U.S. patent application number 12/157960 was filed with the patent office on 2009-05-07 for microorganisms for imaging and/or treatment of tumors.
Invention is credited to Nanhai Chen, Yuman Fong, Aladar A. Szalay, Yong A. Yu, Qian Zhang.
Application Number | 20090117034 12/157960 |
Document ID | / |
Family ID | 40002958 |
Filed Date | 2009-05-07 |
United States Patent
Application |
20090117034 |
Kind Code |
A1 |
Chen; Nanhai ; et
al. |
May 7, 2009 |
Microorganisms for imaging and/or treatment of tumors
Abstract
Modified viruses encoding transporter proteins and methods for
preparing the modified viruses are provided. Vaccines that contain
the viruses are provided. The viruses also can be used in
diagnostic methods, such detection and imaging of tumors. The
viruses also can be used in methods of treatment of diseases, such
as proliferative and inflammatory disorders, including as
anti-tumor agents.
Inventors: |
Chen; Nanhai; (San Diego,
CA) ; Szalay; Aladar A.; (Highland, CA) ; Yu;
Yong A.; (San Diego, CA) ; Zhang; Qian; (San
Diego, CA) ; Fong; Yuman; (New York, NY) |
Correspondence
Address: |
K&L Gates LLP
3580 Carmel Mountain Road, Suite 200
San Diego
CA
92130
US
|
Family ID: |
40002958 |
Appl. No.: |
12/157960 |
Filed: |
June 13, 2008 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60934768 |
Jun 15, 2007 |
|
|
|
Current U.S.
Class: |
424/1.17 ;
424/155.1; 424/199.1; 424/9.2; 424/9.3; 424/9.4; 424/9.6; 424/93.2;
435/235.1 |
Current CPC
Class: |
C07K 14/705 20130101;
G01N 33/574 20130101; A61P 35/00 20180101; A61P 9/00 20180101; C12N
2710/24143 20130101; A61K 35/768 20130101; Y02A 50/471 20180101;
A61P 35/04 20180101; Y02A 50/473 20180101; A61K 35/76 20130101;
A61K 38/00 20130101; A61P 43/00 20180101; A61P 35/02 20180101; C12N
7/00 20130101; C12N 2710/24132 20130101 |
Class at
Publication: |
424/1.17 ;
435/235.1; 424/93.2; 424/199.1; 424/9.6; 424/9.3; 424/9.4;
424/155.1; 424/9.2 |
International
Class: |
A61K 51/02 20060101
A61K051/02; C12N 7/01 20060101 C12N007/01; A61K 35/76 20060101
A61K035/76; A61K 49/04 20060101 A61K049/04; A61K 39/395 20060101
A61K039/395; A61P 35/00 20060101 A61P035/00; A61P 35/04 20060101
A61P035/04; A61P 35/02 20060101 A61P035/02; A61B 10/00 20060101
A61B010/00; A61K 49/00 20060101 A61K049/00; A61K 49/06 20060101
A61K049/06; A61K 39/285 20060101 A61K039/285 |
Claims
1. A recombinant vaccinia virus that encodes a sodium-dependent
transporter protein.
2. The recombinant vaccinia virus of claim 1, wherein the
transporter protein is selected from among a solute carrier 5
family and solute carrier 6 transporter protein.
3. The recombinant vaccinia virus of claim 1, wherein the
transporter protein is a norepinephrine transporter (NET) or is a
sodium-iodide symporter (NIS).
4. The recombinant vaccinia virus of claim 3, wherein the
transporter protein is a norepinephrine transporter that is a human
norepinephrine transporter (hNET).
5. The recombinant vaccinia virus of claim 3, wherein the
transporter protein is a sodium-iodide symporter that is a human
sodium-iodide symporter (hNIS).
6. The recombinant vaccinia virus of claim 1, that is a Lister
strain virus.
7. The recombinant vaccinia virus of claim 6 that is an LIVP strain
virus.
8. The recombinant vaccinia virus of claim 7 that is selected from
among GLV-1h99, GLV-1h100, GLV-1h101, GLV-1h139, GLV-1h146,
GLV-1h150, GLV-1h151, GLV-1h152 and GLV-1h153.
