or overexpress specific CXCR4 isoforms that can be bound by SVV or an SVV-like picornavirus.
  • the invention provides a hybridoma cell line, designated Anti-H446 MMAb 8G10 and having ATCC Accession No. , that produces a monoclonal antibody of the invention was deposited with the Patent Depository of the American Type Culture Collection (ATCC),
  • the invention provides a hybridoma cell line, designated Anti-H82 MMAb 17El 1 and having ATCC Accession No. , that produces a monoclonal antibody of the invention was deposited with the Patent Depository of the American Type Culture Collection (ATCC),
  • viruses are used interchangeably.
  • 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:1 and Figures 1A-1H for the SVV full-length genomic sequence), where the sequence comparison is not limited to a whole-genome analysis, but can be focused on a particular region of the genome, such as the 5'UTR, structural encoding regions, nonstructural encoding regions, and portions thereof.
  • the particular length of the genome for sequence comparison that is adequate to determine relatedness/likeness to SVV is known to one skilled in the art, and the adequate length can very with respect to the percentage of identity that is present.
  • the length for sequence comparison can be, for example, at least 20, 50, 100, 200, 300, 400, 500, 750, 1000, 1500, 2000, or 2500 nucleotides. Where the length is shorter, one skilled in the art understands, for example, that the identity between sequences can be higher in order to consider the two sequences to be related. However, such guidance is qualified at least with respect to considerations of sequence conservation, in that certain regions of the genome are more conserved than others between related species.
  • SVV-like picornaviruses can include cardioviruses.
  • 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.
  • an "attenuated virus” or a “live attenuated virus” are viral mutants that are unable to cause disease or are less able to replicate or grow in a host, but the mutants retain their antigenicity such that they can induce immunogenic protection.
  • an "inactivated” or “killed” virus is a virus that is treated such that it cannot replicate.
  • 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. et al. (1990) "Basic local alignment search tool.” J. MoL Biol. 215:403-410)) or by visual inspection.
  • the BLAST algorithm 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).
  • 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.
  • 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 difference (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.
  • SVV receptor refers to a receptor or co-receptor for SVV binding to a cell or entry into a cell or fusion into a cell.
  • a "SVV receptor” can also be referred to as a "cellular receptor for SVV” or a “cellular receptor of SVV.” Examples of SVV receptors are CXCR4 and 8G10 antigen.
  • SW Seneca Valley Virus
  • SVV is an RNA virus, and with respect to previously characterized picornaviruses, SVV is most closely related to members from the Cardiovirus genus (Scraba, D. et al., "Cardioviruses (Picornaviridae),” in Encyclopedia of Virology, 2 nd Ed., R.G. Webseter and A. Granoff, Editors, 1999) in the Picornaviridae family. The results of sequence analyses between SVV and other Cardioviruses are discussed in International Application No. PCT/US2004/031504), which is hereby incorporated by reference in its entirety.
  • FIG. 4 presents a phlyogenetic relationship tree between members of the Picornavirus family.
  • the genome of SVV consists of one single-stranded positive (+) sense strand RNA molecule having a size of about 7,290 nucleotides (see Figure 1 A-IH).
  • genomic RNA is infectious, and thus can be transfected into cells to bypass the virus-receptor binding and entry steps in the viral life cycle;
  • a long untranslated region (UTR) at the 5 ' end of the genome for SVV, nucleotides 1-666 of SEQ ID NO:1 and a shorter 3' untranslated region (for SVV, nucleotides 7210-7280 of SEQ ID NO:1);
  • the 5' UTR contains a clover-leaf secondary structure known as the internal ribosome entry site (IRES);
  • the rest of the genome encodes a single polyprotein (for SVV, nu
  • the SVV polyprotein is cleaved into a number of smaller proteins ( Figure 5).
  • DNA clones of the genome or portions thereof can be made by reverse- transcription of the SVV RNA genome.
  • the DNA clones can be subcloned into expression plasmids, such that the expression plasmids can express any one of the SVV proteins or a portion thereof.
  • expression plasmids can be generated such that more than one SVV protein can be expressed as a single larger protein.
  • the following table lists the nucleotides of SEQ ID NO:1 that encode the SVV proteins.
  • the table also lists the amino acid sequences of the SVV proteins with respect to the polyprotein sequence listed in SEQ ID NO:2.
  • the Internal Ribosome Entry Sequence (IRES) of SVV has been compared to other Picornaviruses, and it has been determined that the SVV IRES is Type IV, whereas Cardiovirus IRES's are Type II.
  • IRES Internal Ribosome Entry Sequence
  • multiple unique picornaviruses have been discovered at the USDA that are more similar to SVV than SVV is to other Cardioviruses. Therefore, these unique picornaviruses (which are SVV-like picornaviruses) and SVV will either be designated to be members of new genus of the Picornaviridae family or to be members of the genus Cardiovirus.
  • SVV-like picornaviruses discovered at the USDA are about 95-98% identical to SVV at the nucleotide level, and antisera against one virus (MN 88-36695) neutralizes SVV, and this virus is reactive to antisera that neutralizes SVV.
  • MN 88-36695 antisera against one virus
  • These SVV-like picornaviruses were isolated from pigs, and thus, pigs are likely a permissive host for SVV and other SVV-like viruses.
  • SVV-like picornaviruses isolated from pigs include, but are not limited to, 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.
  • SVV-like picornaviruses may also include cardioviruses closely related to SVV (as determined by sequence analysis or by cross- reactivity to antibodies raised against SVV antigens).
  • SW Cellular Receptors In general, cell and tissue tropism of a virus is determined by specific cell surface receptor(s), internalization or post-internalization events. For SVV, the transfection of genomic RNA into nonpermissive cells results in virus production in the cells, indicating that the tropism is likely a pre-internalization event governed by the presence of cell surface receptors (see Example 1).
  • transcription profiling was used to compare differential expression of genes encoding for cell surface proteins of permissive cell lines compared to non-permissive cell lines.
  • Candidate genes expressed in permissive cell lines but not expressed in non- permissive cell lines were expressed in non-permissive cells to determine if the gene confers permissivity to infection by SVV.
  • a cellular receptor (or co-receptor) for SVV has been identified using an expression cloning approach.
  • the cellular receptor identified is CXCR4.
  • Confirmation that CXCR4 is a receptor for SVV is shown in Example 1.
  • Differential analysis between the gene expression profiles from permissive and non-permissive cell lines indicated twelve initial receptor candidates. These twelve candidates were expressed in non-permissive cell lines to determine whether expression of the candidate could change the non-permissive cell line to become permissive to SVV infection (confer permissiveness to infection by SVV).
  • CXCR4 is expressed on a variety of different cell-types that include cell-types that are non-permissive for SVV infection, it may be the case that a specific CXCR4 isoform is responsible for SVV tumor-specific tropism and/or that there are other SVV receptor(s) (or co-receptor(s)) for SVV binding, entry, and/or fusion.
  • Cellular receptors for SVV can also be identified by using a broad expression cloning approach, where cDNAs are transfected and expressed in a non-permissive cell line to determine whether expression of the cDNA can provide permissiveness to SVV infection.
  • the cDNAs can be transfected into the non-permissive cells in the form of an expression library, where the identity (i.e., the sequence or name of the protein product encoded by the cDNA) of the cDNA are unknown prior to transfection.
  • the identity of the cDNAs are known are prior to transfection. Whether the sequence of the cDNA is known or unknown prior to transfection, transfected cells can be screened in a high-throughput manner (see Figure 6 and Example 1).
  • mouse monoclonal antibodies were raised to cell surface proteins of permissive cells as described in Example 3.
  • a monoclonal antibody provided by the invention for example, 8G10 mAb produced by a hybridoma cell having
  • Accession No. recognizes an antigen (for example, 8G10 antigen or 17El 1 antigen) that is a SVV cellular receptor (or co-receptor) as demonstrated by, for example, the ability of the mAb to block virus entry into permissive cells in a dose dependent manner (see Figure 21) and to recognize proteins on the surface of permissive cells but not non-permissive cells (see Figures 18A - 18D).
  • 8G10 antigen or 17El 1 antigen a SVV cellular receptor (or co-receptor) as demonstrated by, for example, the ability of the mAb to block virus entry into permissive cells in a dose dependent manner (see Figure 21) and to recognize proteins on the surface of permissive cells but not non-permissive cells (see Figures 18A - 18D).
  • the 8G10 and 17El 1 antigens have not yet been identified.
  • Integrins are proteins that span the cell membrane to link the extracellular space with the inside of a cell. Integrins serve as cellular receptors for other picornaviruses.
  • An amino acid motif (LDV) known to bind integrin ⁇ 4 ⁇ l is found in a surface-exposed loop of SVV VP2, a capsid protein. Virus mutated at this site could not be rescued from culture.
  • the 8G10 and 17El 1 mAbs recognize and form a complex with a surface protein on SVV permissive cells, thereby inhibiting SVV entry into the cells.
  • the surface protein functions as an SVV receptor.
  • the monoclonal antibody may be a chimeric antibody.
  • a chimeric antibody is monoclonal antibody comprising constant region fragments from a species different from the species in which the monoclonal antibody was raised.
  • the 8G10 mAb was raised in a mouse and thus is a murine mAb; a chimeric 8G10 mAb would comprise constant region fragments from a different species.
  • the monoclonal antibody may be a humanized antibody.
  • a humanized antibody is a non-human monoclonal antibody in which human protein sequences have been substituted for all of the non-human protein sequences except for the non-human complementarity determining regions (CDRs) of both the heavy and light chains (see U.S. Patent No. 5,824,307, U.S. Patent Application Publication Nos. US 2006/0228350, US 2005/0048617, US 2005/0042664).
  • human protein sequences may be substituted for all of the murine protein sequences except for the murine CDRs of both the light and heavy chains.
  • a monoclonal antibody of the invention may be used to determine whether a subject with a tumor will respond to SVV treatment.
  • the monoclonal antibody may be linked to a detectable label and used to assay a tumor biopsy sample in vitro, or administered to the subject to image the tumor in vivo.
  • a tumor is predicted to respond to SVV treatment if it is determined that the monoclonal antibody
  • a monoclonal antibody of the invention may be used in a method for delivering a compound to a tumor cell, comprising linking the compound to the antibody.
  • Non-limiting examples of such compounds include therapeutic agents, such as a radioisotope, a toxin or a chemotherapeutic agent, and imaging agents such as a fluorophore or a radioisotope.
  • the invention provides methods of treating a tumor in a subject, comprising administering to the subject an effective amount of a monoclonal antibody which specifically binds and forms a complex with an antigen located on the surface of a tumor cell and thereby inhibits SVV entry into the cell, the antigen being an antigen to which a monoclonal antibody produced by a hybridoma cell designated Anti-H446 MMAb 8G10 deposited as ATCC Accession No. , or a hybridoma cell designated Anti-H82 MMAb
  • the antibody is a monoclonal antibody produced by a hybridoma cell designated Anti-H446
  • MMAb 8G10 deposited as ATCC Accession No. , or a hybridoma cell designated Anti-
  • the antibody is a chimeric antibody. In one embodiment, the antibody is a humanized antibody. In one embodiment, the antibody comprises a human constant region and a heavy and light chain variable region, wherein the heavy and light chain variable region comprises heavy and light chain framework regions and heavy and light chain complementarity determining regions (CDRs), at least a portion of the heavy and light chain framework regions being derived from a human antibody, and the CDRs comprising heavy-chain CDRs light-chain CDRs derived from a monoclonal antibody produced by a hybridoma cell designated Anti-
  • CDRs heavy-chain CDRs light-chain CDRs
  • H446 MMAb 8G10 deposited as ATCC Accession No. , or a hybridoma cell designated
  • CXCR4 C-X-C motif receptor 4; also known as fusion and CD 184) is a seven- transmembrane (7-TM) G-protein-coupled receptor (GPCR) that interacts with the chemokine stromal cell-derived factor- 1 alpha (SDF- l ⁇ ; also known as CXCL 12).
  • GPCR G-protein-coupled receptor
  • SDF- l ⁇ chemokine stromal cell-derived factor- 1 alpha
  • CXCR4 has been shown to act as a co- receptor during infection of human cells by T-trophic HIV-I.
  • the gpl20 of T- trophic HIV-I interacts with CD4 and induces the formation of a trimolecular complex between gpl20, CD4, and CXCR4. This interaction is thought to be critical for subsequent conformational changes in the gpl20/gp41 that ultimately lead to the exposure of HIV gp41 fusion peptide and initiation of virus-cell fusion.
  • Human CXCR4 comprises 352 amino acids (see GenBank Accession No. P61703; also known as isoform 1 , isoform a, or isoform ⁇ ) and the predicted molecular weight (MW) of non-glycosylated human CXCR4 is 39,746 Daltons. The predicted MW of glycosylated human CXCR4 is about 45-47 kDa.
  • CXCR4 also called isoform 2 or isoform b
  • isoform 2 also called isoform b
  • CXCR4 There are numerous post-translation modifications of CXCR4 that can account for many of the CXCR4 isoforms. Observed post-translation modifications of CXCR4 include, but are not limited to, N-glycosylation (Chabot et al, J. Virol. (2000) 74:4404-13), disulfide formation (Chabot et al, J. Virol. (1999) 73:6598-609), tyrosine sulfation (Farzan et al, J 1 Biol. Chem.
  • CXCR4 isoforms can be identified by immunob lotting analysis of cell extracts (see Sloane, A. et al., Immunology and Cell Biology (2005) 83:129-143; which is hereby incorporated by reference in its entirety, and at least for its teachings on how to identify CXCR4 isoforms; and Example 2).
  • an immunoblot analysis can be conducted with equal amounts of cellular or membrane extracts of cell lines permissive and non- permissive to SVV infection.
  • Cell extracts can be resolved in both denaturing and non- denaturing gel systems in one and/or two dimensions. Extracts can be run on such gels and transferred to a membrane, such as nitrocellulose.
  • the nitrocellulose can then be probed with antibodies against CXCR4 in order to compare the presence and/or absence as well as changes in intensity of different molecular weight bands that indicate the different CXCR4 iso forms (see Table below).
  • the nitrocellulose can also be probed using a VOPBA (Virus Overlay Protein Binding Assay).
  • VOPBA Virus Overlay Protein Binding Assay
  • the nitrocellulose can be probed with SVV particles followed by anti-SVV antibody.
  • the extracts can also be analyzed by immunoprecipitation - extracts are mixed with SVV and potential SVV-CXCR4 isoforms co- complexes are precipitated with anti-SVV antibody.
  • the co-complexes are then resolved on a gel and transferred to a membrane (such as nitrocellulose), and the membrane is then detected using anti-CXCR4 antibodies to determine which isoform(s) were precipitated.
  • Specific molecular weight bands resolved on a gel that correspond to specific CXCR4 isoforms can be excised such that the isoforms can be used as immunogens for antibody generation, including monoclonal antibody generation.
  • the bands can also be digested with trypsin such that mass spectrometry can be used to confirm that the band is CXCR4.
  • post-translational modifications of the isoform can be characterized by using protein modification enzymes such as glycosylases.
  • Chromatographic separation can comprise the identification of protein isoforms that have been separated by means of ion-exchange, affinity, size-exclusion, or chromato focusing. Affinity interactions with lectins also offers separation possibilities.
  • the 34 kDa species is a non-glycosylated monomer.
  • the 68 kDa species is a non-glycoslyated dimer.
  • the 40, 47, 73 and 80 kDa species are glycosylated, most likely at the N-terminus ⁇ i.e., extracellular region) of CXCR4.
  • the 40 kDa species is a glycosylated monomer.
  • the 80 kDa species is a glycosylated dimer.
  • the 73 kDa species may be a dimer comprising a 40 kDa glycosylated monomer and a 34 kDa non-glycosylated monomer.
  • the MW of CXCR4 isomers that are glycosylated may vary slightly (for example, from about 1 to about 5 kDa) between cell-types because different cell-types may provide different glycosylation patterns on a protein.
  • CXCR4 isoforms of about 62 kDa, of about 75-80 kDa, and of about 95-100 kDa have also been reported. These isoforms may be CXCR4-ubiquitin complexes. A 54 kDa isoform has also been reported. The invention is not limited to the isoforms described and referred to herein.
  • the invention provides methods for identifying which
  • CXCR4 isoform(s) is capable of binding to SVV.
  • Extracts can be prepared from cells that are permissive and non-permissive for SVV infection.
  • the extracts can be whole cell extracts, or extracts of specific cell compartments, such as a membrane extract.
  • the extracts from the permissive and non-permissive cell-lines can be compared in order to determine whether there are any CXCR4 isoforms that are only present in the extracts from permissive cell-lines.
  • the extracts from the permissive and non-permissive cell-lines can be compared in order to determine whether there are any CXCR4 isoforms that are overexpressed only in permissive cell-lines, where overexpression of a CXCR4 isoform may allow or contribute to permissivity.
  • Extracts from non-permissive and permissive cell lines of a particular tumor can be compared to determine whether there are any CXCR4 isoforms that are only present or overexpressed in the extracts from permissive cell-lines of a particular tumor. For example, a comparison can be conducted between non-permissive and permissive SCLC cells (see Table below) to determine if any of the permissive SCLC cells express a CXCR4 isoform that is not expressed or underexpressed in any of the non-permissive SCLC cells.
  • Comparisons are not limited to tumor cell-types, but can also include comparisons between a permissive tumor- cell type that is metastatic (or even metastatic at a particular site) and a non-permissive tumor-cell type that is metastatic.
  • a comparison can be conducted between a permissive SCLC cell-type that is metastatic at the bone marrow (i.e., NCI-H209) and a non- permissive SCLC cell-type that is metastatic at the bone marrow (i.e, NCI-H2195). It may be the case that there are different CXCR4 isoforms expressed in different types of tumors that provide permissiveness to SVV infection.
  • Comparisons can be conducted, for example, by immunoblot assays.
  • Cells that are permissive or non-permissive for SVV infection are listed in the Table below.
  • the Table below also indicates the tumor cell-type, the state of the cancer (i.e, if metastatic), and if metastatic, the site of metastasis.
  • SCLC multidrug resistant
  • SCLC Cancer Carcinoma
  • SCLC Cancer Carcinoma
  • SCLC Cancer Carcinoma
  • PAoSMC Porcine Adult Normal, Heart, Aorta Smooth Muscle Cells 88
  • PK(15) Porcine Adult Normal, Kidney 1144, 129
  • HUVEC Human Adult Normal, Umbilical Vein Endothelial Cells >10,000
  • SCLC Carcinoma
  • SCLC Carcinoma
  • SCLC Cancer Carcinoma
  • SCLC Cancer Carcinoma
  • NSCLC Carcinoma
  • NSCLC Carcinoma
  • NSCLC Carcinoma
  • NSCLC Carcinoma
  • NSCLC Carcinoma
  • NSCLC Carcinoma
  • APL Blood, Leukemia
  • CML Cancer Leukemia
  • SCLC Carcinoma
  • PAOEC Porcine Adult Normal, Heart, Aorta Endothelial Cells >10,000
  • PCAEC Porcine Adult Normal, Heart, Coronary Endothelial Cells >10,000
  • PPAEC Porcine Adult Normal, Lung, Endothelial Cells >10,000
  • SCLC Carcinoma
  • SCLC Carcinoma
  • SCLC Cancer Carcinoma
  • SCLC Cancer Carcinoma
  • SCLC Cancer Carcinoma
  • Comparisons can be between any permissive cell and any non-permissive cell. Comparisons can also be conducted between non-permissive cell-lines that do not express CXCR4 and these cell-lines when transfected with a CXCR4 expression construct. Comparisons can also include, but are not limited to, selections from the following pairs listed in the Table below:
  • An SVV-specific CXCR4 isoform can be used to identify whether a subject has a tumor that can be treated with SVV therapy. For example, tumor cells from a subject can be isolated in order to determine whether the cells express a particular SVV-specific CXCR4 isoform (this can be assayed, for example, by immunob lotting or by FACS analysis with an antibody specific to the SVV-specific CXCR4 isoform).
  • the 8G10 antigen has not yet been identified.
  • the 8G10 mAb can be used to DitteredDcipitated the 8G10 antigen from membrane extracts prepared from cells that are permissive for SVV infection.
  • the isolated antigen can be digested with a protease, such as trypsin, such that mass spectrometry can be used to identify the antigen.
  • cell extracts can be resolved in both denaturing and non-denaturing gel systems in one and/or two dimensions.
  • An immunoblot of the gel with the 8G10 mAb can be used to identify the protein on the gel that corresponds to the protein recognized by the 8G10 mAb on the immunoblot.
  • the identified protein can be extracted from the gel and subjected to mass spectrometry to identify the antigen.
  • the proteins comprising the proteome of an SVV-permissive cell can be analyzed for 8G10 mAb reactivity on a protein array.
  • the 8G10 antigen can be analyzed for binding to SVV, as described for CXCR4.
  • the 8G10 antigen is expressed on a variety of cell types, including cell types that are non-permissive for SVV infection, it may be the case, as discussed above for CXCR4, that a specific isoform of the 8G10 antigen is responsible for SVV tumor-specific tropism and/or that there is another unidentified SVV receptor(s) (or co- receptor(s)) for SVV binding, entry, and/or fusion.
  • cell type tropism of SVV may result from (1) expression of a particular 8G10 antigen(s), (2) overexpression of a particular 8G10 antigen(s), (3) expression of a particular 8G10 antigen isoform(s), and/or (4) overexpression of a particular 8G10 antigen isoform(s).
  • An SVV-specific 8G10 antigen can be used to determine whether a subject has a tumor that can be treated with SVV therapy.
  • tumor cells from a subject can be isolated in order to determine whether the cell express an SVV-specific 8G10 antigen (this can be assayed, for example, by immunoblotting or by FACS analysis with an antibody specific to the 8G10 antigen (i.e., the 8G10 mAb)).
  • SVV-specific receptors can account for SVV permissiveness in a particular type of tumor, including a tumor cell-type and a tumor tissue type. It may also be possible that such SVV-specific SVV receptors can account for SVV permissiveness in a particular stage of tumor (i.e, benign, malignant, or metastatic). An SVV receptor can provide permissiveness by virtue that it is expressed in a cell or by virtue that it is overexpressed or upregulated as compared to a cell that is not permissive.
  • an SVV receptor When an SVV receptor is identified, it is asked whether this receptor is also a tumor-specific marker (biomarker), i.e., whether the receptor (or isoform of the receptor) is expressed in a particular tumor cell-type, tumor tissue-type, or stage of tumor and not expressed in the corresponding normal cell-type or tissue-type or different stage of tumor; or whether the antigen is overexpressed only on a particular tumor cell and not overexpressed in the corresponding normal cell.
  • a tumor-specific marker i.e., whether the receptor (or isoform of the receptor) is expressed in a particular tumor cell-type, tumor tissue-type, or stage of tumor and not expressed in the corresponding normal cell-type or tissue-type or different stage of tumor
  • the antigen is overexpressed only on a particular tumor cell and not overexpressed in the corresponding normal cell.
  • the presence of cellular receptors to the virus can serve as predictive biomarkers if the mechanism of tropism is receptor- mediated.
  • predictive biomarkers such
  • an SVV receptor is also a tumor-specific marker, then the receptor can be a target for cancer therapy. It may also be the case that a particular SVV receptor does not confer SVV tropism, but rather the expression level of an SVV receptor confers permissiveness. For example, it is possible that higher or lower expression levels are required for SVV permissiveness depending on the SVV receptor. For example, in the case of an SVV-specific CXCR4 isoform, there is a very good correlation between CXCR4 protein expression as measured by FACS (fluorescence activated cell sorter) with 12G5 antibody and the EC50s of SVV on SCLC cell lines (see Table 5 below).
  • FACS fluorescence activated cell sorter
  • 12G5 can detect the 52, 47, 42, 40, and 34 kDa CXCR4 iso forms. It may be the case that one of these isoforms is specifically expressed in permissive cells and not expressed in non-permissive cells - where only the specifically expressed isoform in permissive cells can bind to SVV. Alternatively, it is possible that more than one of these iso forms can bind to SVV and are specifically expressed in permissive cells. Alternatively, it is possible that isoforms that can bind to SVV are expressed in both permissive and non-permissive cells, and a threshold level of expression of isoform(s) that can bind to SVV accounts for permissivity.
