Clonal strains of attenuated vaccinia viruses and methods of use thereof

• US 20120308484A1
• Filed: 04/13/2012
• Published: 12/06/2012
• Est. Priority Date: 04/15/2011
• Status: Active Grant

• Current Assignee: Genelux Corporation
• Original Assignee: Genelux Corporation
• Inventors: Szalay, Aladar A., Yu, Yong A., Zhang, Qian, Chen, Nanhai G.
• Granted Patent: US 11,149,254 B2
• Time in Patent Office: Days
• US Class Current: 424/9.3
• CPC Class Codes: A61K 35/768 Oncolytic viruses not provided for in groups A61K35/761 - A61K35/766
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 1/18 for pancreatic disorders, e.g. pancreatic enzymes
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 13/08 of the prostate
• CPC Class Codes: A61P 15/00 Drugs for genital or sexual disorders for disorders of sex hormones A61P5/24; Contraceptives
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/04 specific for metastasis
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 16/22 against growth factors ; against growth regulators
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C12N 2710/24121 Viruses as such, e.g. new isolates, mutants or their genomic sequences
• CPC Class Codes: C12N 2710/24132 Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
• CPC Class Codes: C12N 2710/24143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00

Abstract

Clonal strains of vaccinia viruses are provided. Also provided are methods of identifying and isolating attenuated and oncolytic clonal strains from virus preparations. Modified recombinant forms of the clonal strains also are provided. The clonal strains and virus preparations can be used for diagnostic and therapeutic methods, in particular for therapy and diagnosis or monitoring treatment of proliferative disorders, including neoplastic diseases, such as, but are not limited to, solid tumors and blood cancers.

Claims

1. An isolated clonal LIVP strain other than a clonal strain whose genome comprises the sequence of nucleotides set forth in SEQ ID NO:

10.

2. The isolated clonal LIVP strain of claim 1 that has greater anti-tumorigenicity and/or reduced toxicity compared to GLV-1h68, wherein GLV-1h68 has a sequence of nucleotides set forth in SEQ ID NO:

9.

3. An LIVP preparation produced by propagating the isolated clonal LIVP strain of claim 1.

4. The isolated clonal LIVP strain of claim 1, further comprising in its genome non-viral heterologous nucleic acid or a deletion of an open reading frame.

5. The isolated clonal LIVP strain of claim 1 that has reduced toxicity compared to the virus designated GLV-1h68 having a sequence of nucleotides set forth in SEQ ID NO:

9.

6. The isolated clonal LIVP strain of claim 1 that has greater anti-tumorigencity compared to the virus strain designated GLV-1h68 having a sequence of nucleotides set forth in SEQ ID NO:

9.

7. The isolated clonal LIVP strain of claim 1 that has reduced toxicity and greater anti-tumorigenicity compared to the virus designated GLV-1h68 having a sequence of nucleotides set forth in SEQ ID NO:

9.

8. An isolated clonal strain of claim 1 that comprises a sequence of nucleotides that has at least 85% sequence identity with the sequence of nucleotides set forth in SEQ ID NO:

10 but does not comprise the sequence of nucleotides set forth in SEQ ID NO;

10.

9. The isolated clonal strain of claim 1, comprising a sequence of nucleotides selected from:

a) nucleotides 10,073-180,095 of SEQ ID NO;

1, nucleotides 11,243-182,721 of SEQ ID NO;

2, nucleotides 6,264-181,390 of SEQ ID NO;

4, nucleotides 7,044-181,820 of SEQ ID NO;

5, nucleotides 6,674-181,409 of SEQ ID NO;

6, nucleotides 6,716-181,367 of SEQ ID NO;

7 or nucleotides 6,899-181,870 of SEQ ID NO;

8;

or

b) a sequence of nucleotides that has at least 99% sequence identity to a sequence of nucleotides 10,073-180,095 of SEQ ID NO;

1, nucleotides 11,243-182,721 of SEQ ID NO;

2, nucleotides 6,264-181,390 of SEQ ID NO;

4, nucleotides 7,044-181,820 of SEQ ID NO;

5, nucleotides 6,674-181,409 of SEQ ID NO;

6, nucleotides 6,716-181,367 of SEQ ID NO;

7 or nucleotides 6,899-181,870 of SEQ ID NO;

8.

10. The isolated clonal strain of claim 1, comprising a sequence of nucleotides set forth in SEQ ID NOS:

1, 2, 4, 5, 6, 7 or 8, a sequence of nucleotides that has at least 99% sequence identity to a sequence of nucleotides set forth in SEQ ID NO;

1, 2, 4, 5, 6, 7 or 8.