9. The recombinant vaccinia virus of claim 1, wherein nucleic acid
encoding the transporter protein is inserted in the hemagglutinin
(HA), thymidine kinase (TK) or F14.5 gene or locus.
10. The recombinant vaccinia virus of claim 1, wherein the virus
encodes a therapeutic agent.
11. The recombinant vaccinia virus of claim 10, wherein the
therapeutic agent is an anti-cancer agent or anti-angiogenic
agent.
12. The recombinant vaccinia virus of claim 10, wherein the
therapeutic agent is inserted into a different gene or locus from
the transporter gene and is inserted in the hemagglutinin (HA),
thymidine kinase (TK) or F14.5 gene or locus.
13. The recombinant vaccinia virus of claim 10, wherein the
therapeutic agent is selected from among a cytokine, a chemokine,
an immunomodulatory molecule, an antigen, an antibody or fragment
thereof, antisense RNA, prodrug converting enzyme, siRNA,
angiogenesis inhibitor, a toxin, an antitumor oligopeptide, a
mitosis inhibitor protein, an antimitotic oligopeptide, an
anti-cancer polypeptide antibiotic, and tissue factor.
14. The recombinant virus of claim 13 wherein the therapeutic agent
is an antibody or fragment thereof that is a single chain antibody
(scFv).
15. The recombinant virus of claim 13, wherein the therapeutic
agent is an anti-VEGF single chain antibody, a plasminogen K5
domain, a human tissue factor-.alpha.v.beta.3-integrin RGD fusion
protein, interleukin-24 or an IL-6-IL-6 receptor fusion
protein.
16. A combination, comprising: a recombinant virus of claim 1; and
a substrate transported into a cell that expresses the transporter;
and/or an anti-cancer compound.
17. The combination of claim 16, wherein the substrate is
detectable.
18. The combination of claim 16, wherein the substrate emits an
electromagnetic signal or induces such emission.
19. The combination of claim 16, wherein the substrate is
radiolabeled.
20. The combination of claim 16, wherein the substrate is
conjugated to a cytotoxic agent.
21. The combination of claim 20, wherein conjugation is direct or
via a linker.
22. The combination of claim 20, wherein the cytotoxic agent is a
radiolabel, a cytotoxin or a cytotoxic drug.
23. The combination of claim 20, wherein the cytotoxic agent is
selected from among double-chain ricin, ricin A chain, abrin, abrin
A chain, saporin, modeccin, modeccin A chain, Pseudomonas
aeruginosa exotoxin, Cholera toxin, Shigella toxin, E. coli heat
labile toxin, Diptheria toxin, doxorubicin, daunomycin,
fluorouracil, methotrexate, taxol, ricin A, colchicine,
cytochalasins, monensin, ouabain, mitoxanthrone, vindesine,
vinblastine, vincristine or enterotoxin.
24. The combination of claim 16, wherein the substrate is
conjugated to an anticancer agent.
25. The combination of claim 16, wherein the combination comprises
the virus and an anti-cancer compound 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 anti-cancer antibody, an anti-cancer antibiotic,
an immunotherapeutic agent, a bacterium and a combination of any of
the preceding thereof.
26. The combination of claim 25, wherein the anti-cancer compound
is selected from among cisplatin, carboplatin, gemcitabine,
irinotecan, an anti-EGFR antibody and an anti-VEGF antibody.
27. The combination of claim 16, wherein the substrate and virus
are formulated as a single composition or separately in two
compositions.
28. The combination of claim 16, wherein the anti-cancer compound
and virus are formulated as a single composition or separately in
two compositions.
29. A kit, comprising the combination of claim 16; and optionally
instructions for administration of the composition(s).
30. A pharmaceutical composition, comprising a recombinant virus of
claim 1 in a pharmaceutically acceptable carrier.
31. The pharmaceutical composition of claim 30 that is formulated
for local or systemic administration.
32. A vaccine, comprising the recombinant virus of claim 1.
33. The vaccine of claim 32 that is a smallpox vaccine.
34. A method of imaging or detecting a tumor, an inflammation or a
wound within a subject comprising: administering a vaccinia virus
of claim 1 to a subject having a tumor; administering a detectable
substrate that is transported into a cell that expresses the
transporter encoded by the virus; and detecting the accumulation of
the substrate, whereby the tumor, wound and/or inflammation is
imaged or detected.