  • the invention provides a method for treating SCLC by administering to a subject SVV (or an SVV-like picornavirus), wherein the subject is afflicted with a tumor comprising a SCLC cell that specifically expresses or overexpresses one or more CXCR4 isoforms that can bind to SVV (or an SVV-like picornavirus).
  • Therapy can include, for example, a small molecule, an antibody, an antibody conjugate, SVV, a derivative of SVV (including a genetically modified virus, a viral chimera using portions of SVV), a peptide or protein fragment of SVV (including a portion of the capsid that can bind to CXCR4 such that the peptide/protein can compete or inhibit SDF- l ⁇ binding to CXCR4), an anti-idiotypic antibody or other engineered protein representing the portion of SVV that binds to an SVV receptor, or any combination thereof.
  • a small molecule an antibody, an antibody conjugate, SVV, a derivative of SVV (including a genetically modified virus, a viral chimera using portions of SVV), a peptide or protein fragment of SVV (including a portion of the capsid that can bind to CXCR4 such that the peptide/protein can compete or inhibit SDF- l ⁇ binding to CXCR4), an anti-idiotyp
  • the invention provides methods for treating cancer in a subject where the subject is afflicted with a tumor cell that expresses or overexpresses an SVV receptor that is capable of binding SVV.
  • the tumor cell expresses or overexpresses an SVV-specific receptor and the receptor is not expressed or is expressed at lower levels in a normal or non-tumor cell.
  • the SVV receptor can be an SVV- specific CXCR4 isoform that has, for example, a MW of about 110, 101, 90-95, 80-83, 68- 75, 62, 52, 47, 42, 40, or 34 kilodaltons.
  • the cancer can be treated by administering to a subject having cancer an SVV particle or an SVV-like picornavirus particle, such that a cancer cell in the subject is infected and killed by the particle (for example, infection by the SVV or SVV-like picornavirus particle can cause the cancer cell to apoptose).
  • a cancer cell in the subject is infected and killed by the particle (for example, infection by the SVV or SVV-like picornavirus particle can cause the cancer cell to apoptose).
  • the cancer can be treated by administering to a subject having cancer an SVV protein or peptide (or SVV-like protein or peptide) that can bind to an SVV receptor (for example, an SVV protein or peptide that can bind to a CXCR4 isoform such that it competes with SDF- l ⁇ for binding);
  • an SVV protein or peptide that can bind to an SVV receptor
  • the SVV or SVV-like picornavirus protein or peptide can be VPl, VP2, VP3, VP4, or any portion or combination thereof.
  • a large number of tumors express CXCR4, including but not limited to, B-CLL, AML, B-lineage ALL, intraocular lymphoma, Non-Hodgkin lymphoma, follicular center lymphoma, CML, multiple myeloma, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, thyroid cancer, colorectal cancer, oral squamous carcinoma, cervical cancer, neuroblastoma, kidney cancer, glioma, astrocytoma, rhabdomyosarcoma, SCLC, and melanoma.
  • CXCR4 including but not limited to, B-CLL, AML, B-lineage ALL, intraocular lymphoma, Non-Hodgkin lymphoma, follicular center lymphoma, CML, multiple myeloma, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, thyroid cancer, colorectal cancer, oral squamous carcinoma, cervical cancer, neuro
  • Primary cells or cell-lines of these tumors (and other tumors that express CXCR4) can be assessed for CXCR4 isoform overexpression (or upregulation) as compared to normal cells.
  • Primary cells or cell-lines of these tumors (and other tumors that express CXCR4) can be assessed for CXCR4 isoform specific expression as compared to normal cells, i.e., the normal cells do not express a CXCR4 isoform(s) that is expressed in the tumor cell.
  • Primary cells or cell-lines of these tumors can be assessed for CXCR4 isoform specific expression in a particular stage of the tumor, i.e., is there a specific CXCR4 isoform expressed (or overexpressed) in a metastatic tumor as compared to a benign tumor (or malignant vs. benign, or metastatic vs. malignant, etc.).
  • a CXCR4 isoform(s) is a tumor biomarker (i.e, if the isoform(s) is specifically expressed on a tumor cell or is specifically overexpressed or upregulated in a tumor cell, or is specifically expressed/overexpressed in a particular stage of a tumor), then the isoform(s) are tested to see whether they can bind an SVV or an SVV-like picornavirus particle.
  • Isoforms that are tumor biomarkers are targets for cancer therapy
  • isoforms that are tumor biomarkers and can also bind SVV or an SVV-like picornavirus are targets for SVV or SVV-like picornavirus mediated cancer therapy.
  • an SVV receptor is identified to be a tumor biomarker and if it does not bind to SVV or to an SVV-like picornavirus, SVV or an SVV-like picornavirus can be mutated such that the tropism of the virus is altered to become specific for the isoform (see PCT/US2004/031504 for methods for altering tropism).
  • an SVV receptor is identified to be a tumor biomarker and it does bind to SVV or to an SVV-like picornavirus
  • SVV or an SVV-like picornavirus can also be used as gene- therapy vectors such that they can infect the tumor cell and express a heterologous gene that can trigger apoptosis, inhibit cell cycle progression, or otherwise kill the tumor or otherwise inhibit tumor replication.
  • SVV or an SVV-like picornaviruses can be used as vehicles to deliver toxins or poisons to tumor cells by virtue of tumor-cell specific tropism; SVV or an SVV-like picornavirus can be used to specifically target a particular type of tumor or stage of tumor if the tumor or stage of tumor expresses an SVV receptor that can bind to SVV or an SVV-like picornavirus.
  • CXCR4 has been shown to play a central role in both chemotaxis and HIV-I entry into T lymphocytes. It is generally understood that CXCR4 is a co-receptor for T-trophic (T-cell trophic) HIV-I strains (X4 isolates), and CCR5 is a co-receptor for M-trophic (monocyte/macrophage trophic) HIV-I strains (R5 isolates).
  • T-tropic strains can infect macrophages (or MDMs - monocyte-derived macrophages), but at a much lower efficiency that than of M-trophic strains.
  • HIV-I strains that are dual-tropic, in that they can utilize either CXCR4 or CCR5 as a co- receptor, and therefore can infect T-cells, monocytes and macrophages that express one or both of these co-receptors and CD4.
  • Macrophages can be infected more efficiently be M-tropic HIV-I strains even though macrophages express both CXCR4 and CCR5.
  • Western blot analyses of total cell extracts and surface proteins from multiple sets of monocytes and macrophages demonstrate substantial differences between CXCR4 molecules. (See Lapham et ah, Nature Med. (1999) 5:303-8.)
  • CXCR4 is mainly a monomer in monocytes and a species of higher molecular weight (90 kDa) in macrophages.
  • SVV is not oncolytic for non-tumor cells, and therefore is not oncolytic for normal T-cells, monocytes, or macrophages
  • SVV can still be used in methods to treat or inhibit HIV infection. If SVV can bind the CXCR4 isoform(s) expressed on T-cells, monocytes, and macrophages, then SVV can be used to inhibit HIV infection - for example, by competing with HIV for binding to CXCR4.
  • SVV viral RNA was transfected into non-permissive cell lines and virus production was assayed.
  • Non-permissive cell were transfected with SVV viral RNA (a ⁇ -galactosidase reporter plasmid was used to determine the trans fection efficiency of the non-permissive cell lines; SVV viral RNA was transfected into cell lines that had a 20% or higher transfection efficiency).
  • the cells were cultured for about 18 hours before crude viral lysates (CVLs) were prepared. The viral lysates were Dittered on PER.C6 cells. The results are shown in Table 6 below, and indicate that SVV tropism is primarily receptor mediated because trans fection of viral RNA allows virus production in nonpermissive cell lines.
  • ⁇ Negative results for DMS53 may indicate that there is an intracellular mechanism or another receptor/co-receptor that contributes to tropism.
  • NCI-H446 used interchangeably herein with H446
  • H446 human lung carcinoma cell line
  • NCI-H446 is SVV permissive.
  • NCI-H446 cells were injected into mice, then the mice were screened for the development of antibodies to the NCI- H446 cell surface proteins.
  • mice 8-10 week old female BALB/c were injected with 3 million NCI-H446 cells suspended in 0.3 mL PBS every 2 - 3 weeks (Table 7).
  • mice were bled and serum was monitored for antibodies in virus neutralization or virus blocking assay.
  • Mice whose sera blocked SVV-OOl induced cytotoxicity were injected with 1 million H446 cells intravenously.
  • mice were immunized by injecting 200 ⁇ g of H82 membrane extract mixed with equal amounts of Freund's complete adjuvant subcutaneously. With two weeks between the injections, the mice were reinjected with the same amount of antigen mixed with equal amounts of Freund's incomplete adjuvant subcutaneously. Ten days after the third and fourth injections, mice were bled, and the sera were used to monitor for virus entry blocking antibodies in a neutralization assay.
  • mice whose sera blocked the virus induced cytotoxicity in H82 cells were injected with 200 ⁇ g of membrane extract in PBS intravenously. Three days following the final injection, the mice were sacrificed and spleens were collected. The spleenocytes were used to generate hybridomas.
  • NCI-H446 cells were seeded in a 48 well plate two days prior to infection. Serum was added to each well (150 ⁇ l/well; 1 :4 or 1 :8 dilution). Thirty minutes after addition of serum, 150 ⁇ l of virus (100 TCID50/well; TCID50 means 50% Tissue Culture Infective Dose) was added to the appropriate wells and the cells were incubated for 1 hour. The cells were then washed twice, then 500 ⁇ l of complete cell culture media was added to the cells and the cells were monitored for up to three days post-infection.
  • TCID50 50% Tissue Culture Infective Dose
  • Antibody-producing spleen cells were isolated from mice whose serum blocked viral infection of NCI-H446 cells in the assay described above. To produce a monoclonal antibody producing hybridoma cell, the spleen cell is fused with a long-lived myeloma cell. Single-cell suspensions of spleenocytes were made and fused with SP2/O myeloma cells at a ratio of 5:1 (5e7 spleenocytes and Ie7 myeloma cells) using polyethylene glycol.
  • the fusion mixture was resuspended in RPMI 1640 containing 18% FBS and HAT (hypoxanthine, aminopterin, thymidine) and plated into twenty five 96-well tissue culture dishes. Seven days post- fusion, the media was replaced with fresh RPMI 1640 containing HAT. Twelve days post- fusion, 100 ⁇ L of culture supernatant was collected from each well and tested for inhibition of virus-induced GFP expression or CPE in virus blocking assays.
  • HAT hypoxanthine, aminopterin, thymidine
  • Hybridoma clones were screened with the virus blocking assay to identify hybridoma cells that produce monoclonal antibodies that block infection. Two rounds of screening were carried out. For the first approach described above, the virus blocking assay was carried out by seeding NCI-H446 cells (30,000 cells/well) in a 96-well plate one day prior to infection. For the second approach, H446 and Per.C6 cells were used. On the day of infection, the media was removed from the cells and the cells were incubated with 100 ⁇ l of hybridoma culture supernatant for 40 minutes at room temperature.
  • the hybridoma clones secreting virus entry blocking monoclonal antibodies (>25 for each fusion) were subjected to single cell cloning and expanded. Isotype of monoclonal antibodies was determined using an antibody isotyping kit (Roche Pharmaceuticals). A few select clones were cultured in CELLineTM flasks (BD Biosciences) in serum-free medium and the culture supernatant was used for purification of monoclonal antibodies by size exclusion chromatography. The purified antibodies were used in Western blot and virus entry blocking experiments.
  • SVV tagged with green fluorescent protein was used in the virus blocking assay.
  • NCI-H446 cells were seeded (30,000 cells/well) in 24-well dishes one day prior to infection. On the day of infection, the culture media was removed from the cells, and the cells were incubated with 100 ⁇ l of hybridoma culture supernatant for 40 minutes at room temperature.
  • SVV-GFP was added at 200 vp/cell (PPC) and the cells were incubated at 37 0 C for one hour.
  • PER.C6 cell line Another human cell line that is SVV permissive is the PER.C6 cell line.
  • PER.C6 cells were treated with purified 8G10 mAb then analyzed for SVV-GFP infection. As shown in Figures 14A - 14D, SVV-GFP entry into PER.C6 cells was reduced in the presence of 8G10 mAb.
  • EXAMPLE 4 IMMUNOBLOT DETECTION AND IMMUNOPRECIPITATION OF MEMBRANE PROTEINS BY MONOCLONAL ANTIBODIES
  • Cell membrane proteins recognized by the monoclonal antibodies of the invention represent candidates for SVV cellular receptors or co-receptors that mediate SVV entry into cells.
  • the membrane was washed three times with TBS-T (TBS with 0.05% Tween20) containing 5% milk for lhr at room temperature.
  • TBS-T TBS with 0.05% Tween20
  • the membrane was washed three times with Ix TBS-T and incubated with 8G10 clone culture supernatant at room temperature for two hours.
  • the membrane was washed three times with TBS-T and incubated with sheep anti-mouse HRP-conjugated antibody (GE-Healthcare) at room temperature for one hour.
  • the blot was washed three times and developed using ECL Plus kit (GE-Healthcare).
  • the beads were then boiled in Laemmli sample buffer with 4OmM DTT for 5 minutes and resolved on a 4-12% Bis/Tris gradient gel.
  • the proteins were transferred onto PVDF membrane.
  • the membrane was blocked with TBS-T (TBS with 0.05% Tween20) containing 5% milk for a minimum of lhr at room temperature.
  • TBS-T TBS with 0.05% Tween20
  • 17El 1 1 : 100
  • the membrane was washed three times with TBS-T and incubated with sheep anti-mouse IgM HRP conjugated antibody (Novus) at room temperature for one hour.
  • the blot was washed three times and developed using ECL Plus kit (GE-Healthcare).
  • H446 cells were grown to -95% confluence as per the ATCC passaging instructions. The cells were washed with DPBS (Invitrogen) and dislodged with a cell scraper. The cells were again washed, two times using DPBS with centrifugation at 1500 RPM for 5 minutes. Samples were then resuspended in DPBS with protease inhibitors (Pierce) and vortexed.
  • the suspension was then passed through a 21 gauge needle 10 times on ice.
  • the homogenate was sonicated 3x10 cycles of 10 watts (Fisher model 60 sonic dismembranator) on ice.
  • the homogenate was then centrifuged at 10,000xg for 10 minutes. The supernatant was removed (top 75% to prevent contamination with pellet material) and spun at 100,000xg for 1 hour.
  • the pellet (containing total membranes) was resuspended in Extraction buffer (7M Urea, 2M Thiourea, 4% CHAPS, 1% IPG Buffer, 4OmM DTT and 2X Halt Protease Inhibitor Cocktail).
  • the suspension was vortexed for 10 seconds and incubated for 30 minutes at room temperature on a nutator.
  • this extract was spun at 5,000xg for 5 minutes to remove insoluble material. The supernatant was then aliquoted and stored at - 8O 0 C. The protein concentration was determined by Bio-Rad RC/DC assay using the manufacturer's instructions. 200-400ug of membrane extract was diluted to 450ul with 2OmM DTT (GE Healthcare) and 350ul destreak rehydration solution (w/1% 3-11 IPG Buffer) (GE Healthcare). The sample was then applied to a 24cm 3-1 INL IPG strip (GE Healthcare) and overlaid with dry strip cover fluid (GE Healthcare). The IPG strip was then focused in a 24cm strip holder in the Ettan IPGPhor III IEF system (GE Healthcare).
  • the focused IPG strips were equilibrated using SDS equilibration buffer as per the manufacturer's instructions. Equilibrated IPG strips were overlayed on top of 4-12% gradient gels (JULE) along with molecular weight markers (Biorad) and sealed with agarose overlay solution (GE Healthcare). Gels were run in IX Tris Glycine running buffer (Invitrogen) at 1 watt per gel overnight in an Ettan Daltsix electrophoresis unit (GE Healthcare). Resolved gels were then fixed for silver stain analysis and stained according to the manufacturer's instructions (Invitrogen). Alternatively, gels were transferred to PVDF (GE Healthcare) at 40OmAMP for 1 hour in transfer buffer using a semi-dry transfer unit (GE Healthcare).
  • PVDF membranes were then stained with MemCode (Pierce) to confirm the transfer.
  • Membranes were blocked in 5% non-fat dry milk in TBST (Teknova) for a minimum of 2 hours, and then stained overnight at 4° C with the monoclonal antibody 8G10 at a dilution of 1 : 1000 in block on a rocker.
  • the blots were washed with TBST for 3 times 5 minutes on an orbital shaker and incubated with 2° HRP antibodies (GE Healthcare) at 1 :5000 in block for 1 hour at room temperature.
  • the membranes were then washed 4 times for 30 minutes in TBST.
  • ECLp lus GE Healthcare
  • Results are shown in Figure 16C.
  • the western blot was aligned to the duplicate gel, and a piece of gel corresponding to the position of western blot signal on the membrane was cut. Sequence identification via LC MS/MS was performed by Proteomic Research Services (PRS).
  • a cell line flask system was used to produce concentrated monoclonal antibody produced by hybridoma clone 8G10.
  • the 8G10-F10 clone was cultured in cell line flasks for 8 - 10 days ( Figure 17).
  • the culture supernatant was collected by pelleting the cells three times at 1500 rpm.
  • the supernatant was then dialyzed against PBS (four buffer changes).
  • the dialyzed solution containing the concentrated 8G10 mAb was stored at -2O 0 C in PBS containing 50% glycerol.
  • the cells were seeded in Lab-Tek II chamber slides (Nalgene Nunc). The next day, the cells were fixed with freshly made 4 % paraformaldehyde in PBS for 30 min at room temperature. The cells were washed 3 times for 5 min each with PBS. The cells were incubated in blocking solution (PBS with 1% normal goat serum) for 30 min to block nonspecific binding of the antibodies. The cells were washed three times with PBS and incubated with 8G10 or 17El 1 for 60 min at room temperature. The cells were washed with PBS and incubated with donkey anti-mouse IgG (heavy and light chain) conjugated to Alexa fluor 594 (Invitrogen) and incubated for 60 min at room temperature.
  • the cells were washed three times and mounted with mount medium containing DAPI dye (Vector Laboratories). The cells were visualized under fluorescent confocal microscope and photographed. To determine if the antigens are expressed intracellularly, cells were permeabilized with 0.1% Triton X-100 prior to staining ( Figures 18E - 18H).
  • EXAMPLE 7 EFFECT OF 8G10 MONOCLONAL ANTIBODY ON SVV-MEDIATED
  • the 8G10 mAb significantly blocks SVV entry into NCI-H446 cells which are otherwise permissive to SVV infection.
  • NCI-H446 cells were plated in a 96-well dish (30,000 cells/well) two days prior to infection. On the day of infection, media was removed from the cells, and RPMI media (100 ⁇ l) containing 8G10 mAb (80 ⁇ g/ ml) or mouse IgM isotype control (80 ⁇ g/ml) was added to the cells, followed by incubation at room temperature for 40 minutes.
  • SVV-OOl 1000 - 0.00001 PPC was added to the cells, followed by incubation at 37 0 C for 1 hour. The cells were then washed three times with PBS. Complete media (200 ⁇ l) containing 80 ⁇ g/ml 8G10 mAb or 40 ⁇ g/ml mouse IgM isotype control was added to the cells. The cells were incubated at 37 0 C. Cell survival was monitored at 48 hours post-infection; results are shown in Figure 19. In the absence of 8G10 mAb, the EC50 (amount of virus necessary to kill 50% of cells) of SVV-OOl was 0.0988 virus particles/cell. In the presence of 8G10, the EC50 increased to 6.872 virus particles/cell. The dose-response results show that SVV-OOl -mediated cell killing is reduced in the presence of 8GlO mAb.
  • EXAMPLE 8 EFFECT OF 8G10 AND 17El 1 MONOCLONAL ANTIBODIES ON
  • H446 cells were seeded in 24-well tissue culture dishes one day prior. On the day of the experiment, medium was replaced with medium containing varying amounts (0, 2, 5, 10, 20, 40 and 80 ⁇ g/mL) of 8G10 or 40 ⁇ g/mL of IgM isotype control antibody and incubated at room temperature for 40 minutes. SVV-OOl was added at 1 or 10 particles/cell, and cells were incubated at 37° C for one hour. The cells were washed three times with PBS and 1 mL of RPMI containing 10% FBS was added. The cells and media were harvested at 14 hours post-infection.
  • CVL was generated by three cycles of freeze-thaw, and virus titer determined by tissue culture infectivity assay (TCID50) in PER.C6 cell.
  • TCID50 tissue culture infectivity assay
  • additional cell lines including H446, H 1299, HEK293, H69AR, PER.C6, Hl 87, H82 and Hl 184 were tested. The blocking effect was evident with both 8G10 ( Figure 21A) and 17El 1 ( Figure 21B).
  • Xenografts of SVV permissive or non-permissive cells were analyzed by immunohistochemistry (IHC) for expression of SVV receptors.
  • IHC immunohistochemistry
  • 8G10 antigen and integrin ⁇ 4 are expressed in SVV permissive cells (H446), but not expressed in non-permissive cells (H460).
  • the markers were also found to co-stain the same cells from an invasive edge of a xenograft tumor from a permissive cell line ( Figure 23).
  • the 8G10 mAb was used to analyze human SCLC tissue microarrays (TMA) ( Figure 24). As shown in Table 8 below, almost 60% of 34 SCLC tissues stained ++ or +++ with 8G10 mAb. Other tumor types also stained positive and virtually none of the normal major organ tissues stained positive.
  • Table 8 Summary of IHC results of SCLC TMA using 8G10 mAb
  • SVV was tested in patients with solid tumors expressing at least one neuroendocrine marker. SVV was administered systemically as a one-hour intravenous infusion. Viral load in serum was monitored using an infectivity-based assay (TCIDso/mL) and a quantitative real-time RT-PCR assay (RNA copies/mL). Patient- specific patterns of viral amplification and serum kinetics of SVV-OOl were observed, indicative of different patterns of viral replication. These patterns of kinetics ranged from a lack detectable infectious virus and genomic RNA at all time points post-dosing to rapid amplification up to greater than 10 7 TCID 5 o/mL and 10 9 RNA copies/mL in serum (Figure 26A).
  • mice demonstrated transient low-level replication of SVV, with self-limiting viremia in normal tissue.
  • the pharmacokinetic profile in the murine model was found to be predictive of clinical data. For example, replication of SVV was observed in tumor cells, and viremia was resolved by a neutralizing anti-SVV antibody response in the presence of a tumor ( Figure 28).
  • the murine model, as well as porcine and non-human primate models had toxicity profiles that were predictive of clinical data. For example, the toxicity was limited to transient changes in white blood cells/platelets.
  • a pre-clinical tumor model showed that SVV replicates in tumor cells despite an immune response in the animal ( Figures 3 IA - 3 IB). SVV was also shown to have a high therapeutic index ( Figures 32A - 32B), with at least a one million-fold therapeutic window in pre-clinical mouse models.
  • EXAMPLE 12 PHARMACOKINETICS OF INTRAVENOUSLY ADMINISTERED SVV IN PATIENTS WITH SOLID TUMORS OF NEUROENDOCRINE DIFFERENTIATION
  • Seneca Valley Virus is a newly discovered picornavirus with natural oncolytic selectivity towards human tumor cells of neuroendocrine (NE) differentiation.
  • NE neuroendocrine
  • a dose- escalation Phase I clinical trial was initiated for patients with solid tumors expressing at least one NE marker.
  • SVV was administered systemically as a one hour i.v. infusion.
  • Viral load in serum was monitored using an infectivity-based assay (TCID50/mL) and a quantitative real-time RT-PCR assay (RNA copies/mL). Patient- specific patterns of viral amplification and serum kinetics of SVV were observed indicative of different patterns of viral replication.
  • the dose-escalation was 10 7 - 10 11 viral particles of SVV/kg (the dose was 10 7 , 10 8 , 10 9 , 10 10 , and then 10 11 ).
  • the solid tumor of the subject receiving SVV was expressing at least one neuroendocrine marker. Then, safety, toxicity and dosage was evaluated. Viral kinetics and clearance were evaluated.
  • Viral Kinetics in Serum were monitored in serum utilizing an infectivity (IFT)-based TCID50 assay on PER.C6® cells and a quantitative, realtime reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Patient-specific patterns of viral replication were observed.
  • Viral Neutralization in Serum was determined by incubation of serum dilutions with 100 TCID50 SVV prior to infection of PER.C6 ® cells. Neutralizing titer was reported as the greatest dilution of serum capable of neutralizing greater than 50% of replicate wells. Patient-specific patterns of viral neutralization were observed.
  • Subject Serum RNA is Degraded (see Figure 36).
  • RNA from Subject's serum on study days 4, 17 (shown) and 25 was subjected to RT-PCR for three regions of the SVV genome ranging from 961 to 1557 bp. To determine quantitation limits, the samples were spiked with log dilutions of full-length viral RNA. Large fragments could be amplified from serum RNA on day 4, but not on days 17 or 25, indicating lack of full length circulating viral RNA at later time points.
  • Anti-SVV Immunohistochemistry see Figure 37, 400X. A subject succumbed to progressive disease on day 28 post-infusion. Tissues were collected at autopsy and fixed in 10% neutral-buffered formalin. Sections were stained by H&E and using mouse anti-serum to SVV. Specific anti-SVV staining was observed in a liver metastasis, but not surrounding normal liver tissue or in a panel of normal organs, including adrenal gland and pancreas.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Virology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Communicable Diseases (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The invention provides a hybridoma cell and a monoclonal antibody produced therefrom, wherein the monoclonal antibody specifically binds and forms a complex with an antigen on the surface of a tumor cell and thereby inhibits Seneca Valley Virus (SVV) entry into the tumor cell. The invention further provides SVV receptors, for example, CXCR4 and the antigen recognized by the monoclonal antibody of the invention. The invention also provides methods of using the monoclonal antibody to determine whether SVV will enter a tumor cell and for determining whether a subject with a tumor will respond to SVV treatment.