11. A cell culture or isolated cell, comprising a clonal strain of claim 10.

12. An isolated clonal strain produced by:

propagating the cell or cell culture of claim 11; and

isolating an LIVP clone from a resulting cell.

13. A recombinant LIVP virus strain, comprising:

a genome of an LIVP clonal strain of claim 1; and

nucleic acid encoding a heterologous gene product in the genome.

14. The recombinant LIVP virus strain of claim 13, wherein the nucleic acid encoding the heterologous gene product is inserted into or in place of a non-essential gene or region in the genome of the virus.

15. The recombinant LIVP virus strain of claim 13, wherein the nucleic acid encoding the heterologous gene product is inserted at the hemagglutinin (HA), thymidine kinase (TK), F14.5L, vaccinia growth factor (VGF), A35R, N1L, E2L/E3L, K1L/K2L, superoxide dismutase locus, 7.5K, C7-K1L, B13R+B14R, A26L or 14L gene loci in the genome of the virus.

16. The recombinant LIVP virus strain of claim 13, wherein the heterologous gene product is a therapeutic or diagnostic agent.

17. The recombinant LIVP virus strain of claim 13, wherein the heterologous gene product is selected from among an anticancer agent, an antimetastatic agent, an antiangiogenic agent, an immunomodulatory molecule, an antigen, a cell matrix degradative gene, genes for tissue regeneration and reprogramming human somatic cells to pluripotency, enzymes that modify a substrate to produce a detectable product or signal or are detectable by antibodies, proteins that can bind a contrasting agent, genes for optical imaging or detection, genes for PET imaging and genes for MRI imaging.

18. The recombinant LIVP virus strain of claim 13, wherein the heterologous gene product is a therapeutic agent selected from among a hormone, a growth factor, cytokine, a chemokine, a costimulatory molecule, ribozymes, a transporter protein, a single chain antibody, an antisense RNA, a prodrug converting enzyme, an siRNA, a microRNA, a toxin, an antitumor oligopeptide, a mitosis inhibitor protein, an antimitotic oligopeptide, an anti-cancer polypeptide antibiotic, an angiogenesis inhibitor, a tumor suppressor, a cytotoxic protein, a cytostatic protein and a tissue factor.

19. The recombinant LIVP virus strain of claim 13, wherein the heterologous gene product is a diagnostic agent that is a detectable protein or a protein that induces a detectable signal.

20. The recombinant LIVP virus strain claim 19, wherein the heterologous gene product is a diagnostic agent selected from among a luciferase, a fluorescent protein, a bioluminescent protein, a receptor or transporter protein that binds to and/or transports a contrast agent, chromophore, compound or ligand that can be detected.

21. The recombinant LIVP virus strain of claim 13, wherein the heterologous gene product is selected from among a granulocyte macrophage colony stimulating factor (GM-CSF), monocyte chemotactic protein-1 (MCP-1), interleukin-6 (IL-6), interleukin-24 (IL-24), interferon gamma-induced protein 10 (IP-10), lymphotoxin inducible expression competes with HSV glycoprotein D for HVEM a receptor expressed on T-lymphocytes (LIGHT), p60 superantigen, OspF, OspG, signal transducer and activator of transcription protein (STAT1alpha), STATIbeta, plasminogen k5 domain (hK5), pigment epithelium-differentiation factor (PEDF), single chain anti-VEGF antibody, single chain anti-DLL4 antibody, single chain anti-fibroblast activation protein (FAP), NM23, cadherin 1 (ECAD or cdhl), relaxin 1 (RLN1), matrix metallopeptidase 9 (MMP9), erythropoietin (EPO), microRNAl26 (miR-126), microRNA 181, microRNA 335, manganese superoxide dismutase (MnSOD), E3 ubiquitin protein ligase 1 (HACE1), natriuretic peptide precursor A (nppa1), carboxypeptidase G2 (CPG2), alcohol dehydrogenase (ADH), CDC6, bone morphogenetic protein 4 (BMP4), green click beetle luciferase, a lux operon, an infrared fluorescent protein, a flavin reductase protein, mNeptune far-red fluorescent protein, green fluorescent protein (GFP), red fluorescent protein (RFP),-coelenterazine-binding protein (CBP), human epinephrine receptor (hNET), a sodium iodide symporter (NIS) protein, a cytochrome p450 family enzyme, allostatin A receptor (AlstR), Pepl Receptor (PEPR-1), LAT-4, sterol 14 alpha-demethylase (Cyp51), transferring receptor (TR), ferritin, divalent metal transporter (DMT), Magnetotactic A (MagA), cisplatin influx transporter (CTR1), newt AG (nAG), Oct4, NANOG, Ngn3, Pdx1 and Mafa.