35. The method of claim 34, wherein the substrate is detected by
fluorescence imaging, magnetic resonance imaging (MRI),
single-photon emission computed tomography (SPECT), positron
emission tomography (PET), scintigraphy, gamma camera, a .beta.+
detector, a .gamma. detector or a combination thereof.
36. A method of treatment, comprising administering a vaccinia
virus of claim 1 to a subject to effect treatment.
37. The method of claim 36, wherein the subject is treated for a
tumor, cancer or metastasis.
38. The method of claim 36, further comprising: administering a
substrate that is transported into a cell that expresses the
transporter encoded by the virus, wherein: the substrate is
therapeutic or is conjugated to a therapeutic agent, whereby
treatment is effected; and the substrate is administered before or
after or simultaneously with the virus.
39. The method of claim 38, wherein the subject has a tumor and the
substrate or substrate conjugate is effective in treating a
tumor.
40. The method of claim 38, wherein the substrate is conjugated to
a cytotoxic agent.
41. The method of claim 40, wherein the cytotoxic agent is a
radiolabel, a cytotoxin or a cytotoxic drug.
42. The method of claim 41, wherein the cytotoxic agent is selected
from among double-chain ricin, ricin A chain, abrin, abrin A chain,
saporin, modeccin, modeccin A chain, Pseudomonas aeruginosa
exotoxin, Cholera toxin, Shigella toxin, E. coli heat labile toxin
and Diptheria toxin, doxorubicin, daunomycin, 5-fluorouracil,
methotrexate, taxol, ricin A, colchicine, cytochalasins, monensin,
ouabain, mitoxanthrone, vindesine, vinblastine, vincristine or
enterotoxin.
43. The method of claim 38, wherein the substrate is conjugated to
an anticancer agent.
44. The method of claim 36, wherein the virus is administered
systemically, intravenously, intraarterially, intratumorally,
endoscopically, intralesionally, intramuscularly, intradermally,
intraperitoneally, intravesicularly, intraarticularly,
intrapleurally, percutaneously, subcutaneously, orally,
parenterally, intranasally, intratracheally, by inhalation,
intracranially, intraprostaticaly, intravitreally, topically,
ocularly, vaginally, or rectally.
45. The method of claim 36, further comprising administering an
anticancer agent or treatment, wherein the anti-cancer agent or
treatment is administered before or after or simultaneously with
the virus or the virus and substrate.
46. The method of claim 45, wherein: an anticancer agent is
administered; and the anticancer agent is 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 anti-cancer antibody, an anti-cancer antibiotic,
an immunotherapeutic agent, hyperthermia or hyperthermia therapy, a
bacterium, radiation therapy and a combination of any of the
preceding thereof.
47. The method of claim 46, wherein the anticancer agent is
selected from among cisplatin, carboplatin, gemcitabine,
irinotecan, an anti-EGFR antibody and an anti-VEGF antibody.
48. The method of claim 45, wherein the anticancer agent is
administered simultaneously or intermittently with the virus or
with the virus and/or substrate.
49. The method of claim 45, wherein the virus and the anticancer
agent are administered as a single composition or as two
compositions, or the virus, substrate and anticancer agent are
administered in a single composition or in two compositions
containing two of the substrate, virus and anticancer agent, or in
three compositions.
50. The method of claim 37, wherein the subject is treated for a
human tumor selected from among a bladder tumor, breast tumor,
prostate tumor, carcinoma, basal cell carcinoma, biliary tract
cancer, bladder cancer, bone cancer, brain cancer, CNS cancer,
glioma tumor, cervical cancer, choriocarcinoma, colon and rectum
cancer, connective tissue cancer, cancer of the digestive system,
endometrial cancer, esophageal cancer, eye cancer, cancer of the
head and neck, gastric cancer, intra-epithelial neoplasm, kidney
cancer, larynx cancer, leukemia, liver cancer, lung cancer,
lymphoma, Hodgkin's lymphoma, Non-Hodgkin's lymphoma, melanoma,
myeloma, neuroblastoma, oral cavity cancer, ovarian cancer,
pancreatic cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer,
renal cancer, cancer of the respiratory system, sarcoma, skin
cancer, stomach cancer, testicular cancer, thyroid cancer, uterine
cancer, and cancer of the urinary system.