Description

MONOCLONAL ANTIBODY THAT RECOGNIZES A SENECA VALLEY VIRUS (SW) CELLULAR RECEPTOR AND USES THEREOF
[0001] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
[0002] A portion of the disclosure of this patent document 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 Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
BACKGROUND OF THE INVENTION
[0003] Seneca Valley virus ("SVV") is a picornavirus that can selectively kill some types of tumors in humans but is not cytotoxic to normal human cells (see International Application No. PCT/US2004/031504 and U.S. Serial No. 60/506,182, both of which are hereby incorporated by reference in their entireties, including SVV nucleotide and amino acid sequences obtained from the SVV isolate ATCC Patent Deposit Number PTA-5343). For example, 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. In vivo results indicate that SVV is not toxic to normal cells, in that up to 1x1014 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.
SUMMARY OF THE INVENTION
[0004] The invention relates in part to compositions and methods stemming from the understanding that SVV entry into cells is a receptor (or co-receptor)-mediated process, and that such a receptor may confer cell-type tropism of SVV.
[0005] The invention provides discovery that CXCR4 is a cellular receptor for SVV, and that at least some cells normally non-permissive for SVV infection can be made permissive by expressing or overexpressing CXCR4. Although CXCR4 is expressed on a wide-variety of cell-types, different cell-types express different CXCR4 isoforms, different sets of CXCR4 isoforms, and/or different protein levels of particular CXCR4 isoforms. Without being bound by theory, cell-type tropism of SVV may result from (1) expression of a particular CXCR4 isoform(s), and/or (2) overexpression of a particular CXCR4 isoform(s).
[0006] The invention further provides the discovery that a monoclonal antibody raised against cell surface proteins expressed on an SVV permissive cell can reduce SVV yield in a dose dependent manner. The antigen to which the monoclonal antibody binds represents a candidate for an SVV cellular receptor.
[0007] The invention provides SVV receptors which may confer cell-type tropism of SVV. The SVV receptors, as well as the monoclonal antibody, provided by the invention can be used, for example, as biomarkers for prescreening and stratification of patients.
[0008] The invention provides a hybridoma cell line, designated Anti-H446 MMAb 8G10 and having ATCC Accession No. , that produces a monoclonal antibody of the invention was deposited with the Patent Depository of the American Type Culture Collection (ATCC),
10801 University Blvd., Manassas, VA, 20110, on , under the provisions of the
Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of a Patent Procedure.
[0009] The invention provides a hybridoma cell line, designated Anti-H82 MMAb 17El 1 and having ATCC Accession No. , that produces a monoclonal antibody of the invention was deposited with the Patent Depository of the American Type Culture Collection (ATCC),
10801 University Blvd., Manassas, VA, 20110, on , under the provisions of the
Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of a Patent Procedure.
[0010] The invention provides monoclonal antibodies. In one aspect, a monoclonal antibody is produced by the hybridoma cell designated Anti-H446 MMAb 8G10 deposited as ATCC
Accession No. . In another aspect, a monoclonal antibody is produced by the hybridoma cell designated Anti-H82 MMAb 17El 1 deposited as ATCC Accession No. . In another aspect, a monoclonal antibody is provided, which specifically binds and forms a complex with an antigen located on the surface of a tumor cell and thereby inhibits SVV entry into the cell, the antigen being an antigen to which a monoclonal antibody produced by a hybridoma cell designated Anti-H446 MMAb 8G10 deposited as ATCC Accession No. specifically binds. In another aspect, a monoclonal antibody is provided, which specifically binds and forms a complex with an antigen located on the surface of a tumor cell and thereby inhibits SVV entry into the cell, the antigen being an antigen to which a monoclonal antibody produced by a hybridoma cell Anti-H82 MMAb 17El 1 deposited as ATCC Accession No. specifically binds.
[0011] In one embodiment, an antibody fragment is provided consisting essentially of an antigen-binding domain of the monoclonal antibody of any one the disclosed monoclonal antibodies. In another embodiment, the antibody is an chimeric antibody. In another embodiment, the antibody is a humanized monoclonal antibody. In another embodiment, the antibody is a murine monoclonal antibody. In another embodiment, the antibody is a human monoclonal antibody.
[0012] In one embodiment, the antibody, or an antigen-binding fragment thereof, comprises a detectable label. In another embodiment, the antibody, or an antigen-binding fragment thereof, is linked to a moiety capable of producing a detectable signal.
[0013] A method is provided for determining whether SVV will enter a tumor cell, the method comprising: (a) contacting a tumor cell with a monoclonal antibody, or an antigen- binding fragment thereof, produced by (i) a hybridoma cell designated Anti-H446 MMAb
8G10 deposited as ATCC Accession No. , or (ii) a hybridoma cell designated Anti-H82
MMAb 17El 1 deposited as ATCC Accession No. ; and (b) determining whether the monoclonal antibody or fragment thereof binds to the tumor cell, wherein a determination of binding indicates that SVV will enter the tumor cell. In one embodiment, the tumor cell is contained within a tumor or a tumor sample. In another embodiment, the method is carried out in vitro. In another embodiment, the method is carried out in vivo. In one embodiment, the determining comprises direct detection of the monoclonal antibody. In another embodiment, the determining comprises indirect detection of the monoclonal antibody. In another embodiment, the determining comprises detecting a chromogenic signal, a fluorescent signal, a luminogenic signal or a radioactive signal.
[0014] A method is provided for determining whether a subject with a tumor will respond to SVV treatment, the method comprising: (a) contacting the tumor with a monoclonal antibody, or an antigen-binding fragment thereof, produced by (i) a hybridoma cell designated Anti-
H446 MMAb 8G10 deposited as ATCC Accession No. , or (ii) a hybridoma cell designated Anti-H82 MMAb 17El 1 deposited as ATCC Accession No. ; and (b) determining whether the monoclonal antibody binds to the tumor, wherein a determination of binding indicates that the subject will respond to SVV treatment, and wherein the response comprises an arrest, delay, inhibition or reversal in the progression of the cancer. In one embodiment, the contacting is in vitro.
[0015] A method is provided for delivering a compound to a tumor cell in a subject, the method comprising (a) linking the compound to an antibody produced by (i) a hybridoma cell designated Anti-H446 MMAb 8G10 deposited as ATCC Accession No. , or (ii) a hybridoma cell designated Anti-H82 MMAb 17El 1 deposited as ATCC Accession No. , or a fragment thereof; and (b) administering the antibody to the subject. In one embodiment, the linking is via a covalent bond.
[0016] A kit is provided for determining whether SVV will enter a tumor cell, the kit comprising: (a) a monoclonal antibody, or an antigen-binding fragment thereof, produced by (i) a hybridoma cell designated Anti-H446 MMAb 8G10 deposited as ATCC Accession No. , or (ii) a hybridoma cell designated Anti-H82 MMAb 17El 1 deposited as ATCC
Accession No. ; and (b) at least one negative control cell sample, wherein the monoclonal antibody or fragment thereof does not bind to the cell sample. In one embodiment, the kit further comprises at least one positive control cell sample to which the monoclonal antibody or fragment thereof binds. In another embodiment, the kit further comprises instructions for preparing a tumor sample. In another embodiment, the monoclonal antibody or the fragment thereof is linked to a detectable signal or a moiety capable of producing a detectable signal.
[0017] A method is provided for identifying stem cells, the method comprising: (a) contacting a cell with the monoclonal antibody, or an antigen-binding fragment thereof, produced by (i) a hybridoma cell designated Anti-H446 MMAb 8G10 deposited as ATCC
Accession No. , or (ii) a hybridoma cell designated Anti-H82 MMAb 17El 1 deposited as
ATCC Accession No. ; and (b) determining whether the monoclonal antibody binds to the cell, wherein binding of the antibody to the cell in (b) indicates that the cell is a stem cell.
[0018] An antigen is provided that is specifically bound by the monoclonal antibody, or an antigen-binding fragment thereof, produced by (i) a hybridoma cell designated Anti-H446
MMAb 8G10 deposited as ATCC Accession No. , or (ii) a hybridoma cell designated
Anti-H82 MMAb 17El 1 deposited as ATCC Accession No. . In one embodiment, the antigen is linked to a detectable moiety. [0019] A pharmaceutical composition is provided comprising a disclosed monoclonal antibody, and a pharmaceutically acceptable carrier.
[0020] A method is provided for treating cancer in a subject, the method comprising administering to a subject an effective amount of a monoclonal antibody which specifically binds and forms a complex with an antigen located on the surface of a tumor cell and thereby inhibits SVV entry into the cell, the antigen being an antigen to which a monoclonal antibody produced by (i) a hybridoma cell designated Anti-H446 MMAb 8G10 deposited as ATCC
Accession No. , or (ii) a hybridoma cell designated Anti-H82 MMAb 17El 1 deposited as
ATCC Accession No. specifically binds. In one embodiment, the antibody is a monoclonal antibody produced by (i) a hybridoma cell designated Anti-H446 MMAb 8G10 deposited as ATCC Accession No. , or (ii) a hybridoma cell designated Anti-H82 MMAb
17El 1 deposited as ATCC Accession No. . In another embodiment, the antibody is a chimeric antibody. In another embodiment, the antibody is a humanized monoclonal antibody. In another embodiment, the antibody comprises a human constant region and a heavy and light chain variable region, wherein the heavy and light chain variable region comprises heavy and light chain framework regions and heavy and light chain complementarity determining regions (CDRs), at least a portion of the heavy and light chain framework regions being derived from a human antibody, and the CDRs comprising heavy- chain CDRs light-chain CDRs derived from a monoclonal antibody produced by (i) a hybridoma cell designated Anti-H446 MMAb 8G10 deposited as ATCC Accession No. , or (ii) a hybridoma cell designated Anti-H82 MMAb 17El 1 deposited as ATCC Accession No. .
BRIEF DESCRIPTION OF THE FIGURES
[0021] Figures IA - IH: The genomic sequence (SEQ ID NO:1) and encoded polyprotein sequence (SEQ ID NO:2) of SVV. Specific features of the SVV genomic sequence, such as specific coding regions for proteins cleaved from the polyprotein sequence are described herein. The SVV genome sequence was obtained from the SVV isolate that has been deposited with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Virginia, 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: July 25, 2003. [0022] Figures 2A - 2D: The nucleic acid sequence of the SVV genome (SEQ ID NO:1). [0023] Figure 3: The amino acid sequence of the SVV polyprotein (SEQ ID NO:2).
[0024] Figure 4: An analysis of the epidemiology of SVV. SVV is a unique virus, phylogenetically similar to Cardioviruses, but classified in a separate tree.
[0025] Figure 5: The protein products made from proteolytic processing of a picornavirus polyprotein.
[0026] Figure 6: A screening assay that tests the ability of cDNAs to confer SVV permissiveness on cell lines originally non-permissive for SVV infection. In a slight variation of the method shown in the Figure, cDNAs can be transfected en masse to non- permissive cells in culture prior to aliquoting the cells onto a multi-well plate.
[0027] Figure 7: Results of an expression cloning experiment to determine whether the expression of a particular cellular receptor in the non-permissive cell-line Hep3b confers SVV permissiveness to the cells. As shown in the Figure, only CXCR4 conferred permissiveness to the same degree as the positive control (the permissive cell line PER.C6). The SVV cellular receptor candidates were identified based upon the results of differential gene expression profiles of non-permissive and permissive cell lines.
[0028] Figures 8A - 8B: The anti-CXCR4 antibody 12G5 can inhibit SVV infection.
[0029] Figure 9: The amino acid sequence of human CXCR4 (SEQ ID NO:3; GenBank Accession No. P61073). Residues 1-39, 100-110, 176-200 and 262-285 are extracellular domain residues (i.e., the approximate residues for the four extracellular domains). Potential N-linked glycosylation sites at least include positions 11-13 and 176-178. At least residues 2-9, 7, 11, 12, 14, 15, 18, 20, 21, 176, 187 and 268 may play a role in SDF-I α binding.
[0030] Figures 1OA - 1OB: Serum collected from mice following a second injection (Figure 10A) or a fourth injection (Figure 10B) of NCI-H446 cells was screened with a virus blocking assay to determine the presence of antibodies specific for cell surface proteins as described in Example 3. Mice were identified as numbers 2-1 through 2-14. Serum was added to the cells at a dilution of 1 :4 or 1 :8. Gray boxes represent wells in which a cytopathic effect (CPE) was observed. White boxes represent wells in which no CPE was observed.
[0031] Figure 11: Generation of hybridoma clones and screening for virus entry blocking monoclonal antibodies. Mice were immunized with H446 cells or membrane extracts of H82, and spleenocytes from the mice were used for fusion with myeloma cells to generate hybridomas which were selected in the presence of HAT medium. Hybridomas secreting antibody which blocked viral infection were selected and subjected to single cell cloning.
[0032] Figures 12A - 12B: Supernatants of selected hybridoma clones were tested in duplicate in a virus blocking assay as described in Example 3. For each positive hybridoma clone, ten different single cell clones were tested in a virus blocking assay for the presence of monoclonal antibodies that block virus entry into NCI-H446 cells. Dark blue wells indicate that viral replication was blocked, and indicate the presence in the supernatant of monoclonal antibodies that block SVV entry into NCI-H446 cells.
[0033] Figures 13A - 13B: Supernatant from hybridoma clone 8G10 was incubated with NCI-H446 cells, then SVV-GFP (200 PPC) was added to the cells to determine if the monoclonal antibody in the supernatant blocked virus entry into the cells. Cells were analyzed by fluorescence microscopy. Figure 13A shows virus uptake in NCI-H446 cells in the absence of monoclonal antibody. Figure 13B shows a significant reduction in virus uptake in the presence of 8G10 monoclonal antibody.
[0034] Figures 14A - 14D: Monoclonal antibody purified from the supernatant of hybridoma clone 8G10 was incubated with PER.C6 cells, then SVV-GFP (200 PPC) was added to the cells to determine if the mAb blocked SVV-GFP entry into the cells. Fluorescence microscopy images (Figures 14A - 14B) show a reduction in SVV-GFP uptake in the presence of 8G10 mAb. Fig. 14C and Fig. 14D.
[0035] Figures 15A - 15C: Proteins were octyl D-glucoside extracted from membranes of NCI-H446 and PER.C6 cells. Figure 15A shows an SDS-PAGE analysis of the extracted membrane proteins (50 μg/lane). The SDS-PAGE gel was stained with Coomassie blue. Figure 15B is a Western blot analysis of the membrane proteins (20 μg/lane) with 8G10 mAb (IgM isotype; 1 :10 dilution). Figure 15C, NCI-H446 membrane proteins (25 μg/lane) were analyzed by Western blot with monoclonal antibodies (IgM isotype, 1 :10 dilution) produced by several different hybridoma clones.
[0036] Figures 16A - 16C: Figure 16A, Immunoprecipitation of proteins recognized by 17El 1. Immunoprecipitation of H446 proteins using the monoclonal antibody 17El 1. The arrow points to the specific protein immunoprecipitated by 17El 1. Figure 16B, Immunoprecipitation of proteins recognized by 8G10. Immunoprecipitation of S35 labeled H446 proteins using the monoclonal antibody 8G10. Decay accelerating factor was used as positive control in immunoprecipitation reactions. Figure 16C, Two-dimensional gel electrophoresis and Western blot analysis.
[0037] Figure 17: Analysis of concentrated 8G10 mAb produced by the cell line flask system. Samples from the first cell line flask (lanes 1 (1 μ.1 sample), 2 (2 μl sample) and 3 (3 μl sample)) and the second cell line flask (lanes 4 (1 μl sample), 5 (2 μl sample) and 6 (4 μl sample)) were compared to an IgM isotype control (lanes 7 (1 μl control), 8 (2 μl control) and 9 (4 μl control)).
[0038] Figures 18A - 18L: To determine the cellular location of the 8G10 antigen, SVV permissive cells (NCI-H446 and PER.C6) and SVV non-permissive cells (NCI-H460 and Hep3B) were analyzed by immunofluorescence staining as described in Example 6. Cells were analyzed under non-permeabilizing (Figures 18A - 18D) or permeabilizing (Figures 18E - 18H) conditions. Cell nuclei are stained with DAPI (blue) and the red fluorescence indicates the location of the bound 8G10 mAb. Cells were also stained with a control IgM isotype antibody. The cells were visualized under confocal microscopy. 8G10 shows distinct surface staining of permissive cells, but not non-permissive cells. Figures 181 - 18L, Immunofluorescence for detection of protein recognized by 17El 1. Cells were stained with 17El 1 as well as a control IgM isotype antibody (data not shown). 17El 1 gave surface staining as well as cytoplasmic staining of permissive cells, but not non-permissive cells.
[0039] Figure 19: Results of a dose-response study to determine SVV-OOl -mediated killing of NCI-H446 cells in the absence (triangles) and presence (squares) of 8G10 mAb. See Example 5 for details.
[0040] Figures 2OA - 2OB: 8G10 treatment lowers yields of SVV-OOl . The cells were treated with 8G10 at 25, 75 μg/mL or isotype control antibody (75 μg/mL) and incubated at room temperature for one 40 minutes. The cells were then infected with SVV-OOl at 1 virus particle/cell and incubated at 37°C for one hour. The cells were washed three times and further incubated for 14 hours. The virus yields were assayed on PER.C6 cells and shown as total yields (TCID50/mL- Figure 20A) and percent of IgM isotype control (Figure 20B).
[0041] Figures 21A - 21B: Figure 21A, 8G10 treatment lowers yields of SVV-OOl. H446 cells were treated with 8G10 at varying amounts and infected with 1 or 10 virus particles/cell. The cells were washed three times and further incubated for 14 hours. The virus yields were assayed on PER.C6 cells and shown as percent of IgM isotype control. Figure 21B, Virus yield assays in the presence or absence of 17El 1 and 8G10. Shown is a representative competition experiment with 8G10 and 17El 1 blocking viral production on H446 cells.
[0042] Figure 22: Immunohistochemistry staining of xenografts of SVV permissive (H446) and non-permissive (H460) cells shows that permissive cells are infected by SVV and express 8G10 antigen and integrin α4, while non-permissive cells are not infected by SVV and do not express detectable levels of 8G10 antigen or integrin α4.
[0043] Figure 23: Immunohistochemistry analysis of cell markers. All markers co-stain the same cells on an invasive edge of a xenograft tumor from a permissive cell line. Ki-67 is expressed in proliferating cells. NCAM; neuronal cell adhesion molecule.
[0044] Figure 24: Results of immunohistochemistry analysis conducted with the 8G10 mAb on human SCLC tissue microarrays. The 8G10 mAb strongly stained SCLC tissue (+++; upper panels), with no staining of the control tissue (-; lower panels).
[0045] Figure 25: Schematic of a proposed mechanism of receptor-mediated binding of SVV to permissive cells.
[0046] Figures 26 A - 26B: Figure 26 A depicts the viral titer of SVV as detected by RT- PCR in the serum of four patients who received an intravenous infusion of SVV. Figure 26B depicts the titer of anti-SVV neutralizing antibodies generated in each patient.
[0047] Figures 27A - 27B: SVV replication is tumor-selective and continues in the presence of immune response. Figure 27A depicts immunohistochemistry analysis of tissue samples obtained from one patient. The metastatic tumor cells stained positive for viral capsid, while surrounding normal tissue was not stained. Figure 27B depicts the serum levels of SVV and neutralizing antibodies present in the patient of Figure 27A.
[0048] Figure 28: Kinetics of Seneca Valley Virus (SVV-OOl) in serum of A/J mice following intravenous injection. SVV-OOl was injected intravenously at 109 vp/kg into female A/J mice. Study groups included normal mice or mice bearing subcutaneous tumors (n=3/group/time point) formed from the murine neuroblastoma line NlE-115, which is permissive to SVV-OOl infection. Plotted are infectious viral titers and neutralizing antibody titers (±SD) in serum for each study group following injection on Day 0.
[0049] Figures 29A - 29B: Following a 109 vp/kg intravenous bolus dose of SVV, SVV concentration was measured in the tissues of tumor-bearing mice (Figure 29A) and normal mice (no tumor) (Figure 29B). SVV replication is tumor selective. [0050] Figure 30: Immunohistochemistry analysis of SVV replication in tissues from a tumor-bearing mouse. Positive staining for SVV replication can be seen in the tumor sample; staining is not detected in other tissues.