22. The recombinant LIVP virus strain of claim 13, wherein the nucleic acid encoding the heterologous gene product is operably linked to a promoter.

23. The recombinant LIVP virus strain of claim 22, wherein the promoter is a mammalian promoter or a viral promoter.

24. The recombinant LIVP virus strain of claim 22, wherein the promoter is selected from among P_7.5k, P_11k, P_SE, P_SEL, P_SL, H5R, TK, P28, C11R, G8R, F17R, I3L, I8R, A1L, A2L, A3L, H1L, H3L, H5L, H6R, H8R, D1R, D4R, D5R, D9R, D11L, D12L, D13L, M1L, N2L, P4b and K1 promoters.

25. A composition, comprising an isolated clonal virus of claim 1.

26. A composition, comprising the recombinant LIVP virus strain of claim 13.

27. The composition of claim 25 that is a pharmaceutical composition.

28. The composition of claim 26 that is a pharmaceutical composition.

29. A method of treating a proliferative disorder in a subject comprising administering the pharmaceutical composition of claim 27.

30. The method of claim 29, wherein the proliferative disease is cancer.

31. The method of claim 30, wherein the cancer is breast cancer, prostate cancer, ovarian cancer, lung cancer, colon cancer or pancreatic cancer.

32. The method of any of claim 29, wherein the subject is a human.

33. The method of claim 29, wherein the subject is a non-human animal that is selected from among a horse, cat, dog, cow, pig, sheep, goat, mouse, rabbit, chicken, rat, and guinea pig

34. The method of claim 29, further comprising another treatment selected from among surgery, radiation therapy, immunosuppressive therapy and administration of an anticancer agent, wherein the further treatment is effected before, after, simultaneously or intermittently with the virus.

35. The method of claim 34, wherein the further treatment is administration of an anticancer agent selected from among a cytokine, a chemokine, a growth factor, a photosensitizing agent, a toxin, an anti-cancer antibiotic, a chemotherapeutic compound, a radionuclide, an angiogenesis inhibitor, a signaling modulator, an anti-metabolite, an anti-cancer vaccine, an anti-cancer oligopeptide, a mitosis inhibitor protein, an antimitotic oligopeptide, an anticancer antibody, an anti-cancer antibiotic, an immunotherapeutic agent and a combination of any of the preceding thereof.

36. A method of detecting a tumor or metastasis in a subject, comprising:

administering a clonal strain of claim 19; and

detecting the detectable protein or a protein that induces a detectable signal, whereby detection indicates the presence of the tumor or metastasis in the subject.

37. The method of claim 36, wherein detection is effected by imaging the subject.

38. The method of claim 36, wherein detection is effected by detecting the protein or signal in a tissue or body fluid sample.

39. The method of claim 36, wherein the detectable protein or protein that induces a detectable signal is selected from among a luciferase, a fluorescent protein, a bioluminescent protein, a receptor or transporter protein that binds to and/or transports a contrast agent, chromophore, compound or ligand that can be detected.

40. The method of claim 36, wherein the detectable protein or detectable signal is detected by low-light imaging, fluorescence spectroscopy, x-ray imaging, magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MRS), positron emission tomography (PET) or single-photon emission computed tomography (SPECT).

41. A method of selecting an LIVP clonal strain for cancer therapy and diagnosis, comprising:

(i) providing a first LIVP virus sample that contains a mixture of virus particles with different genomic sequences;

(ii) selecting and isolating single clonal isolates from the sample;

(iii) assaying each clonal isolate for toxicity;

(iv) assaying each clonal isolate for anti-tumorigenicity; and

(v) selecting a clonal strain that exhibits greater anti-tumorigenicity and reduced toxicity compared to a reference LIVP virus strain, whereby the clonal isolates that exhibit reduced toxicity and greater anti-tumorigenicity are identified as LIVP clonal strains for cancer therapy or diagnosis.

42. The method of claim 41, wherein the reference LIVP virus is an attenuated recombinant LIVP virus.

44. The method of claim 41, wherein the first LIVP virus sample is an LIVP virus strain that comprises an LIVP having a genome set forth in SEQ ID NO:

10, or a genome that is at least 99% identical to SEQ ID NO;

10.

45. The method of claim 41, wherein single clonal isolates from the sample are selected by a plaque assay.

46. The method of claim 45, wherein the largest plaques in the plaque assay are selected.

43. The method of 41, wherein the reference LIVP virus is the virus designated GLV-1h68 whose genome comprises the sequence of nucleotides set forth in SEQ ID NO:

9 (GLV-1h68).