51. The method of claim 37, wherein the subject is treated for a
tumor and the tumor is an ovarian tumor, a breast tumor, a
pancreatic tumor, a colon tumor, or a lung tumor.
52. The method of claim 37, wherein the subject is treated for a
tumor and the tumor is selected from among lymphosarcoma,
osteosarcoma, mammary tumors, mastocytoma, brain tumor, melanoma,
adenosquamous carcinoma, carcinoid lung tumor, bronchial gland
tumor, bronchiolar adenocarcinoma, fibroma, myxochondroma,
pulmonary sarcoma, neurosarcoma, osteoma, papilloma,
retinoblastoma, Ewing's sarcoma, Wilm's tumor, Burkitt's lymphoma,
microglioma, neuroblastoma, osteoclastoma, oral neoplasia,
fibrosarcoma, osteosarcoma and rhabdomyosarcoma, genital squamous
cell carcinoma, transmissible venereal tumor, testicular tumor,
seminoma, Sertoli cell tumor, hemangiopericytoma, histiocytoma,
chloroma, granulocytic sarcoma, corneal papilloma, corneal squamous
cell carcinoma, hemangiosarcoma, pleural mesothelioma, basal cell
tumor, thymoma, stomach tumor, adrenal gland carcinoma, oral
papillomatosis, hemangioendothelioma, cystadenoma, follicular
lymphoma, intestinal lymphosarcoma, fibrosarcoma, and pulmonary
squamous cell carcinoma, leukemia, hemangiopericytoma, ocular
neoplasia, preputial fibrosarcoma, ulcerative squamous cell
carcinoma, preputial carcinoma, connective tissue neoplasia,
mastocytoma, hepatocellular carcinoma, lymphoma, pulmonary
adenomatosis, pulmonary sarcoma, Rous sarcoma,
reticulo-endotheliosis, fibrosarcoma, nephroblastoma, B-cell
lymphoma, lymphoid leukosis, retinoblastoma, hepatic neoplasia,
lymphosarcoma, plasmacytoid leukemia, swimbladder sarcoma (in
fish), caseous lumphadenitis, lung carcinoma, insulinoma, lymphoma,
sarcoma, neuroma, pancreatic islet cell tumor, gastric MALT
lymphoma and gastric adenocarcinoma.
53. The method of claim 36, further comprising administering an
anti-viral agent.
54. The method of claim 53, wherein the antiviral agent is selected
from among cidofovir, alkoxyalkyl esters of cidofovir, imatinib,
gancyclovir, acyclovir and Tecovirimat.
55. The method of claim 36, further comprising: administering a
detectable substrate that is transported into a cell that expresses
the transporter encoded by the virus; and detecting the substrate
or accumulation of the substrate, whereby the therapeutic treatment
of the tumor is monitored.
56. The method of claim 38, further comprising: detecting the
substrate or accumulation of the substrate, whereby the therapeutic
treatment of the tumor is monitored.
57. A method of detection of a tumor or metastasis and treatment a
tumor and/or metastases within a subject, comprising: administering
a vaccinia virus of claim 1 to a subject having a tumor, wherein
the virus optionally encodes an anti-tumor therapeutic agent;
optionally administering a therapeutic agent to the subject for
treatment of a tumor; administering a detectable substrate that is
transported into a cell that expresses the transporter encoded by
the virus; and detecting substrate or the accumulation of the
substrate to detect a tumor or metastases and to effect treatment
thereof.
58. The method of claim 57, wherein detection is monitored as a
function of time, whereby treatment is monitored.
59. An isolated cell, comprising a recombinant virus of claim 1.
Description
RELATED APPLICATIONS
[0001] Benefit of priority is claimed under 35 U.S.C. .sctn.119(e)
to U.S. Provisional Application Ser. No. 60/934,768, to Nanhai
Chen, Yuman Fong, Aladar A. Szalay, Yong A. Yu and Qian Zhang,
filed on Jun. 15, 2007, entitled "MICROORGANISMS FOR IMAGING AND/OR
TREATMENT OF TUMORS." The subject matter of this application is
incorporated by reference in its entirety.