[0051] Figures 31A - 31B: Figure 31A shows the increasing titer of anti-SVV neutralizing antibodies, indicating an immune response in the mouse. Figure 3 IB show the concentration of SVV in mouse tissues. SVV replicates in tumor cells despite the immune response.
[0052] Figures 32A - 32B: Figure 32A, In vivo efficacy of Seneca Valley Virus-001 (SVV-OOl) following systemic administration. A single intravenous injection of saline vehicle or SVV-OOl at 107 or 108 vp/kg (virus particles per kg) was given to athymic mice bearing pre-established human small-cell lung H446 xenograft tumors (n=10 per group) on study day 1 (denoted by \). Mean tumor volumes and 95% confidence intervals are plotted as a function of time. Figure 32B, Body weight change in A/J mice following intravenous injection of Seneca Valley Virus (SVV-OOl). Male and female A/J mice were injected with saline vehicle (n=25 per sex) or SVV-OOl at 1 x 1014 virus particles per kg (n=25 per sex) on study day 1. Plotted is body weight change relative to pre-dose weight over time.
[0053] Figure 33: Infectivity (IFT)-based TCID50 assay was used to monitor viral load in serum of patients with solid tumors expressing at least one neuroendocrine marker.
[0054] Figure 34: Quantitative, real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was used to monitor viral load in serum of patients with solid tumors expressing at least one neuroendocrine marker.
[0055] Figure 35: Viral neutralization in serum of patients with solid tumors expressing at least one neuroendocrine matker.
[0056] Figure 36: In one exemplary patient, serum RNA is degraded at later time points, indicating lack of full-length circulating viral RNA at later time points.
[0057] Figure 37: Anti-SVV immunohistochemistry in patient tissue shows specific anti- SVV staining in a liver metastasis, but not in surrounding normal liver tissue or in a panel of normal organs.
DETAILED DESCRIPTION OF THE INVENTION
[0058] The invention provides for the discovery that a monoclonal antibody, raised against surface proteins of an SVV permissive cell, blocks SVV infection of permissive cells. The invention further provides for the discovery that the antigen of the monoclonal antibody functions as a cellular receptor (or co-receptor) for SVV.
[0059] The invention further provides that upon identification of SVV receptors as tumor markers (i.e., where an SVV receptor is expressed or over-expressed in a tumor cell-type as compared to a normal cell of that cell-type (the normal cell either does not express the SVV receptor or expresses the SVV receptor at a lower level than the tumor cell); or where an SVV receptor is only expressed or over-expressed in a particular stage of a tumor), therapeutic molecules can be generated to target the SVV receptor. If it is determined that SVV or an SVV-like picornavirus can bind to the SVV receptor that is expressed or overexpressed in a tumor cell-type as compared to the normal cell-type, then SVV or the SVV-like picornavirus can be used to target and kill the tumor. Additionally, if it is determined that SVV or an SVV-like picornavirus can bind to the SVV receptor that is expressed or overexpressed in a tumor cell-type as compared to the normal cell-type, then SVV or the SVV-like picornavirus can be used in diagnostic methods to detect the tumor in a subject. Further, because certain strains of HIV use CXCR4 as a co-receptor, SVV or components or derivatives thereof can be used in methods for inhibiting or decreasing HIV infection in a subject. Cells that express CXCR4 and can be infected by HIV can also be examined to see whether these cells express or overexpress specific CXCR4 isoforms that can be bound by SVV or an SVV-like picornavirus.
[0060] The invention provides a hybridoma cell line, designated Anti-H446 MMAb 8G10 and having ATCC Accession No. , that produces a monoclonal antibody of the invention was deposited with the Patent Depository of the American Type Culture Collection (ATCC),
10801 University Blvd., Manassas, VA, 20110, on , under the provisions of the
Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of a Patent Procedure.
[0061] The invention provides a hybridoma cell line, designated Anti-H82 MMAb 17El 1 and having ATCC Accession No. , that produces a monoclonal antibody of the invention was deposited with the Patent Depository of the American Type Culture Collection (ATCC),
10801 University Blvd., Manassas, VA, 20110, on , under the provisions of the
Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of a Patent Procedure.
[0062] Terms [0063] The terms "virus," "viral particle," "virus particle," and "virion" are used interchangeably.
[0064] 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:1 and Figures 1A-1H for the SVV full-length genomic sequence), where the sequence comparison is not limited to a whole-genome analysis, but can be focused on a particular region of the genome, such as the 5'UTR, structural encoding regions, nonstructural encoding regions, and portions thereof. The particular length of the genome for sequence comparison that is adequate to determine relatedness/likeness to SVV is known to one skilled in the art, and the adequate length can very with respect to the percentage of identity that is present. The length for sequence comparison can be, for example, at least 20, 50, 100, 200, 300, 400, 500, 750, 1000, 1500, 2000, or 2500 nucleotides. Where the length is shorter, one skilled in the art understands, for example, that the identity between sequences can be higher in order to consider the two sequences to be related. However, such guidance is qualified at least with respect to considerations of sequence conservation, in that certain regions of the genome are more conserved than others between related species. Additionally, if an antiserum generated from a virus can neutralize SVV infection of an SVV permissive cell line, then the virus is considered to be an SVV-like picornavirus. Additionally, if an antiserum generated from a virus can neutralize SVV infection of an SVV permissive cell line, and that antiserum can also bind to other viruses (for example, if the antiserum can be used in indirect immunofluorescence assays to detect virus), then the other viruses that can be bound by the antiserum are considered to be SVV-like picornaviruses. For purposes of the invention, SVV-like picornaviruses can include cardioviruses. 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.
[0065] An "attenuated virus" or a "live attenuated virus" are viral mutants that are unable to cause disease or are less able to replicate or grow in a host, but the mutants retain their antigenicity such that they can induce immunogenic protection.
[0066] An "inactivated" or "killed" virus is a virus that is treated such that it cannot replicate. [0067] 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. et al. (1990) "Basic local alignment search tool." J. MoL Biol. 215:403-410)) or by visual inspection. The BLAST algorithm 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).
[0068] 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 difference (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.
[0069] As used herein, a "SVV receptor" refers to a receptor or co-receptor for SVV binding to a cell or entry into a cell or fusion into a cell. As used herein, a "SVV receptor" can also be referred to as a "cellular receptor for SVV" or a "cellular receptor of SVV." Examples of SVV receptors are CXCR4 and 8G10 antigen.
[0070] Seneca Valley Virus (SW) Genome and Proteins
[0071] SVV is an RNA virus, and with respect to previously characterized picornaviruses, SVV is most closely related to members from the Cardiovirus genus (Scraba, D. et al., "Cardioviruses (Picornaviridae)," in Encyclopedia of Virology, 2nd Ed., R.G. Webseter and A. Granoff, Editors, 1999) in the Picornaviridae family. The results of sequence analyses between SVV and other Cardioviruses are discussed in International Application No. PCT/US2004/031504), which is hereby incorporated by reference in its entirety. Since the time of the sequence analysis of SVV described in PCT/US2004/031504, the Picornavirus Study Group is considering whether to classify SVV as a member of a new genus. Figure 4 presents a phlyogenetic relationship tree between members of the Picornavirus family.
[0072] The genome of SVV consists of one single-stranded positive (+) sense strand RNA molecule having a size of about 7,290 nucleotides (see Figure 1 A-IH). As SVV is a picornavirus, it has a number of features that are conserved in all picornaviruses: (i) genomic RNA is infectious, and thus can be transfected into cells to bypass the virus-receptor binding and entry steps in the viral life cycle; (ii) a long untranslated region (UTR) at the 5 ' end of the genome (for SVV, nucleotides 1-666 of SEQ ID NO:1) and a shorter 3' untranslated region (for SVV, nucleotides 7210-7280 of SEQ ID NO:1); (iii) the 5' UTR contains a clover-leaf secondary structure known as the internal ribosome entry site (IRES); (iv) the rest of the genome encodes a single polyprotein (for SVV, nucleotides 667-7209 of SEQ ID NO:1 encode the polyprotein (SEQ ID NO:2)) and (v) both ends of the genome are modified, the 5' end by a covalently attached small, basic protein, "Vpg," and the 3' end by polyadenylation (nucleotides 7281-7290 of SEQ ID NO:1).
[0073] In a host cell, the SVV polyprotein is cleaved into a number of smaller proteins (Figure 5). DNA clones of the genome or portions thereof can be made by reverse- transcription of the SVV RNA genome. The DNA clones can be subcloned into expression plasmids, such that the expression plasmids can express any one of the SVV proteins or a portion thereof. Additionally, expression plasmids can be generated such that more than one SVV protein can be expressed as a single larger protein. The following table lists the nucleotides of SEQ ID NO:1 that encode the SVV proteins. The table also lists the amino acid sequences of the SVV proteins with respect to the polyprotein sequence listed in SEQ ID NO:2.
Table 1: SW Genome/Protein Features
Figure imgf000016_0001
Figure imgf000017_0001
[0074] SW Epidemiology
[0075] 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. However, with additional 5' sequence information since obtained, a meeting of the Picornavirus Study Group has considered whether to classify SVV in a separate new genus. Specifically, from the additional 5' sequence information, the Internal Ribosome Entry Sequence (IRES) of SVV has been compared to other Picornaviruses, and it has been determined that the SVV IRES is Type IV, whereas Cardiovirus IRES's are Type II. Additionally, multiple unique picornaviruses have been discovered at the USDA that are more similar to SVV than SVV is to other Cardioviruses. Therefore, these unique picornaviruses (which are SVV-like picornaviruses) and SVV will either be designated to be members of new genus of the Picornaviridae family or to be members of the genus Cardiovirus.
[0076] Several of the SVV-like picornaviruses discovered at the USDA are about 95-98% identical to SVV at the nucleotide level, and antisera against one virus (MN 88-36695) neutralizes SVV, and this virus is reactive to antisera that neutralizes SVV. These SVV-like picornaviruses were isolated from pigs, and thus, pigs are likely a permissive host for SVV and other SVV-like viruses. Examples of SVV-like picornaviruses isolated from pigs include, but are not limited to, 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. SVV-like picornaviruses may also include cardioviruses closely related to SVV (as determined by sequence analysis or by cross- reactivity to antibodies raised against SVV antigens).
[0077] SW Cellular Receptors [0078] In general, cell and tissue tropism of a virus is determined by specific cell surface receptor(s), internalization or post-internalization events. For SVV, the transfection of genomic RNA into nonpermissive cells results in virus production in the cells, indicating that the tropism is likely a pre-internalization event governed by the presence of cell surface receptors (see Example 1).
[0079] Expression Cloning
[0080] In one approach to identify cellular receptors involved in binding and/or internalization of SVV, transcription profiling was used to compare differential expression of genes encoding for cell surface proteins of permissive cell lines compared to non-permissive cell lines. Candidate genes (expressed in permissive cell lines but not expressed in non- permissive cell lines) were expressed in non-permissive cells to determine if the gene confers permissivity to infection by SVV.
[0081] A cellular receptor (or co-receptor) for SVV has been identified using an expression cloning approach. The cellular receptor identified is CXCR4. Confirmation that CXCR4 is a receptor for SVV is shown in Example 1. Differential analysis between the gene expression profiles from permissive and non-permissive cell lines indicated twelve initial receptor candidates. These twelve candidates were expressed in non-permissive cell lines to determine whether expression of the candidate could change the non-permissive cell line to become permissive to SVV infection (confer permissiveness to infection by SVV). Without being bound by theory, because CXCR4 is expressed on a variety of different cell-types that include cell-types that are non-permissive for SVV infection, it may be the case that a specific CXCR4 isoform is responsible for SVV tumor-specific tropism and/or that there are other SVV receptor(s) (or co-receptor(s)) for SVV binding, entry, and/or fusion.
[0082] Cellular receptors for SVV can also be identified by using a broad expression cloning approach, where cDNAs are transfected and expressed in a non-permissive cell line to determine whether expression of the cDNA can provide permissiveness to SVV infection. In one embodiment, the cDNAs can be transfected into the non-permissive cells in the form of an expression library, where the identity (i.e., the sequence or name of the protein product encoded by the cDNA) of the cDNA are unknown prior to transfection. In another embodiment, the identity of the cDNAs are known are prior to transfection. Whether the sequence of the cDNA is known or unknown prior to transfection, transfected cells can be screened in a high-throughput manner (see Figure 6 and Example 1). [0083] Monoclonal Antibody
[0084] In another approach to identify cell surface proteins that may be involved in binding and/or internalization of SVV, mouse monoclonal antibodies (mAbs) were raised to cell surface proteins of permissive cells as described in Example 3. A monoclonal antibody provided by the invention (for example, 8G10 mAb produced by a hybridoma cell having
ATCC Accession No. , and 17El 1 mAb produced by a hybridoma cell having ATCC
Accession No. ) recognizes an antigen (for example, 8G10 antigen or 17El 1 antigen) that is a SVV cellular receptor (or co-receptor) as demonstrated by, for example, the ability of the mAb to block virus entry into permissive cells in a dose dependent manner (see Figure 21) and to recognize proteins on the surface of permissive cells but not non-permissive cells (see Figures 18A - 18D). The 8G10 and 17El 1 antigens have not yet been identified.
[0085] Integrins
[0086] Integrins are proteins that span the cell membrane to link the extracellular space with the inside of a cell. Integrins serve as cellular receptors for other picornaviruses. An amino acid motif (LDV) known to bind integrin α4βl is found in a surface-exposed loop of SVV VP2, a capsid protein. Virus mutated at this site could not be rescued from culture.
[0087] 8G10 and 17E11 Monoclonal Antibodies
[0088] The 8G10 and 17El 1 mAbs recognize and form a complex with a surface protein on SVV permissive cells, thereby inhibiting SVV entry into the cells. Thus, the surface protein functions as an SVV receptor.
[0089] In one embodiment of the invention, the monoclonal antibody may be a chimeric antibody. A chimeric antibody is monoclonal antibody comprising constant region fragments from a species different from the species in which the monoclonal antibody was raised. For example, the 8G10 mAb was raised in a mouse and thus is a murine mAb; a chimeric 8G10 mAb would comprise constant region fragments from a different species. In another embodiment, the monoclonal antibody may be a humanized antibody. A humanized antibody is a non-human monoclonal antibody in which human protein sequences have been substituted for all of the non-human protein sequences except for the non-human complementarity determining regions (CDRs) of both the heavy and light chains (see U.S. Patent No. 5,824,307, U.S. Patent Application Publication Nos. US 2006/0228350, US 2005/0048617, US 2005/0042664). For example, to generate a humanized version of the murine 8G10 mAb, human protein sequences may be substituted for all of the murine protein sequences except for the murine CDRs of both the light and heavy chains.
[0090] A monoclonal antibody of the invention may be used to determine whether a subject with a tumor will respond to SVV treatment. For example, the monoclonal antibody may be linked to a detectable label and used to assay a tumor biopsy sample in vitro, or administered to the subject to image the tumor in vivo. A tumor is predicted to respond to SVV treatment if it is determined that the monoclonal antibody
[0091] A monoclonal antibody of the invention may be used in a method for delivering a compound to a tumor cell, comprising linking the compound to the antibody. Non-limiting examples of such compounds include therapeutic agents, such as a radioisotope, a toxin or a chemotherapeutic agent, and imaging agents such as a fluorophore or a radioisotope.
[0092] In one aspect, the invention provides methods of treating a tumor in a subject, comprising administering to the subject an effective amount of a monoclonal antibody which specifically binds and forms a complex with an antigen located on the surface of a tumor cell and thereby inhibits SVV entry into the cell, the antigen being an antigen to which a monoclonal antibody produced by a hybridoma cell designated Anti-H446 MMAb 8G10 deposited as ATCC Accession No. , or a hybridoma cell designated Anti-H82 MMAb
17El 1 deposited as ATCC Accession No. specifically binds. In one embodiment, the antibody is a monoclonal antibody produced by a hybridoma cell designated Anti-H446
MMAb 8G10 deposited as ATCC Accession No. , or a hybridoma cell designated Anti-
H82 MMAb 17El 1 deposited as ATCC Accession No. . In one embodiment, the antibody is a chimeric antibody. In one embodiment, the antibody is a humanized antibody. In one embodiment, the antibody comprises a human constant region and a heavy and light chain variable region, wherein the heavy and light chain variable region comprises heavy and light chain framework regions and heavy and light chain complementarity determining regions (CDRs), at least a portion of the heavy and light chain framework regions being derived from a human antibody, and the CDRs comprising heavy-chain CDRs light-chain CDRs derived from a monoclonal antibody produced by a hybridoma cell designated Anti-
H446 MMAb 8G10 deposited as ATCC Accession No. , or a hybridoma cell designated
Anti-H82 MMAb 17El 1 deposited as ATCC Accession No. .
[0093] CXCR4 Isoforms [0094] CXCR4 (C-X-C motif receptor 4; also known as fusion and CD 184) is a seven- transmembrane (7-TM) G-protein-coupled receptor (GPCR) that interacts with the chemokine stromal cell-derived factor- 1 alpha (SDF- lα; also known as CXCL 12). In addition to its main role as a chemokine receptor, CXCR4 has been shown to act as a co- receptor during infection of human cells by T-trophic HIV-I. In T cells, the gpl20 of T- trophic HIV-I interacts with CD4 and induces the formation of a trimolecular complex between gpl20, CD4, and CXCR4. This interaction is thought to be critical for subsequent conformational changes in the gpl20/gp41 that ultimately lead to the exposure of HIV gp41 fusion peptide and initiation of virus-cell fusion.
[0095] Human CXCR4 comprises 352 amino acids (see GenBank Accession No. P61703; also known as isoform 1 , isoform a, or isoform α) and the predicted molecular weight (MW) of non-glycosylated human CXCR4 is 39,746 Daltons. The predicted MW of glycosylated human CXCR4 is about 45-47 kDa. There is also an alternatively spliced form of CXCR4 (also called isoform 2 or isoform b) that comprises 356 amino acids (see GenBank Accession Nos. P61073-2, P30991-2). However, neither predicted glycosylation nor alternative splicing of CXCR4 mRNA account for all of the observed CXCR4 isoforms.
[0096] There are numerous post-translation modifications of CXCR4 that can account for many of the CXCR4 isoforms. Observed post-translation modifications of CXCR4 include, but are not limited to, N-glycosylation (Chabot et al, J. Virol. (2000) 74:4404-13), disulfide formation (Chabot et al, J. Virol. (1999) 73:6598-609), tyrosine sulfation (Farzan et al, J1 Biol. Chem. (2002) 277:29484-9; Farzan et al, CeU (1999) 96:667-76), serine chondroitin sulfation (Farzan (2002)), oligomerization (Lapham et α/.. Nat. Med. (1999) 5:303-8), and proteolysis (Lapidot and Petit, Exp. Hematol. (2002) 30:973; Levesque et al, J. Clin. Invest. (2003) 111 :187-96; Valenzuela-Fernandez et al, J. Biol. Chem. (2002) 277:15677-89); these references are hereby incorporated by reference in their entirety. These post-translational modifications affect approximately 9% of the extracellular residues of CXCR4.