[0002] This application is related to International Application No.
(Attorney Dkt. No. 0119356-00132/112PC) to Nanhai Chen, Yuman Fong,
Aladar A. Szalay, Yong A. Yu and Qian Zhang, filed on Jun. 13,
2008, entitled "MICROORGANISMS FOR IMAGING AND/OR TREATMENT OF
TUMORS," which also claims priority to U.S. Provisional Application
Ser. No. 60/934,768. The subject matter of this application is
incorporated by reference in its entirety.
INCORPORATION BY REFERENCE OF A SEQUENCE LISTING PROVIDED ON
COMPACT DISCS
[0003] An electronic version on compact disc (CD-R) of the Sequence
Listing is filed herewith in duplicate (labeled Copy # 1 and Copy #
2), the contents of which are incorporated by reference in their
entirety. The computer-readable file on each of the aforementioned
compact discs, created on Jun. 13, 2008, is identical, 992
kilobytes in size, and entitled 112SEQ.001.txt.
FIELD OF THE INVENTION
[0004] Modified recombinant viruses for diagnosis and therapy are
provided. Diagnostic and therapeutic methods using the modified
recombinant viruses also are provided.
BACKGROUND
[0005] Cancers, such as pancreatic cancer and malignant pleural
mesothelioma, are highly aggressive diseases. The annual incidence
in the United States was estimated to be .about.40,000 cases for
pancreatic cancer and .about.4,000 cases for malignant mesothelioma
in the year 2004, with increasing incidence worldwide for
mesothelioma, especially in industrialized nations due to the
etiology of this disease from asbestos exposure (Bianchi and
Bianchi (2007) Ind Health 45: 379-87). Both of these tumors are
highly resistant to standard therapies, with 5-year survival rates
of only 5% for pancreatic cancer and 9% for mesothelioma. Even with
combined surgery, chemotherapy and radiation, only a small minority
of patients are rendered disease-free for a prolonged period of
time (Adusumilli et al. (2006) J Gene Med 8:603-15.
[0006] Oncolytic viral therapy has been studied and tested over the
past century, and many viral types, including adenovirus, herpes
simplex virus, Newcastle disease virus, myxoma virus, vaccinia
virus and vesicular stomatitis virus, are being investigated as
novel agents for the treatment of human cancer (Woo et al. (2006)
Curr Opin Investig Drugs 7:549-59). Accordingly, effective tumor
diagnostic and therapeutic viral agents that are highly selective
for tumors are needed. In addition, there exists a need to provide
reagents and methods for tracking and monitoring viral
distribution, tumor targeting, proliferation and persistence in
oncolytic viral therapies by noninvasive imaging, which provide
important safety, efficacy and toxicity data. Such real-time
monitoring also would provide useful viral-dose and administration
schedule information for optimization of therapy and would obviate
the need for multiple and repeated tissue biopsies.
SUMMARY
[0007] Provided are recombinant viruses, particularly, vaccinia
virus, such as LIVP, that accumulates in tumors or other
immunoprivileged tissues, such as wounds and inflamed tissues, and
not accumulate to toxic levels in other tissues. These viruses
encoded a protein that enhances uptake or retention of a compound
that emits a signal that permits detection, such as by non-optical
imaging. Proteins that enhance uptake or retention include
transporter proteins. These viruses also can be used for treatment
of tumors, wounded tissues and inflammations within a subject. The
compound that is taken up or retained can be a therapeutic compound
or can be modified, such as by conjugate to a therapeutic compound,
to have therapeutic activity. The viruses can be used for
detection, detection and treatment, detection and monitoring of
treatment. Methods for detection, detection and treatment,
detection and monitoring of treatment are provided as are uses of
recombinant viruses, such as vaccinia viruses for detection,
detection and treatment, detection and monitoring of treatment.