[0097] CXCR4 isoforms can be identified by immunob lotting analysis of cell extracts (see Sloane, A. et al., Immunology and Cell Biology (2005) 83:129-143; which is hereby incorporated by reference in its entirety, and at least for its teachings on how to identify CXCR4 isoforms; and Example 2). For example, an immunoblot analysis can be conducted with equal amounts of cellular or membrane extracts of cell lines permissive and non- permissive to SVV infection. Cell extracts can be resolved in both denaturing and non- denaturing gel systems in one and/or two dimensions. Extracts can be run on such gels and transferred to a membrane, such as nitrocellulose. The nitrocellulose can then be probed with antibodies against CXCR4 in order to compare the presence and/or absence as well as changes in intensity of different molecular weight bands that indicate the different CXCR4 iso forms (see Table below). The nitrocellulose can also be probed using a VOPBA (Virus Overlay Protein Binding Assay). In a VOPBA, the nitrocellulose can be probed with SVV particles followed by anti-SVV antibody. The extracts can also be analyzed by immunoprecipitation - extracts are mixed with SVV and potential SVV-CXCR4 isoforms co- complexes are precipitated with anti-SVV antibody. The co-complexes are then resolved on a gel and transferred to a membrane (such as nitrocellulose), and the membrane is then detected using anti-CXCR4 antibodies to determine which isoform(s) were precipitated.
[0098] Specific molecular weight bands resolved on a gel that correspond to specific CXCR4 isoforms can be excised such that the isoforms can be used as immunogens for antibody generation, including monoclonal antibody generation. The bands can also be digested with trypsin such that mass spectrometry can be used to confirm that the band is CXCR4. Further, post-translational modifications of the isoform can be characterized by using protein modification enzymes such as glycosylases.
[0099] Other methods for identifying isoforms include, but are not limited to, chromatographic separation, electrophoretic separation (with subsequent immunoblotting as described above), lectin determination, release of oligosaccharides, and protease digestion. Chromatographic separation can comprise the identification of protein isoforms that have been separated by means of ion-exchange, affinity, size-exclusion, or chromato focusing. Affinity interactions with lectins also offers separation possibilities. (For more detail on these techniques, see: Hage, D. S., "Affinity chromatography: a review of clinical applications," Clin. Chem. (1999) 45(5): 593-615; Tang, Z. and Karnes, H.T., "Coupling immunoassays with chromatographic separation techniques," Biomed. Chromatogr. (2000) 14:442-449; Mackiewicz, A. and Kushner, L, "Affinity electrophoresis for studies of mechanisms regulating glycosylation of plasma proteins," Electrophoresis, 10: 830-835; Rudd, P.M. et ah, "A high-performance liquid chromatography based strategy for rapid, sensitive sequencing of N-linked oligosaccharide modification to proteins in sodium dodecyl sulphate polyacrylamide electrophoresis gel band," Proteomics, 1 : 285-294; Hounsell, E. F. (1997) "Method of glycoconjugate analysis," in Glycoscience: status and perspectives, eds. Gabius, H. and Gabius, S., Chapman & Hall, London, pp. 15-29; all of which are incorporated by reference.) [00100] It has been determined that there are at least eleven different CXCR4 isoforms as reflected by differences in molecular weight (there are at least twelve isoforms when the alternative transcript variant is included). The Table below (adapted from Sloane (2005)) shows which anti-CXCR4 antibodies can detect which isoforms (the references cited in the Table are hereby incorporated by reference in their entirety and at least for their disclosure regarding the relevant antibody):
Table 2: Antibodies Reactive with CXCR4 Isoforms
Figure imgf000023_0001
Figure imgf000024_0001
"ND" = not determined; "pAb" = D ittered- antibody
[00101] The 34 kDa species is a non-glycosylated monomer. The 68 kDa species is a non-glycoslyated dimer. The 40, 47, 73 and 80 kDa species are glycosylated, most likely at the N-terminus {i.e., extracellular region) of CXCR4. The 40 kDa species is a glycosylated monomer. The 80 kDa species is a glycosylated dimer. The 73 kDa species may be a dimer comprising a 40 kDa glycosylated monomer and a 34 kDa non-glycosylated monomer. The MW of CXCR4 isomers that are glycosylated may vary slightly (for example, from about 1 to about 5 kDa) between cell-types because different cell-types may provide different glycosylation patterns on a protein.
[00102] CXCR4 isoforms of about 62 kDa, of about 75-80 kDa, and of about 95-100 kDa have also been reported. These isoforms may be CXCR4-ubiquitin complexes. A 54 kDa isoform has also been reported. The invention is not limited to the isoforms described and referred to herein.
[00103] Potential CXCR4 Isoform(s) Specific to SW
[00104] In one embodiment, the invention provides methods for identifying which
CXCR4 isoform(s) is capable of binding to SVV. Extracts can be prepared from cells that are permissive and non-permissive for SVV infection. The extracts can be whole cell extracts, or extracts of specific cell compartments, such as a membrane extract. The extracts from the permissive and non-permissive cell-lines can be compared in order to determine whether there are any CXCR4 isoforms that are only present in the extracts from permissive cell-lines. The extracts from the permissive and non-permissive cell-lines can be compared in order to determine whether there are any CXCR4 isoforms that are overexpressed only in permissive cell-lines, where overexpression of a CXCR4 isoform may allow or contribute to permissivity.
[0100] Extracts from non-permissive and permissive cell lines of a particular tumor can be compared to determine whether there are any CXCR4 isoforms that are only present or overexpressed in the extracts from permissive cell-lines of a particular tumor. For example, a comparison can be conducted between non-permissive and permissive SCLC cells (see Table below) to determine if any of the permissive SCLC cells express a CXCR4 isoform that is not expressed or underexpressed in any of the non-permissive SCLC cells. Comparisons are not limited to tumor cell-types, but can also include comparisons between a permissive tumor- cell type that is metastatic (or even metastatic at a particular site) and a non-permissive tumor-cell type that is metastatic. For example, a comparison can be conducted between a permissive SCLC cell-type that is metastatic at the bone marrow (i.e., NCI-H209) and a non- permissive SCLC cell-type that is metastatic at the bone marrow (i.e, NCI-H2195). It may be the case that there are different CXCR4 isoforms expressed in different types of tumors that provide permissiveness to SVV infection.
[0101] Comparisons can be conducted, for example, by immunoblot assays. Cells that are permissive or non-permissive for SVV infection are listed in the Table below. The Table below also indicates the tumor cell-type, the state of the cancer (i.e, if metastatic), and if metastatic, the site of metastasis.
Table 3: In Vitro Cytolytic Potency and Selectivity of SW
Cell Line Species Stage State Organ Type Metastatic Site EC50*
PERMISSIVE
Y79 Human Adult Cancer Eye, Retina Retinoblastoma 0 00035, 0 0007
NCI-H446 Human Adult Metastatic Lung Variant Small Cell Pleural effusion 0 0012, 0 002,
Cancer Lung Carcinoma 0 0007
(SCLC)
N1 E-115 Murine Adult Cancer Brain Neuroblastoma 0 0028, 0 001
NCI-M 770 Human Adult Metastatic Lung Non-Small Cell Lung Lymph Node 0 00724
Cancer Carcinoma (NSCLC)
NCI-H82 Human Adult Metastatic Lung Variant Small Cell Pleural effusion 0 015
Cancer Lung Carcinoma
(SCLC)
PER C6® Human Fetal Cancer Eye, Retina Retinoblast 0 02, 0 0049
NCI-H69AR Human Adult Cancer Lung Small Cell Lung 0 035, 0 05
Carcinoma, multidrug resistant (SCLC)
SK-NEP-1 Human Adult Metastatic Kidney Wilms' Tumor Pleural effusion 0 03
Cancer
IMR-32 Human Adult Cancer Brain Neuroblastoma 0 035, 0 0059,
0 05
NCI-H187 Human Adult Metastatic Lung Classic Small Cell Pleural effusion 0 00343
Cancer Lung Carcinoma
(SCLC) Cell Line Species Stage State Organ Type Metastatic Site EC50*
NCI-H209 Human Adult Metastatic Lung Small Cell Lung Bone Marrow 0 04
Cancer Carcinoma (SCLC)
NCI-M 184 Human Adult Metastatic Lung Small Cell Lung Lymph Node O 155
Cancer Carcinoma (SCLC)
D283 Med Human Adult Metastatic Brain, Medulloblastoma Peritoneum 0 25
Cancer Cerebellum
SK-N-AS Human Adult Metastatic Brain Neuroblastoma Bone Marrow 0 474
Cancer
BEK PCB3E1 Bovine Fetal Normal, Ad5 Kidney Ad5E1 transformed 0 99 transformed
ST Porcine Fetal Normal, Testis 5 9 immortalized
NCI-M 299 Human Adult Metastatic Lung Large Cell Lung Lymph Node 7 66, 4 8
Cancer Carcinoma
DMS 153 Human Adult Metastatic Lung Small Cell Lung Liver 9 2
Cancer Carcinoma (SCLC)
NCI-H295R Human Adult Cancer Adrenal Gland, Adrenocortical 16 5
Cortex Carcinoma
BEK Bovine Fetal Normal, Kidney 17 55 immortalized
PPASMC Porcine Adult Normal, Lung, Smooth Muscle Cells 18 4
Primary Pulmonary
Artery
PCASMC Porcine Adult Normal, Heart, Coronary Smooth Muscle Cells 11 9
Primary Artery
PAoSMC Porcine Adult Normal, Heart, Aorta Smooth Muscle Cells 88
Primary
NCI-H526 Human Adult Metastatic Lung Variant Small Cell Bone Marrow 46 4
Cancer Lung Carcinoma
(SCLC)
OVCAR-3 Human Adult Cancer Ovary Adenocarcinoma 39
ESK-4 Porcine Fetal Normal, Kidney Fibroblast 60
Immortalized
S W- 13 Human Adult Cancer Adrenal Gland, Small Cell <100
Cortex Adenocarcinoma
293 Human Fetal Normal, Ad5 Kidney Ad5 transformed 0 036, 184 8 transformed
Hs 578T Human Adult Cancer Breast Carcinoma 273
Hs 1 Tes Human Fetal Normal, Testis 416
Immortalized
LOX IMVI Human Adult Cancer Skin Melanoma 569
PK(15) Porcine Adult Normal, Kidney 1144, 129
Immortalized
NON
PERMISSIVE
WI-38 Human Fetal Normal, Lung Fibroblast >10,000
Immortalized
IMR-90 Human Fetal Normal, Lung Fibroblast >10,000
Immortalized
MRC-5 Human Fetal Normal, Lung Fibroblast >10,000
Immortalized
HCN-1A Human Adult Normal, Brain, Cortical >10,000
Immortalized Neuron
HMVEC Human Adult Normal, Skin Microvascular >10,000
(neon Primary Endothelial Cells atal)
HMVEC Human Adult Normal, Skin Microvascular >10,000
Primary Endothelial Cells
HUVEC Human Adult Normal, Umbilical Vein Endothelial Cells >10,000
Primary
HRE Human Adult Normal, Kidney Epithelial Cells >10,000
Primary
HRCE Human Adult Normal, Kidney Cortical Epithelial >10,000
Primary Cells
PHH Human Adult Normal, Liver Hepatocyte >10,000
Primary
HCASMC-c Human Adult Normal, Heart, Coronary Smooth Muscle Cells >10,000
Primary Artery
HCAEC Human Adult Normal, Heart, Coronary Endothelial Cells >10,000
Primary Artery
HAEC Human Adult Normal, Heart, Aorta Endothelial Cells >10,000 Cell Line Species Stage State Organ Type Metastatic Site EC50*
Primary
HAoSMC-c Human Adult Normal, Heart, Aorta Smooth Muscle Cells >10,000 Primary
NHA Human Adult Normal, Brain Astrocytes 1713 Primary
HPASMC Human Adult Normal, Lung Smooth Muscle Cells >10,000 Primary
PBMC Human Adult Normal, Peripheral Blood Mononuclear Cells >10,000 Primary
SF-295 Human Adult Cancer Brain Glioblastoma >10,000
U251 Human Adult Cancer Brain Glioblastoma >10,000
SF-539 Human Adult Cancer Brain Glioblastoma >10,000
SNB-19 Human Adult Cancer Brain Glioblastoma >10,000
SF-268 Human Adult Cancer Brain Glioblastoma 3103
U-1 18MG Human Adult Cancer Brain Glioblastoma, >10,000
Astrocytoma
SNB-75 Human Adult Cancer Brain Astrocytoma >10,000 M059K Human Adult Cancer Brain, Glial Cell Malignant 1061
Glioblastoma
KK Human Adult Cancer Brain, Glial Cell Glioblastoma >10,000
HCC-2998 Human Adult Cancer Colon Carcinoma >10,000
KM12 Human Adult Cancer Colon Carcinoma >10,000
HT-29 Human Adult Cancer Colon Adenocarcinoma >10,000
HCT 116 Human Adult Cancer Colon Carcinoma >10,000
HCT-15 Human Adult Cancer Colon Carcinoma >10,000
COLO 205 Human Adult Metastatic Colon Adenocarcinoma Ascites >10,000
Cancer
SW620 Human Adult Metastatic Colon Colorectal Carcinoma Lymph Node 6503 , >10,000
Cancer
PC3M-2AC6 Human Adult Cancer Prostate >10,000
PC3M-2AC6 + Human Adult Cancer Prostate ND
2-AP
PC-3 Human Adult Metastatic Prostate Adenocarcinoma Bone >10,000 Cancer
LNCaP FGC Human Adult Metastatic Prostate Adenocarcinoma Lymph Node >10,000 Cancer
DU 145 Human Adult Metastatic Prostate Adenocarcinoma Brain >10,000 Cancer
Hep3B Human Adult Cancer Liver Hepatocellular >10,000 Carcinoma
Hep G2 Human Adult Cancer Liver Hepatocellular >10,000 Carcinoma
786-0 Human Adult Cancer Kidney Clear Cell >10,000 Adenocarcinoma
TK- 10 Human Adult Cancer Kidney Carcinoma >10,000
RXF 393 Human Adult Cancer Kidney Carcinoma >10,000
Uθ-31 Human Adult Cancer Kidney Carcinoma >10,000
SN12C Human Adult Cancer Kidney Carcinoma >10,000
A-498 Human Adult Cancer Kidney Carcinoma >10,000
ACHN Human Adult Cancer Kidney Carcinoma >10,000
SW839 Human Adult Cancer Kidney Renal Clear Cell >10,000 Adenocarcinoma
Cakι-1 Human Adult Metastatic Kidney Clear Cell Skin >10,000 Cancer Adenocarcinoma 5637 Human Adult Cancer Bladder Carcinoma >10,000
NCI-H1339 Human Adult Cancer Lung >10,000
NCI-H1514 Human Adult Cancer Lung >10,000
A549 Human Adult Cancer Lung Carcinoma >10,000
S8 Human Adult Cancer Lung Carcinoma >10,000
NCI-H727 Human Adult Cancer Lung Carcinoid >10,000
NCI-H835 Human Adult Cancer Lung Carcinoid >10,000
UMC-11 Human Adult Cancer Lung Carcinoid >10,000
DMS 1 14 Human Adult Cancer Lung Small Cell Lung >10,000 Carcinoma (SCLC) Cell Line Species Stage State Organ Type Metastatic Site EC50*
DMS 53 Human Adult Cancer Lung Small Cell Lung >10,000
Carcinoma (SCLC)
NCI-H69 Human Adult Cancer Lung Small Cell Lung >10,000
Carcinoma (SCLC)
NCI-H2195 Human Adult Metastatic Lung Small Cell Lung Bone Marrow >10,000
Cancer Carcinoma (SCLC)
DMS 79 Human Adult Metastatic Lung Small Cell Lung Pleural effusion >10,000
Cancer Carcinoma (SCLC)
NCI-H146 Human Adult Metastatic Lung Classic Small Cell Bone Marrow >10,000
Cancer Lung Carcinoma
(SCLC)
NCI-H1618 Human Adult Metastatic Lung Classic Small Cell Bone Marrow >10,000
Cancer Lung Carcinoma
(SCLC)
NCI-H345 Human Adult Metastatic Lung Classic Small Cell Bone Marrow >10,000
Cancer Lung Carcinoma
(SCLC)
HOP-62 Human Adult Cancer Lung Non-Small Cell Lung >10,000
Carcinoma (NSCLC)
EKVX Human Adult Cancer Lung Non-Small Cell Lung >10,000
Carcinoma (NSCLC)
HOP-92 Human Adult Cancer Lung Non-Small Cell Lung >10,000
Carcinoma (NSCLC)
NCI-H522 Human Adult Cancer Lung Non-Small Cell Lung >10,000
Carcinoma (NSCLC)
NCI-H23 Human Adult Cancer Lung Non-Small Cell Lung >10,000
Carcinoma (NSCLC)
NCI-H322M Human Adult Cancer Lung Non-Small Cell Lung >10,000
Carcinoma (NSCLC)
NCI-H226 Human Adult Metastatic Lung Squamous Cell Pleural effusion >10,000
Cancer Carcinoma,
Mesothelioma
(NSCLC)
NCI-H460 Human Adult Metastatic Lung Large Cell Lung Pleural effusion >10,000
Cancer Carcinoma
HeLa, HeLa Human Adult Cancer Cervix Adenocarcinoma >10,000
CCRF-CEM Human Adult Cancer Peripheral Acute Lymphoblastic >10,000
Blood, T Leukemia (ALL) lymphoblast
MOLT-4 Human Adult Cancer Peripheral Acute Lymphoblastic >10,000
Blood, T Leukemia (ALL) lymphoblast
RPMI 8226 Human Adult Cancer Peripheral Plasmacytoma, >10,000
Blood, B Myeloma lymphocyte
SR Human Adult Metastatic Lymphoblast Large Cell Pleural effusion >10,000
Cancer Lymphoblastic
Lymphoma
HL-60(TB) Human Adult Cancer Peripheral Acute Promyelocytic >10,000
Blood, Leukemia (APL)
Promyleoblast
K-562 Human Adult Metastatic Bone Marrow Chronic Myelogenous Pleural effusion >10,000
Cancer Leukemia (CML)
UACC-257 Human Adult Cancer Skin Melanoma >10,000
M14 Human Adult Cancer Skin Melanoma >10,000
UACC-62 Human Adult Cancer Skin Melanoma 6614
SK-MEL-2 Human Adult Cancer Skin Malignant Melanoma >10,000
SK-MEL-28 Human Adult Cancer Skin Malignant Melanoma >10,000
A375 S2 Human Adult Cancer Skin Malignant Melanoma >10,000
SK-MEL-28 Human Adult Cancer Skin Malignant Melanoma >10,000
SK-MEL-5 Human Adult Metastatic Skin Malignant Melanoma Lymph Node >10,000
Cancer
MALME-3M Human Adult Metastatic Skin Malignant Melanoma Lung >10,000
Cancer
BT-549 Human Adult Cancer Breast Ductal Carcinoma >10,000
NCI/ADR-RES Human Adult Cancer Breast Carcinoma >10,000
MCF7 Human Adult Metastatic Breast Adenocarcinoma Pleural effusion >10,000
Cancer
MDA-M B-231 Human Adult Metastatic Breast Adenocarcinoma Pleural effusion >10,000
Cancer Cell Line Species Stage State Organ Type Metastatic Site EC50*
T-47D Human Adult Metastatic Breast Ductal Carcinoma Pleural effusion >10,000
Cancer
MDA-MB-435 Human Adult Metastatic Breast Ductal Pleural effusion >10,000
Cancer Adenocarcinoma
IGR-OV1 Human Adult Cancer Ovary Carcinoma >10,000
OVCAR-4 Human Adult Cancer Ovary Adenocarcinoma >10,000
OVCAR-5 Human Adult Cancer Ovary Adenocarcinoma >10,000
OVCAR-8 Human Adult Cancer Ovary Adenocarcinoma >10,000
SK-OV-3 Human Adult Metastatic Ovary Adenocarcinoma Ascites >10,000
Cancer
BxPC-3 Human Adult Cancer Pancreas Adenocarcinoma >10,000
AsPC-1 Human Adult Metastatic Pancreas Adenocarcinoma Ascites >1000
Cancer
NCI-H295 Human Adult Cancer Adrenal Gland, Adrenocortical >10,000
Cortex Carcinoma
TT Human Adult Cancer Thyroid Medullary Carcinoma >10,000
C8-D30 Murine Adult Normal Brain, >10,000
Cerebellum
LLC1 Murine Adult Cancer Lung Lewis Lung >10,000
Carcinoma
RM-1 Murine Adult Cancer Prostate >10,000
M LTC- 1 Murine Adult Cancer Testis Leydig Cell Tumor >10,000
KLN 205 Murine Adult Cancer Lung Squamous Cell >10,000
Carcinoma
CMT-64 Murine Adult Cancer Lung Small Cell Lung >10,000
Carcinoma (SCLC)
CMT-93 Murine Adult Cancer Rectum Polyploid Carcinoma >10,000
B 16-FO Murine Adult Cancer Skin Melanoma >10,000
RM-2 Murine Adult Cancer Prostate >10,000
RM-9 Murine Adult Cancer Prostate >10,000
Neuro-2A Murine Adult Cancer Brain Neuroblastoma >10,000
FBRC Bovine Fetal Eye, Retina >10,000
MDBK Bovine Adult Normal, Kidney >10,000
Immortalized
CSL 503 Ovine Adult Normal, Lung Ad5E1 transformed >10,000
Immortalized
OFRC Ovine Adult Normal, Eye, Retina Ad5E1 transformed >10,000
Immortalized
PC- 12 Rat Adult Cancer Adrenal Gland Pheochromocytoma >10,000
Vera Monkey Adult Normal, Kidney >10,000
Immortalized
PAOEC Porcine Adult Normal, Heart, Aorta Endothelial Cells >10,000
Primary
PCAEC Porcine Adult Normal, Heart, Coronary Endothelial Cells >10,000
Primary Artery
PPAEC Porcine Adult Normal, Lung, Endothelial Cells >10,000
Primary Pulmonary
Artery
TBD
NCI-H289 Human Adult Cancer Lung TBD
NCI-H1963 Human Adult Cancer Lung Small Cell Lung TBD
Carcinoma (SCLC)
NCI-H2227 Human Adult Cancer Lung Small Cell Lung TBD
Carcinoma (SCLC)
NCI-H378 Human Adult Metastatic Lung Classic Small Cell Pleural effusion TBD
Cancer Lung Carcinoma
(SCLC)
NCI-H2107 Human Adult Metastatic Lung Small Cell Lung Bone Marrow TBD
Cancer Carcinoma (SCLC)
HCC970 Human Adult Metastatic Lung Small Cell Lung Bone Marrow TBD
Cancer Carcinoma (SCLC)
HCC33 Human Adult Metastatic Lung Small Cell Lung Pleural effusion <1000/TBD
Cancer Carcinoma (SCLC)
BON Human Adult Cancer Pancreas Carcinoid TBD
H1T-T15 Hamster Adult Normal, Pancreas Islets of Langerhans, TBD
Immortalized b-cell Cell Line Species Stage State Organ Type Metastatic Site EC50*
*EC50 determined after
3 days except where noted
[0102] Comparisons can be between any permissive cell and any non-permissive cell. Comparisons can also be conducted between non-permissive cell-lines that do not express CXCR4 and these cell-lines when transfected with a CXCR4 expression construct. Comparisons can also include, but are not limited to, selections from the following pairs listed in the Table below:
Table 4: Exemplary Comparisons to Identify CXCR4 Isoforms Specific for SW in a
Particular Type of Cancer
Figure imgf000030_0001
[0103] An SVV-specific CXCR4 isoform can be used to identify whether a subject has a tumor that can be treated with SVV therapy. For example, tumor cells from a subject can be isolated in order to determine whether the cells express a particular SVV-specific CXCR4 isoform (this can be assayed, for example, by immunob lotting or by FACS analysis with an antibody specific to the SVV-specific CXCR4 isoform).
[0104] 8G10 Antigen
[0105] The 8G10 antigen has not yet been identified. Several techniques known to one of ordinary skill in the art can be used to identify the protein recognized by the 8G10 mAb. For example, the 8G10 mAb can be used to DitteredDcipitated the 8G10 antigen from membrane extracts prepared from cells that are permissive for SVV infection. The isolated antigen can be digested with a protease, such as trypsin, such that mass spectrometry can be used to identify the antigen. In another exemplary technique, cell extracts can be resolved in both denaturing and non-denaturing gel systems in one and/or two dimensions. An immunoblot of the gel with the 8G10 mAb can be used to identify the protein on the gel that corresponds to the protein recognized by the 8G10 mAb on the immunoblot. The identified protein can be extracted from the gel and subjected to mass spectrometry to identify the antigen. In an additional method to identify the 8G10 antigen, the proteins comprising the proteome of an SVV-permissive cell can be analyzed for 8G10 mAb reactivity on a protein array.
[0106] In addition to the examples described above, the 8G10 antigen can be analyzed for binding to SVV, as described for CXCR4.
[0107] If it is determined that the 8G10 antigen is expressed on a variety of cell types, including cell types that are non-permissive for SVV infection, it may be the case, as discussed above for CXCR4, that a specific isoform of the 8G10 antigen is responsible for SVV tumor-specific tropism and/or that there is another unidentified SVV receptor(s) (or co- receptor(s)) for SVV binding, entry, and/or fusion. Without being bound by theory, cell type tropism of SVV may result from (1) expression of a particular 8G10 antigen(s), (2) overexpression of a particular 8G10 antigen(s), (3) expression of a particular 8G10 antigen isoform(s), and/or (4) overexpression of a particular 8G10 antigen isoform(s).
[0108] An SVV-specific 8G10 antigen can be used to determine whether a subject has a tumor that can be treated with SVV therapy. For example, tumor cells from a subject can be isolated in order to determine whether the cell express an SVV-specific 8G10 antigen (this can be assayed, for example, by immunoblotting or by FACS analysis with an antibody specific to the 8G10 antigen (i.e., the 8G10 mAb)).
[0109] Relating SW Receptors to Tumor Specificity
[0110] As described above, it is possible that there are particular 8G10 antigens and/or particular CXCR4 isoforms that function as SVV receptors and provide permissiveness to SVV infection. It may also be possible that such SVV-specific receptors can account for SVV permissiveness in a particular type of tumor, including a tumor cell-type and a tumor tissue type. It may also be possible that such SVV-specific SVV receptors can account for SVV permissiveness in a particular stage of tumor (i.e, benign, malignant, or metastatic). An SVV receptor can provide permissiveness by virtue that it is expressed in a cell or by virtue that it is overexpressed or upregulated as compared to a cell that is not permissive.
[0111] When an SVV receptor is identified, it is asked whether this receptor is also a tumor- specific marker (biomarker), i.e., whether the receptor (or isoform of the receptor) is expressed in a particular tumor cell-type, tumor tissue-type, or stage of tumor and not expressed in the corresponding normal cell-type or tissue-type or different stage of tumor; or whether the antigen is overexpressed only on a particular tumor cell and not overexpressed in the corresponding normal cell. In the oncolytic virus field, the presence of cellular receptors to the virus can serve as predictive biomarkers if the mechanism of tropism is receptor- mediated. Thus, predictive biomarkers, such as SVV receptors, can be used to define patient populations most likely to benefit from treatment, for example a treatment comprising SVV, and to dramatically reduce the time and cost of drug development.
[0112] If an SVV receptor is also a tumor-specific marker, then the receptor can be a target for cancer therapy. It may also be the case that a particular SVV receptor does not confer SVV tropism, but rather the expression level of an SVV receptor confers permissiveness. For example, it is possible that higher or lower expression levels are required for SVV permissiveness depending on the SVV receptor. For example, in the case of an SVV-specific CXCR4 isoform, there is a very good correlation between CXCR4 protein expression as measured by FACS (fluorescence activated cell sorter) with 12G5 antibody and the EC50s of SVV on SCLC cell lines (see Table 5 below).
Table 5: CXCR4 Expression in SCLC
Figure imgf000032_0001
[0113] From Table 2, it is known that 12G5 can detect the 52, 47, 42, 40, and 34 kDa CXCR4 iso forms. It may be the case that one of these isoforms is specifically expressed in permissive cells and not expressed in non-permissive cells - where only the specifically expressed isoform in permissive cells can bind to SVV. Alternatively, it is possible that more than one of these iso forms can bind to SVV and are specifically expressed in permissive cells. Alternatively, it is possible that isoforms that can bind to SVV are expressed in both permissive and non-permissive cells, and a threshold level of expression of isoform(s) that can bind to SVV accounts for permissivity. Thus, in one embodiment, the invention provides a method for treating SCLC by administering to a subject SVV (or an SVV-like picornavirus), wherein the subject is afflicted with a tumor comprising a SCLC cell that specifically expresses or overexpresses one or more CXCR4 isoforms that can bind to SVV (or an SVV-like picornavirus).
[0114] Therapy can include, for example, a small molecule, an antibody, an antibody conjugate, SVV, a derivative of SVV (including a genetically modified virus, a viral chimera using portions of SVV), a peptide or protein fragment of SVV (including a portion of the capsid that can bind to CXCR4 such that the peptide/protein can compete or inhibit SDF- lα binding to CXCR4), an anti-idiotypic antibody or other engineered protein representing the portion of SVV that binds to an SVV receptor, or any combination thereof.
[0115] Thus, in certain embodiments, the invention provides methods for treating cancer in a subject where the subject is afflicted with a tumor cell that expresses or overexpresses an SVV receptor that is capable of binding SVV. In one embodiment, the tumor cell expresses or overexpresses an SVV-specific receptor and the receptor is not expressed or is expressed at lower levels in a normal or non-tumor cell. For example, the SVV receptor can be an SVV- specific CXCR4 isoform that has, for example, a MW of about 110, 101, 90-95, 80-83, 68- 75, 62, 52, 47, 42, 40, or 34 kilodaltons. The cancer can be treated by administering to a subject having cancer an SVV particle or an SVV-like picornavirus particle, such that a cancer cell in the subject is infected and killed by the particle (for example, infection by the SVV or SVV-like picornavirus particle can cause the cancer cell to apoptose). The cancer can be treated by administering to a subject having cancer an SVV protein or peptide (or SVV-like protein or peptide) that can bind to an SVV receptor (for example, an SVV protein or peptide that can bind to a CXCR4 isoform such that it competes with SDF- lα for binding); the SVV or SVV-like picornavirus protein or peptide can be VPl, VP2, VP3, VP4, or any portion or combination thereof.
[0116] For example, a large number of tumors express CXCR4, including but not limited to, B-CLL, AML, B-lineage ALL, intraocular lymphoma, Non-Hodgkin lymphoma, follicular center lymphoma, CML, multiple myeloma, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, thyroid cancer, colorectal cancer, oral squamous carcinoma, cervical cancer, neuroblastoma, kidney cancer, glioma, astrocytoma, rhabdomyosarcoma, SCLC, and melanoma. Primary cells or cell-lines of these tumors (and other tumors that express CXCR4) can be assessed for CXCR4 isoform overexpression (or upregulation) as compared to normal cells. Primary cells or cell-lines of these tumors (and other tumors that express CXCR4) can be assessed for CXCR4 isoform specific expression as compared to normal cells, i.e., the normal cells do not express a CXCR4 isoform(s) that is expressed in the tumor cell. Primary cells or cell-lines of these tumors (and other tumors that express CXCR4) can be assessed for CXCR4 isoform specific expression in a particular stage of the tumor, i.e., is there a specific CXCR4 isoform expressed (or overexpressed) in a metastatic tumor as compared to a benign tumor (or malignant vs. benign, or metastatic vs. malignant, etc.). If it is determined that a CXCR4 isoform(s) is a tumor biomarker (i.e, if the isoform(s) is specifically expressed on a tumor cell or is specifically overexpressed or upregulated in a tumor cell, or is specifically expressed/overexpressed in a particular stage of a tumor), then the isoform(s) are tested to see whether they can bind an SVV or an SVV-like picornavirus particle. Isoforms that are tumor biomarkers are targets for cancer therapy, and isoforms that are tumor biomarkers and can also bind SVV or an SVV-like picornavirus are targets for SVV or SVV-like picornavirus mediated cancer therapy.
[0117] If an SVV receptor is identified to be a tumor biomarker and if it does not bind to SVV or to an SVV-like picornavirus, SVV or an SVV-like picornavirus can be mutated such that the tropism of the virus is altered to become specific for the isoform (see PCT/US2004/031504 for methods for altering tropism).
[0118] If an SVV receptor is identified to be a tumor biomarker and it does bind to SVV or to an SVV-like picornavirus, SVV or an SVV-like picornavirus can also be used as gene- therapy vectors such that they can infect the tumor cell and express a heterologous gene that can trigger apoptosis, inhibit cell cycle progression, or otherwise kill the tumor or otherwise inhibit tumor replication. SVV or an SVV-like picornaviruses can be used as vehicles to deliver toxins or poisons to tumor cells by virtue of tumor-cell specific tropism; SVV or an SVV-like picornavirus can be used to specifically target a particular type of tumor or stage of tumor if the tumor or stage of tumor expresses an SVV receptor that can bind to SVV or an SVV-like picornavirus.
[0119] Methods for Treating HIV [0120] In another embodiment of the invention, because SVV binds to CXCR4, SVV can be used as a method for treating or inhibiting HIV virus infection. CXCR4 has been shown to play a central role in both chemotaxis and HIV-I entry into T lymphocytes. It is generally understood that CXCR4 is a co-receptor for T-trophic (T-cell trophic) HIV-I strains (X4 isolates), and CCR5 is a co-receptor for M-trophic (monocyte/macrophage trophic) HIV-I strains (R5 isolates). T-tropic strains can infect macrophages (or MDMs - monocyte-derived macrophages), but at a much lower efficiency that than of M-trophic strains. There are also HIV-I strains that are dual-tropic, in that they can utilize either CXCR4 or CCR5 as a co- receptor, and therefore can infect T-cells, monocytes and macrophages that express one or both of these co-receptors and CD4.
[0121] Macrophages can be infected more efficiently be M-tropic HIV-I strains even though macrophages express both CXCR4 and CCR5. Western blot analyses of total cell extracts and surface proteins from multiple sets of monocytes and macrophages demonstrate substantial differences between CXCR4 molecules. (See Lapham et ah, Nature Med. (1999) 5:303-8.) CXCR4 is mainly a monomer in monocytes and a species of higher molecular weight (90 kDa) in macrophages.
[0122] Although SVV is not oncolytic for non-tumor cells, and therefore is not oncolytic for normal T-cells, monocytes, or macrophages, SVV can still be used in methods to treat or inhibit HIV infection. If SVV can bind the CXCR4 isoform(s) expressed on T-cells, monocytes, and macrophages, then SVV can be used to inhibit HIV infection - for example, by competing with HIV for binding to CXCR4.
EXAMPLES
[0123] The examples described below are provided to illustrate the present invention and are not included for the purpose of limiting the invention.
EXAMPLE 1 : THE MECHANISM OF SVV TROPISM IS RECEPTOR-MEDIATED
[0124] To confirm that the mechanism for SVV tropism is primarily receptor mediated, SVV viral RNA was transfected into non-permissive cell lines and virus production was assayed. Non-permissive cell were transfected with SVV viral RNA (a β-galactosidase reporter plasmid was used to determine the trans fection efficiency of the non-permissive cell lines; SVV viral RNA was transfected into cell lines that had a 20% or higher transfection efficiency). After non-permissive cells were transfected with SVV viral RNA, the cells were cultured for about 18 hours before crude viral lysates (CVLs) were prepared. The viral lysates were Dittered on PER.C6 cells. The results are shown in Table 6 below, and indicate that SVV tropism is primarily receptor mediated because trans fection of viral RNA allows virus production in nonpermissive cell lines.
Table 6: SW Tropism is Primarily Receptor Mediated
Figure imgf000036_0001
^Negative results for DMS53 may indicate that there is an intracellular mechanism or another receptor/co-receptor that contributes to tropism.
EXAMPLE 2: CXCR4 IS A RECEPTOR FOR SVV INFECTION
[0125] An expression cloning approach was used to identify a cellular receptor for SVV. Gene expression arrays (Affymetrix Inc., Santa Clara, CA) were performed on permissive and non-permissive cell lines. The arrays showed that there are at least seventeen candidates for an sw cellular receptor. A targeted expression cloning approach was used to determine whether expression of any of the seventeen candidates could confer permissiveness to a non- permissive cell- line (see Figure 6 for a schematic). The results of the targeted expression cloning approach are shown in Figure 7, where CXCR4 expression conferred permissiveness onto Hep3B cells (normally non-permissive).
[0126] To confirm that CXCR4 is a receptor candidate, antibody blocking experiments were conducted. Antibodies that can bind to the extracellular domains of CXCR4 were used to assess whether they could block SVV binding. Hep3B cells were transfected with CXCR4 and CVLs were recovered. The monoclonal antibody 12G5, which is specific to an extracellular portion of CXCR4, was used to block infection of permissive cell lines from the CVLs. Figures 8 A and 8B show the results of these experiments, and indicate that 12G5 can block SVV infection. EXAMPLE 3: DEVELOPMENT OF HYBRIDOMA CLONES AND SCREEING FOR VIRUS ENTRY BLOCKING MONOCLONAL ANTIBODIES
[0127] To identify the cellular protein recognized by SVV-OOl, protective monoclonal antibodies to surface proteins were produced. This approach involved two approaches: (1) immunization of mice with permissive cells, or (2) immunization of mice with membrane extracts of permissive cells to generate monoclonal antibodies that bound to the putative receptor and blocked virus entry into cells.
[0128] In the first approach, the human lung carcinoma cell line NCI-H446 (used interchangeably herein with H446) was used to immunize mice. NCI-H446 is SVV permissive. To identify cell surface proteins on NCI-H446 cells that may act as receptors or co-receptors in receptor-mediated virus entry and confer tropism, NCI-H446 cells were injected into mice, then the mice were screened for the development of antibodies to the NCI- H446 cell surface proteins.
[0129] The mice (8-10 week old female BALB/c) were injected with 3 million NCI-H446 cells suspended in 0.3 mL PBS every 2 - 3 weeks (Table 7). Ten days to two weeks after the second and third injection of cells, mice were bled and serum was monitored for antibodies in virus neutralization or virus blocking assay. Mice whose sera blocked SVV-OOl induced cytotoxicity were injected with 1 million H446 cells intravenously. Three days following final injection, two mice were sacrificed and spleens were collected (Figure 11).
Table 7. Study design for injection of mice with NCI-H446 cells
Figure imgf000037_0001
[0130] All monoclonal antibodies produced to surface proteins of H446 using the first immunization approach were IgM isotype, and therefore, a modified immunization protocol was used in a second approach to generate monoclonal antibodies of IgG isotype. In this approach, mice were immunized by injecting 200 μg of H82 membrane extract mixed with equal amounts of Freund's complete adjuvant subcutaneously. With two weeks between the injections, the mice were reinjected with the same amount of antigen mixed with equal amounts of Freund's incomplete adjuvant subcutaneously. Ten days after the third and fourth injections, mice were bled, and the sera were used to monitor for virus entry blocking antibodies in a neutralization assay. Mice whose sera blocked the virus induced cytotoxicity in H82 cells were injected with 200 μg of membrane extract in PBS intravenously. Three days following the final injection, the mice were sacrificed and spleens were collected. The spleenocytes were used to generate hybridomas.
[0131] To carry out the virus blocking assay, NCI-H446 cells were seeded in a 48 well plate two days prior to infection. Serum was added to each well (150 μl/well; 1 :4 or 1 :8 dilution). Thirty minutes after addition of serum, 150 μl of virus (100 TCID50/well; TCID50 means 50% Tissue Culture Infective Dose) was added to the appropriate wells and the cells were incubated for 1 hour. The cells were then washed twice, then 500 μl of complete cell culture media was added to the cells and the cells were monitored for up to three days post-infection. As shown in Figures 1OA - 1OB, viral infection was blocked (no cytopathic effect (CPE) detected) in several wells, indicating that the serum added to these wells contained antibodies to one or more cell surface proteins that may function as SVV receptors.
[0132] Antibody-producing spleen cells were isolated from mice whose serum blocked viral infection of NCI-H446 cells in the assay described above. To produce a monoclonal antibody producing hybridoma cell, the spleen cell is fused with a long-lived myeloma cell. Single-cell suspensions of spleenocytes were made and fused with SP2/O myeloma cells at a ratio of 5:1 (5e7 spleenocytes and Ie7 myeloma cells) using polyethylene glycol. The fusion mixture was resuspended in RPMI 1640 containing 18% FBS and HAT (hypoxanthine, aminopterin, thymidine) and plated into twenty five 96-well tissue culture dishes. Seven days post- fusion, the media was replaced with fresh RPMI 1640 containing HAT. Twelve days post- fusion, 100 μL of culture supernatant was collected from each well and tested for inhibition of virus-induced GFP expression or CPE in virus blocking assays.
[0133] Individual hybridoma cells were screened for their ability to produce the desired antibody (see Figure 11). Hybridoma clones were screened with the virus blocking assay to identify hybridoma cells that produce monoclonal antibodies that block infection. Two rounds of screening were carried out. For the first approach described above, the virus blocking assay was carried out by seeding NCI-H446 cells (30,000 cells/well) in a 96-well plate one day prior to infection. For the second approach, H446 and Per.C6 cells were used. On the day of infection, the media was removed from the cells and the cells were incubated with 100 μl of hybridoma culture supernatant for 40 minutes at room temperature. Then 100 TCID50 virus/well was added to the cells and the cells were incubated at 370C for one hour. The cells were then washed three times with phosphate buffered saline (PBS) using a plate washer (Bio Tek, Winooski, VT). Following the wash, 200 μl of complete media (RPMI 1640 containing 10% FBS) was added to each well and the cells were incubated at 370C. The wells were monitored under a microscope for lack of CPE everyday for the next three days. For each positive hybridoma clone, ten single cell clones were selected and tested in the virus blocking assay (Figures 12A - 12B). Hybridoma clones producing antibodies that block virus entry are indicated by dark blue wells, indicating that virus replication was blocked. For each fusion, >1000 hybridoma clones were screened in virus blocking assays.
[0134] The hybridoma clones secreting virus entry blocking monoclonal antibodies (>25 for each fusion) were subjected to single cell cloning and expanded. Isotype of monoclonal antibodies was determined using an antibody isotyping kit (Roche Pharmaceuticals). A few select clones were cultured in CELLine™ flasks (BD Biosciences) in serum-free medium and the culture supernatant was used for purification of monoclonal antibodies by size exclusion chromatography. The purified antibodies were used in Western blot and virus entry blocking experiments.
[0135] To visualize blocking of virus entry by the monoclonal antibodies, SVV tagged with green fluorescent protein (SVV-GFP) was used in the virus blocking assay. NCI-H446 cells were seeded (30,000 cells/well) in 24-well dishes one day prior to infection. On the day of infection, the culture media was removed from the cells, and the cells were incubated with 100 μl of hybridoma culture supernatant for 40 minutes at room temperature. SVV-GFP was added at 200 vp/cell (PPC) and the cells were incubated at 370C for one hour. Following the incubation, the cells were washed three times with PBS, then 500 μl of complete media was added to each well and the cells were incubated at 370C. The cells were monitored for GFP expression everyday for the next three days. The cells were visualized under a fluorescent microscope (Zeiss Axiovert 2000) 24 hours post-infection and photographed. Exemplary results are shown in Figures 13A - 13B. Supernatant from hybridoma clone 8G10 blocked SVV-GFP infection in NCI-H446 cells. Only a few GFP-positive cells were evident in wells treated with 8G10. In contrast, there were more GFP-positive cells without prior treatment with 8G10. [0136] Another human cell line that is SVV permissive is the PER.C6 cell line. PER.C6 cells were treated with purified 8G10 mAb then analyzed for SVV-GFP infection. As shown in Figures 14A - 14D, SVV-GFP entry into PER.C6 cells was reduced in the presence of 8G10 mAb.
EXAMPLE 4: IMMUNOBLOT DETECTION AND IMMUNOPRECIPITATION OF MEMBRANE PROTEINS BY MONOCLONAL ANTIBODIES
[0137] Cell membrane proteins recognized by the monoclonal antibodies of the invention represent candidates for SVV cellular receptors or co-receptors that mediate SVV entry into cells. To identify proteins recognized by 8G10 and 17El 1 mAbs, Western blot, immunoprecipitation, and 2-D gel electrophoresis studies were carried out.
[0138] Single dimension gel electrophoresis and Western blot. Membrane proteins from NCI-H446 cells and PER.C6 cells were extracted and separated by SDS-PAGE (Figure 15A), then analyzed by Western blotting with 8G10 or 17El 1 mAb (IgM isotype) (Figure 15B). NCI-H446 cell membrane proteins were further analyzed in Western blots with monoclonal antibodies from several other hybridoma clones (Figure 15C). To carry out western blot studies, octyl-glucoside membrane extracts of H446 (20 μg/lane) were separated on 4-12 Tris-Bis gel, and proteins were transferred onto PVDF membrane. The membrane was washed three times with TBS-T (TBS with 0.05% Tween20) containing 5% milk for lhr at room temperature. The membrane was washed three times with Ix TBS-T and incubated with 8G10 clone culture supernatant at room temperature for two hours. The membrane was washed three times with TBS-T and incubated with sheep anti-mouse HRP-conjugated antibody (GE-Healthcare) at room temperature for one hour. The blot was washed three times and developed using ECL Plus kit (GE-Healthcare).