[0008] Provided herein are recombinant vaccinia viruses that encode
a sodium-dependent transporter protein. Sodium-dependent
transporter proteins include those from the solute carrier 5 and
solute carrier 6 transporter protein families, such as a
norepinephrine transporter (NET) and a sodium-iodide symporter
(NIS), including a human norepinephrine transporter (hNET) and a
human sodium-iodide symporter (hNIS) as well as allelic and species
variants thereof and other variants, including any having at least
about or at least 60, 65, 70, 75, 80, 85, 88, 90, 91, 92, 93, 94,
95, 96, 97, 98 and 99 percent or more sequence identity with those
disclosed herein. These include modified forms that retain
transporter activity sufficient for the methods provided
herein.
[0009] Recombinant vaccinia virus include of any of claims 1-5 that
is a Lister strain viruses, such as the LIVP strain. Nucleic acid
encoding transporter protein can be inserted anywhere in the virus
such that the virus expresses it and replicates in a subject. In
exemplary embodiments, the nucleic acid encoding the transporter
protein is inserted into a nonessential locus or gene, such as the
hemagglutinin (HA), thymidine kinase (TK) or F14.5 gene or locus.
Exemplary of such viruses are those provided herein that include
GLV-1h99, GLV-1h100, GLV-1h101, GLV-1h139, GLV-1h146, GLV-1h150,
GLV-1h151, GLV-1h152 and GLV-1h153. These viruses can be further
modified to encode a therapeutic protein. Generally the encoding
nucleic acid is inserted into a different locus from the nucleic
acid that encodes the transporter protein. Exemplary therapeutic
agents include, but are not limited to an anti-cancer agents and
anti-angiogenic agents. A therapeutic agent, includes, but is not
limited to, a cytokine, a chemokine, an immunomodulatory molecule,
an antigen, an antibody or fragment thereof, antisense RNA, prodrug
converting enzyme, siRNA, angiogenesis inhibitor, a toxin, an
antitumor oligopeptide, a mitosis inhibitor protein, an antimitotic
oligopeptide, an anti-cancer polypeptide antibiotic, and tissue
factor, such as single chain antibody (scFv), including an
anti-VEGF single chain antibody, a plasminogen K5 domain, a human
tissue factor-.alpha.v.beta.3-integrin RGD fusion protein,
interleukin-24 or an IL-6-IL-6 receptor fusion protein and fusion
proteins of substrates for the transporter protein and a
therapeutic agent, such as a chemotherapeutic compound or a toxin.
The viruses can encoded a plurality of therapeutic agents and/or
transporter proteins.
[0010] Also provided are combination that contain one or more of
the recombinant viruses, particularly vaccinia viruses, provided
herein, and a substrate transported into a cell that expresses the
transporter; and/or an anti-cancer compound. Substrates can be
detectable or can induce a detectable signal or can modified to be
detectable or to induce a detectable signal, such as
electromagnetic radiation. The substrate can be radiolabeled; it
can be conjugated to a cytotoxic agent, such as 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 anti-cancer antibody, an anti-cancer antibiotic,
an immunotherapeutic agent, a bacterium and combinations
thereof.
[0011] Conjugation can be chemical or, where the substrate and
cytotoxic agent are proteins can be a fusion protein. Conjugate can
be direct or via a linker. Exemplary cytotoxic agents, include, but
are not limited to, radiolabels, a cytotoxins and chemotherapeutic
drugs and cytotoxic drugs. Exemplary cytotoxic agents, include, but
are not limited to, double-chain ricin, ricin A chain, abrin, abrin
A chain, saporin, modeccin, modeccin A chain, Pseudomonas
aeruginosa exotoxin, Cholera toxin, Shigella toxin, E. coli heat
labile toxin and Diphtheria toxin. doxorubicin, daunomycin,
5-fluorouracil, methotrexate, taxol, ricin A, colchicine,
cytochasins, monensin, ouabain, mitoxanthrone, vindesine,
vinblastine, vincristine, enterotoxin, cisplatin, carboplatin,
gemcitabine, irinotecan, an anti-EGFR antibody and an anti-VEGF
antibody. In addition to conjugation the substrate and cytotoxic
agent can be separate and can be separately administered.
Proteinaceous cytotoxic agents and conjugates also can be expressed
by the virus. In the combinations, the substrate and virus can be
formulated as a single composition or separately in two
compositions. Also, provided are kits that contain the combinations
and optionally reagents and other components for use of the
combinations and instructions for use thereof.