[0139] Several proteins bands with molecular masses ranging from 40 to 175 kDa were seen on the Western blot with a few prominent bands at 70 kDa molecular mass (Figure 15B). The corresponding proteins from the untransferred gel were cut out and used to identify proteins by mass spectrometry. Several proteins hits were obtained with mass spectrometry including heat shock protein 70 (HSP70).
[0140] Immunoprecipitation. For the immunoprecipitation of 8G10 binding proteins, PER.C6 cells were labeled with S35 -Methionine and Cysteine. For immunoprecipitation of 17El 1 binding proteins, H446 cells were left unlabeled. H446 or PER.C6 cells were extracted with RIPA buffer and pre-cleared with Protein-L agarose (Santa Cruz Biotech.). This was followed by incubation of 5 μg of antibody with 500 μg of cleared lysate overnight at 4° C with agitation. The antibody was then precipitated with 50 μL of Protein-L agarose beads. The beads were then boiled in Laemmli sample buffer with 4OmM DTT for 5 minutes and resolved on a 4-12% Bis/Tris gradient gel. The proteins were transferred onto PVDF membrane. The membrane was blocked with TBS-T (TBS with 0.05% Tween20) containing 5% milk for a minimum of lhr at room temperature. The membrane was then incubated with 17El 1 (1 : 100) in blocking solution at 4° C with agitation overnight. The membrane was washed three times with TBS-T and incubated with sheep anti-mouse IgM HRP conjugated antibody (Novus) at room temperature for one hour. The blot was washed three times and developed using ECL Plus kit (GE-Healthcare). Immunoprecipitation using the monoclonal antibody 17El 1 produced a single specific band at -200 kDa (Figure 16A). Efforts to identify this protein are ongoing. Immunoprecipitation of proteins recognized by 8G10 is shown in Figure 16B.
[0141] Two-dimensional gel electorphoresis and Western blot. Membrane extracts of H446 cells were first separated in two sets of two dimensional poly-acrylamide gels, and proteins from one set of gels were transferred to membranes and probed with monoclonal antibodies as follows: H446 cells were grown to -95% confluence as per the ATCC passaging instructions. The cells were washed with DPBS (Invitrogen) and dislodged with a cell scraper. The cells were again washed, two times using DPBS with centrifugation at 1500 RPM for 5 minutes. Samples were then resuspended in DPBS with protease inhibitors (Pierce) and vortexed. The suspension was then passed through a 21 gauge needle 10 times on ice. The homogenate was sonicated 3x10 cycles of 10 watts (Fisher model 60 sonic dismembranator) on ice. The homogenate was then centrifuged at 10,000xg for 10 minutes. The supernatant was removed (top 75% to prevent contamination with pellet material) and spun at 100,000xg for 1 hour. The pellet (containing total membranes) was resuspended in Extraction buffer (7M Urea, 2M Thiourea, 4% CHAPS, 1% IPG Buffer, 4OmM DTT and 2X Halt Protease Inhibitor Cocktail). The suspension was vortexed for 10 seconds and incubated for 30 minutes at room temperature on a nutator. Finally, this extract was spun at 5,000xg for 5 minutes to remove insoluble material. The supernatant was then aliquoted and stored at - 8O0C. The protein concentration was determined by Bio-Rad RC/DC assay using the manufacturer's instructions. 200-400ug of membrane extract was diluted to 450ul with 2OmM DTT (GE Healthcare) and 350ul destreak rehydration solution (w/1% 3-11 IPG Buffer) (GE Healthcare). The sample was then applied to a 24cm 3-1 INL IPG strip (GE Healthcare) and overlaid with dry strip cover fluid (GE Healthcare). The IPG strip was then focused in a 24cm strip holder in the Ettan IPGPhor III IEF system (GE Healthcare). The focused IPG strips were equilibrated using SDS equilibration buffer as per the manufacturer's instructions. Equilibrated IPG strips were overlayed on top of 4-12% gradient gels (JULE) along with molecular weight markers (Biorad) and sealed with agarose overlay solution (GE Healthcare). Gels were run in IX Tris Glycine running buffer (Invitrogen) at 1 watt per gel overnight in an Ettan Daltsix electrophoresis unit (GE Healthcare). Resolved gels were then fixed for silver stain analysis and stained according to the manufacturer's instructions (Invitrogen). Alternatively, gels were transferred to PVDF (GE Healthcare) at 40OmAMP for 1 hour in transfer buffer using a semi-dry transfer unit (GE Healthcare). PVDF membranes were then stained with MemCode (Pierce) to confirm the transfer. Membranes were blocked in 5% non-fat dry milk in TBST (Teknova) for a minimum of 2 hours, and then stained overnight at 4° C with the monoclonal antibody 8G10 at a dilution of 1 : 1000 in block on a rocker. The blots were washed with TBST for 3 times 5 minutes on an orbital shaker and incubated with 2° HRP antibodies (GE Healthcare) at 1 :5000 in block for 1 hour at room temperature. The membranes were then washed 4 times for 30 minutes in TBST. Finally, the membranes were exposed to ECLp lus (GE Healthcare) as per the manufacturer's instructions and exposed to autoradiograph film. Results are shown in Figure 16C. For Mass Spectrometry analysis, the western blot was aligned to the duplicate gel, and a piece of gel corresponding to the position of western blot signal on the membrane was cut. Sequence identification via LC MS/MS was performed by Proteomic Research Services (PRS).
EXAMPLE 5: PRODUCTION OF CONCENTRATED MONOCLONAL ANTIBODY
FROM HYBRIDOMA CLONE 8Gl Q-F 10
[0142] A cell line flask system was used to produce concentrated monoclonal antibody produced by hybridoma clone 8G10. The 8G10-F10 clone was cultured in cell line flasks for 8 - 10 days (Figure 17). The culture supernatant was collected by pelleting the cells three times at 1500 rpm. The supernatant was then dialyzed against PBS (four buffer changes). The dialyzed solution containing the concentrated 8G10 mAb was stored at -2O0C in PBS containing 50% glycerol.
EXAMPLE 6: DETECTION OF 8G10 AND 17El 1 ANTIGENS IN SVV-PERMIS SIVE
CELLS
[0143] To identify the subcellular location of proteins recognized by these two antibodies, and to determine whether the 8G10 and 17El 1 mAb antigens are on the cell surface or intracellular, or both, cells were subjected to immunofluorescence staining. Expression of the antigens was analyzed in SVV permissive cells (NCI-H446 and PER.C6) and SVV non- permissive cells (NCI-H460 and Hep3B). To determine if the antigens are expressed on the cell surface, cells were stained under non-permeabilizing conditions (Figures 18 A - 18D and 181 - 18L). Under non-permeabilizing conditions, only cell surface antigens are available for binding to mAb. The cells were seeded in Lab-Tek II chamber slides (Nalgene Nunc). The next day, the cells were fixed with freshly made 4 % paraformaldehyde in PBS for 30 min at room temperature. The cells were washed 3 times for 5 min each with PBS. The cells were incubated in blocking solution (PBS with 1% normal goat serum) for 30 min to block nonspecific binding of the antibodies. The cells were washed three times with PBS and incubated with 8G10 or 17El 1 for 60 min at room temperature. The cells were washed with PBS and incubated with donkey anti-mouse IgG (heavy and light chain) conjugated to Alexa fluor 594 (Invitrogen) and incubated for 60 min at room temperature. The cells were washed three times and mounted with mount medium containing DAPI dye (Vector Laboratories). The cells were visualized under fluorescent confocal microscope and photographed. To determine if the antigens are expressed intracellularly, cells were permeabilized with 0.1% Triton X-100 prior to staining (Figures 18E - 18H).
[0144] With 8G10, intense membrane staining was observed in permissive cell lines H446 and PER.C6, but no to very little staining was observed in non-permissive cell lines, H460 and Hep3B (Figures 18A - 18D). With 17El 1, surface and cytoplasmic staining was observed in permissive cell lines H446 and H69AR but little to no staining in non-permissive cell lines, as evidenced by the H460 and A549 results in Figures 181 - 18L. As shown in Figures 18A - 18B, SVV permissive cells express a cell surface antigen to which the 8G10 mAb binds. The 8G10 antigen is not detected on the surface of non-permissive cells (Figures 18C - 18D). Permeabilizing conditions enhance the accessibility any intracellular 8G10 antigens.
EXAMPLE 7: EFFECT OF 8G10 MONOCLONAL ANTIBODY ON SVV-MEDIATED
CELL KILLING
[0145] As described in the Examples above, the 8G10 mAb significantly blocks SVV entry into NCI-H446 cells which are otherwise permissive to SVV infection. To determine the effect of 8G10 mAb on SVV-mediated cell killing, NCI-H446 cells were plated in a 96-well dish (30,000 cells/well) two days prior to infection. On the day of infection, media was removed from the cells, and RPMI media (100 μl) containing 8G10 mAb (80 μg/ ml) or mouse IgM isotype control (80 μg/ml) was added to the cells, followed by incubation at room temperature for 40 minutes. SVV-OOl (1000 - 0.00001 PPC) was added to the cells, followed by incubation at 370C for 1 hour. The cells were then washed three times with PBS. Complete media (200 μl) containing 80 μg/ml 8G10 mAb or 40 μg/ml mouse IgM isotype control was added to the cells. The cells were incubated at 370C. Cell survival was monitored at 48 hours post-infection; results are shown in Figure 19. In the absence of 8G10 mAb, the EC50 (amount of virus necessary to kill 50% of cells) of SVV-OOl was 0.0988 virus particles/cell. In the presence of 8G10, the EC50 increased to 6.872 virus particles/cell. The dose-response results show that SVV-OOl -mediated cell killing is reduced in the presence of 8GlO mAb.
EXAMPLE 8: EFFECT OF 8G10 AND 17El 1 MONOCLONAL ANTIBODIES ON
VIRAL YIELD
[0146] To determine the effect of 8G10 on viral production in a selection of SVV-permissive cells, cells were incubated with 25 or 75 μg/ml 8G10 mAb or IgM control for 45 minutes at room temperature, then infected with MOI of 1 for one hour at 370C. The cells were then washed three times with PBS. Infection was continued for 14 hours. Crude virus lysates (CVL) were prepared and tittered in log dilutions on PER.C6 cells. Results are shown in Figure 20.
[0147] H446 cells were seeded in 24-well tissue culture dishes one day prior. On the day of the experiment, medium was replaced with medium containing varying amounts (0, 2, 5, 10, 20, 40 and 80 μg/mL) of 8G10 or 40 μg/mL of IgM isotype control antibody and incubated at room temperature for 40 minutes. SVV-OOl was added at 1 or 10 particles/cell, and cells were incubated at 37° C for one hour. The cells were washed three times with PBS and 1 mL of RPMI containing 10% FBS was added. The cells and media were harvested at 14 hours post-infection. CVL was generated by three cycles of freeze-thaw, and virus titer determined by tissue culture infectivity assay (TCID50) in PER.C6 cell. To determine whether protection by 8G10 and 17El 1 was specific, additional cell lines including H446, H 1299, HEK293, H69AR, PER.C6, Hl 87, H82 and Hl 184 were tested. The blocking effect was evident with both 8G10 (Figure 21A) and 17El 1 (Figure 21B).
EXAMPLE 9: EXPRESSION OF SVV RECEPTORS IN TUMOR SAMPLES
[0148] Xenografts of SVV permissive or non-permissive cells were analyzed by immunohistochemistry (IHC) for expression of SVV receptors. A shown in Figure 22, 8G10 antigen and integrin α4 are expressed in SVV permissive cells (H446), but not expressed in non-permissive cells (H460). The markers were also found to co-stain the same cells from an invasive edge of a xenograft tumor from a permissive cell line (Figure 23).
[0149] The 8G10 mAb was used to analyze human SCLC tissue microarrays (TMA) (Figure 24). As shown in Table 8 below, almost 60% of 34 SCLC tissues stained ++ or +++ with 8G10 mAb. Other tumor types also stained positive and virtually none of the normal major organ tissues stained positive.
Table 8: Summary of IHC results of SCLC TMA using 8G10 mAb
Figure imgf000045_0001
[0150] Based on the findings described in the foregoing Examples, it is a possibility that SVV binding and/or internalization is mediated by a series of events involving SVV receptors, for example, as depicted in Figure 25. As shown in the figure, activated CXCR4 moves to lipid rafts in the cell membrane and the 8G10 antigen is present in the rafts. SDF then activates CXCR4 and causes "inside signaling" to integrins (e.g., α4βl integrins). The integrins are released from the actin cytoskeleton and are free to cluster. Clustered integrins and the 8G10 antigen assembled in the lipid rafts serve as a high affinity receptor for SVV.
EXAMPLE 10: SVV STUDIES IN HUMANS
[0151] SVV was tested in patients with solid tumors expressing at least one neuroendocrine marker. SVV was administered systemically as a one-hour intravenous infusion. Viral load in serum was monitored using an infectivity-based assay (TCIDso/mL) and a quantitative real-time RT-PCR assay (RNA copies/mL). Patient- specific patterns of viral amplification and serum kinetics of SVV-OOl were observed, indicative of different patterns of viral replication. These patterns of kinetics ranged from a lack detectable infectious virus and genomic RNA at all time points post-dosing to rapid amplification up to greater than 107 TCID5o/mL and 109 RNA copies/mL in serum (Figure 26A). The concentration of anti-SVV neutralizing antibodies in patient serum was found to vary with viral titer (Figure 26B). The peak levels of SVV RNA observed in one patient indicated at least 10,000-fold amplification of the input dose. Preliminary proof of concept for SVV was also obtained from this patient with positive immunohistochemical staining for viral capsid in metastatic tumor cells, but not surrounding normal tissue (Figure 27A). Viral replication, as evidenced by the immunohistochemistry signal, occurred in tumor cells despite the presence of a robust neutralizing antibody response (Figure 27B). These data indicate that tumor-selective viral replication occurred in this patient with little to no replication in other patients to date, suggesting that tumor is the primary determinant of SVV amplification in patients.
[0152] Of six patients treated to date with SVV, five patients are alive with no severe side effects following 1 - 6 months of treatment. (One patient died of progressive cancer without SVV dose limiting toxicity.) The results of the studies in humans show that SVV reaches and infects tumor cells following intravenous administration and indicate that a single low-dose is likely to be therapeutic based on the level of replication. There is also evidence of selective, long-term replication in tumor cells. Pharmacokinetic patterns in patients suggest selectivity. Thus far, SVV exhibits a good safety profile with no severe, drug-related adverse effects reported that are attributable to SVV. The safety profile suggests a likelihood that SVV administration will be well tolerated by most or all patients.
EXAMPLE 11 : SVV STUDIES IN NON-HUMAN ANIMALS
[0153] In a murine model, normal mice demonstrated transient low-level replication of SVV, with self-limiting viremia in normal tissue. The pharmacokinetic profile in the murine model was found to be predictive of clinical data. For example, replication of SVV was observed in tumor cells, and viremia was resolved by a neutralizing anti-SVV antibody response in the presence of a tumor (Figure 28). The murine model, as well as porcine and non-human primate models had toxicity profiles that were predictive of clinical data. For example, the toxicity was limited to transient changes in white blood cells/platelets.
[0154] Studies in mouse tissues revealed tumor-selective replication following an intravenous bolus dose of SVV (109 vp/kg) (Figures 29 A - 29B). Immunohistochemistry results show positive staining for SVV replication in the tumor cells, with no detectable staining in normal tissues (Figure 30). The murine model also revealed no dose-limiting toxicity or microscopic pathology up to 1014 vp/kg. A porcine animal model received up to 3 x 1011 vp/kg with no dose-limiting toxicity and no infectious virus detected in tissues 7 days after dosing. A non-human primate model was administered up to 5 x 1011 vp/kg with no dose-limiting toxicity. [0155] A pre-clinical tumor model showed that SVV replicates in tumor cells despite an immune response in the animal (Figures 3 IA - 3 IB). SVV was also shown to have a high therapeutic index (Figures 32A - 32B), with at least a one million-fold therapeutic window in pre-clinical mouse models.
EXAMPLE 12: PHARMACOKINETICS OF INTRAVENOUSLY ADMINISTERED SVV IN PATIENTS WITH SOLID TUMORS OF NEUROENDOCRINE DIFFERENTIATION
[0156] Seneca Valley Virus (SVV) is a newly discovered picornavirus with natural oncolytic selectivity towards human tumor cells of neuroendocrine (NE) differentiation. A dose- escalation Phase I clinical trial was initiated for patients with solid tumors expressing at least one NE marker. SVV was administered systemically as a one hour i.v. infusion. Viral load in serum was monitored using an infectivity-based assay (TCID50/mL) and a quantitative real-time RT-PCR assay (RNA copies/mL). Patient- specific patterns of viral amplification and serum kinetics of SVV were observed indicative of different patterns of viral replication. These patterns of kinetics ranged from a lack detectable infectious virus and genomic RNA at all time points post-dosing to rapid amplification up to greater than 107 TCIDso/mL and 109 RNA copies/mL in serum. The peak levels observed in one patient indicated at least 10,000- fold amplification of the input dose. Preliminary proof of concept for SVV was also obtained from this patient with positive immunohistochemical staining for viral capsid in metastatic tumor cells, but not surrounding normal tissue. Viral replication, as evidenced by the immunohistochemistry signal, occurred in tumor cells despite the presence of a robust neutralizing antibody response. These data indicate that tumor-selective viral replication occurred in this patient with little to no replication in other patients to date, suggesting that tumor is the primary determinant of SVV amplification in patients.
[0157] The dose-escalation was 107 - 1011 viral particles of SVV/kg (the dose was 107, 108, 109, 1010, and then 1011). The solid tumor of the subject receiving SVV was expressing at least one neuroendocrine marker. Then, safety, toxicity and dosage was evaluated. Viral kinetics and clearance were evaluated.
[0158] Viral Kinetics in Serum (see Figures 33 and 34). Viral load was monitored in serum utilizing an infectivity (IFT)-based TCID50 assay on PER.C6® cells and a quantitative, realtime reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Patient-specific patterns of viral replication were observed. [0159] Viral Neutralization in Serum (see Figure 35). Neutralization was determined by incubation of serum dilutions with 100 TCID50 SVV prior to infection of PER.C6 ® cells. Neutralizing titer was reported as the greatest dilution of serum capable of neutralizing greater than 50% of replicate wells. Patient-specific patterns of viral neutralization were observed.
[0160] Subject Serum RNA is Degraded (see Figure 36). RNA from Subject's serum on study days 4, 17 (shown) and 25 was subjected to RT-PCR for three regions of the SVV genome ranging from 961 to 1557 bp. To determine quantitation limits, the samples were spiked with log dilutions of full-length viral RNA. Large fragments could be amplified from serum RNA on day 4, but not on days 17 or 25, indicating lack of full length circulating viral RNA at later time points.
[0161] Anti-SVV Immunohistochemistry (see Figure 37, 400X). A subject succumbed to progressive disease on day 28 post-infusion. Tissues were collected at autopsy and fixed in 10% neutral-buffered formalin. Sections were stained by H&E and using mouse anti-serum to SVV. Specific anti-SVV staining was observed in a liver metastasis, but not surrounding normal liver tissue or in a panel of normal organs, including adrenal gland and pancreas.
[0162] There have been subject- specific patterns of viral kinetics observed. Data suggest that tumor permissivity to SVV virus is primary determinant of viral replication and that little to no replication occurs in normal tissues. There was observed a high level of viral replication in the subject tested. Serum RNA at later time points in the subject was fragmented. RT- PCR signal can potentially be utilized as a marker of ongoing replication in tumor as observed in the subject. There were patient/subject -specific patterns of viral neutralization response observed. The onset of detectable neutralization varied between day 8 and day 25. The peak titer varied between 22 and 218 fold-dilutions. Neutralization response varied with the degree of viral amplification. The tumor-specific IHC signal in liver metastasis of the subject was observed. Proof of concept for SVV: tumor-specific replication in a subject has been shown and demonstrated in this example. There was no staining in normal tissues, including those containing normal neuroendocrine (NE) cells. The ongoing viral replication in tumor despite a strong immune response was observed.