[0012] Also provided are pharmaceutical composition containing the
recombinant viruses provided herein in a pharmaceutically
acceptable carrier. They can be formulated for any type of
administration include local or systemic administration.
[0013] The recombinant viruses provided herein also can be
vaccines, including smallpox vaccines.
[0014] Also provided are methods of imaging or detecting a tumor,
an inflammation or a wound within a subject practiced by
administering any virus provided herein to a subject suspected of a
having a tumor and/or internal wound or inflammation. In practicing
the method, the virus is administered with or sequentially or
intermittently with a substrate that is transported into a cell
that expresses the transporter encoded by the virus. The substrate
or its accumulation can be detected, thereby detecting or imaging a
tumor, wound and/or inflammation. Detection and imaging can be
effected by fluorescence imaging, magnetic resonance imaging (MRI),
single-photon emission computed tomography (SPECT), positron
emission tomography (PET), scintigraphy, gamma camera, a .beta.+
detector, a .gamma. detector and combinations thereof.
[0015] Methods of treatment are provided. The methods are effected
by administering a any virus provided herein to a subject to effect
treatment. Treatment can be for any disease or disorder for which
administration of a virus, particularly a vaccinia virus, is
effective. Such diseases and disorders include, tumors and cancers
and/or metastasis. The method can further include administering a
substrate that is transported into a cell that expresses the
transporter encoded by the virus. The substrate itself can be
therapeutic or it can be conjugated to a therapeutic agent, whereby
treatment is effected. The substrate can be administered before or
after or simultaneously with the virus or it can be encoded by
nucleic acid that is administered, such as another virus or other
vector that encodes it. The substrate can be conjugated to a
cytotoxic agent as described above.
[0016] For the methods of treatment, the virus can be administered
by any suitable route, including systemically, intravenously,
intraarterially, intratumorally, endoscopically, intralesionally,
intramuscularly, intradermally, intraperitoneally,
intravesicularly, intraarticularly, intrapleurally, percutaneously,
subcutaneously, orally, parenterally, mucosally, intranasally,
intratracheally, by inhalation, intracranially, intraprostaticaly,
intravitreally, topically, ocularly, vaginally and rectally. The
virus or virus and substrate can be administered with an anticancer
agent or treatment. The anticancer agent or treatment can be
administered before or after or simultaneously or intermittently
with the virus or the virus and substrate. Anticancer agents
include any noted above, including, but not limited to, 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 anti-cancer antibody, an anti-cancer antibiotic,
an immunotherapeutic agent, hyperthermia or hyperthermia therapy, a
bacterium, radiation therapy and any combination thereof.
[0017] Exemplary anticancer agents include cisplatin, carboplatin,
gemcitabine, irinotecan, an anti-EGFR antibody and an anti-VEGF
antibody. Exemplary anticancer therapies include radiation.
Treatments with anticancer agents or therapies, can be effected
simultaneously or intermittently with the virus or with the virus
and/or substrate in any order. The virus and substrate and
anticancer agent can be administered separately or can be combined
into one or two compositions. Thus, the virus and the anticancer
agent can be administered as a single composition or as two
compositions, or the virus, substrate and anticancer agent can be
administered in a single composition or in two compositions
containing two of the substrate, virus and anticancer agent, or in
three compositions.