Claims

What is claimed is:
1. A hybridoma cell designated Anti-H446 MMAb 8G10 deposited as ATCC Accession No.
2. A hybridoma cell designated Anti-H82 MMAb 17El 1 deposited as ATCC Accession No.
3. A monoclonal antibody produced by the hybridoma cell designated Anti-H446 MMAb
8G10 deposited as ATCC Accession No. .
4. A monoclonal antibody produced by the hybridoma cell designated Anti-H82 MMAb
17El 1 deposited as ATCC Accession No. .
5. A monoclonal antibody, which specifically binds and forms a complex with an antigen located on the surface of a tumor cell and thereby inhibits SVV entry into the cell, the antigen being an antigen to which a monoclonal antibody produced by a hybridoma cell designated Anti-H446 MMAb 8G10 deposited as ATCC Accession No. specifically binds.
6. A monoclonal antibody, which specifically binds and forms a complex with an antigen located on the surface of a tumor cell and thereby inhibits SVV entry into the cell, the antigen being an antigen to which a monoclonal antibody produced by a hybridoma cell Anti-H82 MMAb 17El 1 deposited as ATCC Accession No. specifically binds.
7. An antibody fragment consisting essentially of an antigen-binding domain of the monoclonal antibody of any one of claims 3 - 6.
8. The antibody of any one of claims 3 - 6, wherein the antibody is a chimeric antibody.
9. The antibody of any one of claims 3 - 6, wherein the antibody is a humanized monoclonal antibody.
10. The antibody of any one of claims 3 - 6, wherein the antibody is a murine monoclonal antibody.
11. The antibody of claim any one of claims 3 - 6, wherein the antibody is a human monoclonal antibody.
12. The antibody of any one of claims 3 - 6, or an antigen-binding fragment thereof, wherein the antibody or the fragment comprises a detectable label.
13. The antibody of any one of claims 3 - 6, or an antigen-binding fragment thereof, wherein the antibody or the fragment is linked to a moiety capable of producing a detectable signal.
14. A method for determining whether SVV will enter a tumor cell, the method comprising:
(a) contacting a tumor cell with a monoclonal antibody, or an antigen-binding fragment thereof, produced by (i) a hybridoma cell designated Anti-H446
MMAb 8G10 deposited as ATCC Accession No. , or (ii) a hybridoma cell designated Anti-H82 MMAb 17El 1 deposited as ATCC Accession No. ; and
(b) determining whether the monoclonal antibody or fragment thereof binds to the tumor cell,
wherein a determination of binding indicates that SVV will enter the tumor cell.
15. The method of claim 14, wherein the tumor cell is contained within a tumor or a tumor sample.
16. The method of claim 14, wherein the method is carried out in vitro.
17. The method of claim 14, wherein the method is carried out in vivo.
18. The method of claim 14, wherein the determining comprises direct detection of the monoclonal antibody.
19. The method of claim 14, wherein the determining comprises indirect detection of the monoclonal antibody.
20. The method of claim 14, wherein the determining comprises detecting a chromogenic signal, a fluorescent signal, a luminogenic signal or a radioactive signal.
21. A method for determining whether a subject with a tumor will respond to SVV treatment, the method comprising: (a) contacting the tumor with a monoclonal antibody, or an antigen-binding fragment thereof, produced by (i) a hybridoma cell designated Anti-H446
MMAb 8G10 deposited as ATCC Accession No. , or (ii) a hybridoma cell designated Anti-H82 MMAb 17El 1 deposited as ATCC Accession No. ; and
(b) determining whether the monoclonal antibody binds to the tumor,
wherein a determination of binding indicates that the subject will respond to SVV treatment, and wherein the response comprises an arrest, delay, inhibition or reversal in the progression of the cancer.
22. The method of claim 21, wherein the contacting is in vitro.
23. A method for delivering a compound to a tumor cell in a subject, the method comprising
(a) linking the compound to an antibody produced by (i) a hybridoma cell designated
Anti-H446 MMAb 8G10 deposited as ATCC Accession No. , or (ii) a hybridoma cell designated Anti-H82 MMAb 17El 1 deposited as ATCC Accession No. , or a fragment thereof; and (b) administering the antibody to the subject.
24. The method of claim 23, wherein the linking is via a covalent bond.
25. A kit for determining whether SVV will enter a tumor cell, the kit comprising:
(a) a monoclonal antibody, or an antigen-binding fragment thereof, produced by (i) a hybridoma cell designated Anti-H446 MMAb 8G10 deposited as ATCC
Accession No. , or (ii) a hybridoma cell designated Anti-H82 MMAb
17El 1 deposited as ATCC Accession No. ; and
(b) at least one negative control cell sample, wherein the monoclonal antibody or fragment thereof does not bind to the cell sample.
26. The kit of claim 25, further comprising at least one positive control cell sample to which the monoclonal antibody or fragment thereof binds.
27. The kit of claim 25, further comprising instructions for preparing a tumor sample.
28. The kit of claim 25, wherein the monoclonal antibody or the fragment thereof is linked to a detectable signal or a moiety capable of producing a detectable signal.
29. A method for identifying stem cells, the method comprising:
(a) contacting a cell with the monoclonal antibody, or an antigen-binding fragment thereof, produced by (i) a hybridoma cell designated Anti-H446
MMAb 8G10 deposited as ATCC Accession No. , or (ii) a hybridoma cell designated Anti-H82 MMAb 17El 1 deposited as ATCC Accession No. ; and
(b) determining whether the monoclonal antibody binds to the cell,
wherein binding of the antibody to the cell in (b) indicates that the cell is a stem cell.
30. An antigen that is specifically bound by the monoclonal antibody, or an antigen-binding fragment thereof, produced by (i) a hybridoma cell designated Anti-H446 MMAb
8G10 deposited as ATCC Accession No. , or (ii) a hybridoma cell designated
Anti-H82 MMAb 17El 1 deposited as ATCC Accession No. .
31. The antigen of claim 30, wherein the antigen is linked to a detectable moiety.
32. A pharmaceutical composition comprising an antibody of any one of claims 3 - 6, and a pharmaceutically acceptable carrier.
33. A method for treating cancer in a subject, the method comprising administering to a subject an effective amount of a monoclonal antibody which specifically binds and forms a complex with an antigen located on the surface of a tumor cell and thereby inhibits SVV entry into the cell, the antigen being an antigen to which a monoclonal antibody produced by (i) a hybridoma cell designated Anti-H446 MMAb 8G10 deposited as ATCC Accession No. , or (ii) a hybridoma cell designated Anti-H82
MMAb 17El 1 deposited as ATCC Accession No. specifically binds.
34. The method of claim 33, wherein the antibody is a monoclonal antibody produced by (i) a hybridoma cell designated Anti-H446 MMAb 8G10 deposited as ATCC Accession No. , or (ii) a hybridoma cell designated Anti-H82 MMAb 17E11 deposited as
ATCC Accession No. .
35. The method of claim 33, wherein the antibody is a chimeric antibody.
36. The method of claim 33, wherein the antibody is a humanized monoclonal antibody.
37. The method of claim 33, wherein the antibody comprises a human constant region and a heavy and light chain variable region, wherein the heavy and light chain variable region comprises heavy and light chain framework regions and heavy and light chain complementarity determining regions (CDRs), at least a portion of the heavy and light chain framework regions being derived from a human antibody, and the CDRs comprising heavy-chain CDRs light-chain CDRs derived from a monoclonal antibody produced by (i) a hybridoma cell designated Anti-H446 MMAb 8G10 deposited as
ATCC Accession No. , or (ii) a hybridoma cell designated Anti-H82 MMAb
17El 1 deposited as ATCC Accession No. .
PCT/US2008/056852 2007-03-13 2008-03-13 Monoclonal antibody that recognizes a seneca valley virus (svv) cellular receptor and uses thereof WO2008112891A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US90673507P 2007-03-13 2007-03-13
US60/906,735 2007-03-13

Publications (2)

Publication Number Publication Date
WO2008112891A2 true WO2008112891A2 (en) 2008-09-18
WO2008112891A3 WO2008112891A3 (en) 2008-12-31

Family

ID=39760399

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2008/056852 WO2008112891A2 (en) 2007-03-13 2008-03-13 Monoclonal antibody that recognizes a seneca valley virus (svv) cellular receptor and uses thereof

Country Status (1)

Country Link
WO (1) WO2008112891A2 (en)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017096201A1 (en) 2015-12-02 2017-06-08 Memorial Sloan-Kettering Cancer Center Seneca valley virus (svv) cellular receptor targeted oncotherapy
CN107253978A (en) * 2017-08-13 2017-10-17 中牧实业股份有限公司 Sai Nika paddy virus structural protein antibody ELISA immunity detection reagents
CN111394367A (en) * 2020-03-24 2020-07-10 中国农业科学院兰州兽医研究所 Selcarinovirus recombinant nucleic acid, recombinant vaccine strain, and preparation methods and applications thereof
CN111729091A (en) * 2020-05-15 2020-10-02 金宇保灵生物药品有限公司 Method for testing efficacy of porcine epikavirus inactivated vaccine by using domestic rabbit
CN112433049A (en) * 2019-06-24 2021-03-02 中国动物疫病预防控制中心(农业农村部屠宰技术中心) Reagent for detecting Seneca virus antibody and application thereof
CN117487006A (en) * 2023-12-29 2024-02-02 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） Monoclonal antibody for resisting A-type sai virus, epitope and application

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060159659A1 (en) * 2003-09-26 2006-07-20 Hallenbeck Paul L Seneca valley virus based compositions and methods for treating disease

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060159659A1 (en) * 2003-09-26 2006-07-20 Hallenbeck Paul L Seneca valley virus based compositions and methods for treating disease

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11096972B2 (en) 2015-12-02 2021-08-24 Memorial Sloan-Kettering Cancer Center Seneca Valley Virus (SVV) cellular receptor targeted oncotherapy
WO2017096201A1 (en) 2015-12-02 2017-06-08 Memorial Sloan-Kettering Cancer Center Seneca valley virus (svv) cellular receptor targeted oncotherapy
EP3383496A4 (en) * 2015-12-02 2019-07-31 Memorial Sloan-Kettering Cancer Center Seneca valley virus (svv) cellular receptor targeted oncotherapy
US10537599B2 (en) 2015-12-02 2020-01-21 Memorial Sloan-Kettering Cancer Center Seneca valley virus (SVV) cellular receptor targeted oncotherapy
US11738058B2 (en) 2015-12-02 2023-08-29 Memorial Sloan-Kettering Cancer Center Seneca valley virus (SVV) cellular receptor targeted oncotherapy
CN107253978A (en) * 2017-08-13 2017-10-17 中牧实业股份有限公司 Sai Nika paddy virus structural protein antibody ELISA immunity detection reagents
CN107253978B (en) * 2017-08-13 2020-11-20 中牧实业股份有限公司 Enzyme-linked immunoassay kit for structural protein antibody of Seneca valley virus
CN112433049A (en) * 2019-06-24 2021-03-02 中国动物疫病预防控制中心(农业农村部屠宰技术中心) Reagent for detecting Seneca virus antibody and application thereof
CN112433049B (en) * 2019-06-24 2023-08-18 中国动物疫病预防控制中心(农业农村部屠宰技术中心) Reagent for detecting sai card virus antibody and application thereof
CN111394367A (en) * 2020-03-24 2020-07-10 中国农业科学院兰州兽医研究所 Selcarinovirus recombinant nucleic acid, recombinant vaccine strain, and preparation methods and applications thereof
CN111394367B (en) * 2020-03-24 2021-05-14 中国农业科学院兰州兽医研究所 Selcarinovirus recombinant nucleic acid, recombinant vaccine strain, and preparation methods and applications thereof
CN111729091A (en) * 2020-05-15 2020-10-02 金宇保灵生物药品有限公司 Method for testing efficacy of porcine epikavirus inactivated vaccine by using domestic rabbit
CN117487006A (en) * 2023-12-29 2024-02-02 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） Monoclonal antibody for resisting A-type sai virus, epitope and application
CN117487006B (en) * 2023-12-29 2024-04-12 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） Monoclonal antibody for resisting A-type sai virus, epitope and application

Also Published As

Publication number Publication date
WO2008112891A3 (en) 2008-12-31

Similar Documents

Publication Publication Date Title
US10344096B2 (en) Antibodies and vaccines for use in treating ROR1 cancers and inhibiting metastasis
JP2022104941A (en) Humanized anti-CD73 antibody
KR101989134B1 (en) Anti-IL1RAP Antibodies and their use for treating human
US20080213171A1 (en) Cancerous disease modifying antibodies
US20040180002A1 (en) Cancerous disease modifying antibodies
WO2008112891A2 (en) Monoclonal antibody that recognizes a seneca valley virus (svv) cellular receptor and uses thereof
EP2067791A1 (en) Prophylactic or therapeutic agent for cancer
JP4287147B2 (en) Monoclonal antibodies and cell surface antigens for the detection and treatment of small cell lung cancer (SCLC)
US20030096285A1 (en) Identifying anti-tumor targets or agents by lipid raft immunization and proteomics
NZ717534A (en) Immunoreceptor modulation for treating cancer and viral infections
WO2007047907A2 (en) Dek protein compositions and methods of using the same
KR20040010613A (en) Antibodies against cancer
AU2005219322B2 (en) Pharmaceutical Composition Comprising CXCR3 Inhibitor
US20170260278A1 (en) Methods of inhibiting pathological angiogenesis with doppel-targeting molecules
US20040151665A1 (en) Cancerous disease modifying antibodies
WO2003086456A2 (en) Anti-ck18 monoclonal antibody and therapeutic and diagnostic uses thereof in cancer
JP5881085B2 (en) Composition for treatment and diagnosis of pancreatic cancer
EP3016977A1 (en) Human anti-il-32 antibodies
WO2017095136A9 (en) Methods of inhibiting pathological angiogenesis with doppel-targeting molecules
WO2008055889A1 (en) Compositions and methods of treating oncological, inflammatory and autoimmune diseases mediated by sema4a
JP2008163029A (en) ANTIBODY AGAINST PAP2a, AND DIAGNOSTIC AND THERAPEUTIC USE THEREOF
EP1932857A1 (en) Preventive/remedy for cancer
AU2005225472A1 (en) Cancerous disease modifying antibodies
CN111116743A (en) Hsp90 antibodies and their use against fungal infections
WO2018164441A1 (en) Methods of inhibiting pathological angiogenesis with doppel-targeting molecules

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08782753

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase in:

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC, EPO FORM 1205A DATED 04.02.2010

122 Ep: pct application non-entry in european phase

Ref document number: 08782753

Country of ref document: EP

Kind code of ref document: A2