[0018] Tumors that can be treated by administration of the virus,
or virus and substrate or virus, substrate and anticancer agent or
therapy, include, but are not limited to a bladder tumor, breast
tumor, prostate tumor, carcinoma, basal cell carcinoma, biliary
tract cancer, bladder cancer, bone cancer, brain cancer, CNS
cancer, glioma tumor, cervical cancer, choriocarcinoma, colon and
rectum cancer, connective tissue cancer, cancer of the digestive
system, endometrial cancer, esophageal cancer, eye cancer, cancer
of the head and neck, gastric cancer, intra-epithelial neoplasm,
kidney cancer, larynx cancer, leukemia, liver cancer, lung cancer,
lymphoma, Hodgkin's lymphoma, Non-Hodgkin's lymphoma, melanoma,
myeloma, neuroblastoma, oral cavity cancer, ovarian cancer,
pancreatic cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer,
renal cancer, cancer of the respiratory system, sarcoma, skin
cancer, stomach cancer, testicular cancer, thyroid cancer, uterine
cancer, and cancer of the urinary system, such as lymphosarcoma,
osteosarcoma, mammary tumors, mastocytoma, brain tumor, melanoma,
adenosquamous carcinoma, carcinoid lung tumor, bronchial gland
tumor, bronchiolar adenocarcinoma, small cell lung cancer, non
small cell lung cancers, fibroma, myxochondroma, pulmonary sarcoma,
neurosarcoma, osteoma, papilloma, retinoblastoma, Ewing's sarcoma,
Wilm's tumor, Burkitt's lymphoma, microglioma, neuroblastoma,
osteoclastoma, oral neoplasia, fibrosarcoma, osteosarcoma and
rhabdomyosarcoma, genital squamous cell carcinoma, transmissible
venereal tumor, testicular tumor, seminoma, Sertoli cell tumor,
hemangiopericytoma, histiocytoma, chloroma, granulocytic sarcoma,
corneal papilloma, corneal squamous cell carcinoma,
hemangiosarcoma, pleural mesothelioma, basal cell tumor, thymoma,
stomach tumor, adrenal gland carcinoma, oral papillomatosis,
hemangioendothelioma, cystadenoma, follicular lymphoma, intestinal
lymphosarcoma, fibrosarcoma, and pulmonary squamous cell carcinoma,
leukemia, hemangiopericytoma, ocular neoplasia, preputial
fibrosarcoma, ulcerative squamous cell carcinoma, preputial
carcinoma, connective tissue neoplasia, mastocytoma, hepatocellular
carcinoma, lymphoma, pulmonary adenomatosis, pulmonary sarcoma,
Rous sarcoma, reticulo-endotheliosis, fibrosarcoma, nephroblastoma,
B-cell lymphoma, lymphoid leukosis, retinoblastoma, hepatic
neoplasia, lymphosarcoma, plasmacytoid leukemia, swimbladder
sarcoma (in fish), caseous lumphadenitis, lung carcinoma,
insulinoma, lymphoma, sarcoma, neuroma, pancreatic islet cell
tumor, gastric MALT lymphoma and gastric adenocarcinoma.
[0019] An antiviral agent can be administered simultaneously or
sequentially with any of the above-treatments. Anti-viral agents,
include, but are not limited to, cidofovir, alkoxyalkyl esters of
cidofovir, Gleevec, gancyclovir, acyclovir and ST-26.
[0020] Methods of treatment and detection are provided. The virus
or virus and anti-cancer agent or therapy can be administered
sequentially or simultaneously with a detectable substrate or a
substrate that induces a signal and that is transported into a cell
that expresses the transporter encoded by the virus; and then the
substrate or accumulation of the substrate is detected. Thus,
treatment and detection can be effected. Detection, which includes
imaging, can be used to monitor treatment, particularly, if
detection is effected a plurality of times to monitor the changes
in the pattern of accumulation of a substrate. Effective treatment
would result in less accumulation of substrate or a more localized
accumulation or other pattern correlated with a decrease in tumor
size or metastasis or other indicator of tumor treatment.
[0021] In practicing the methods of treatment or treatment and
detection, the virus can encoded a therapeutic agent, such as an
anti-tumor therapeutic agent. Inclusion of such agent is optional
as the viruses, particularly the vaccinia viruses provided herein,
effect treatment in the absence of further therapeutic agent.
[0022] Also provided are cells, particularly isolated cells,
including tumor cells, that contain any virus provided herein. The
cells can be provided in pharmaceutical compositions for treatment.
Uses of the virus provided herein for treatment of for the
preparation of a pharmaceutical composition for the treatment
cancer or detection of cancer, tumors, metastases, wound and/or
inflammation in a subject. Are provided. Also compositions
containing the viruses for such uses are provided. The compositions
and uses can include a substrate or conjugate containing the
substrate that can be transported into a cell that expresses the
transporter protein encoded by the virus. The compositions
optionally can include an anti-cancer compound in addition to the
virus and/or substrate or conjugate as described above. Anticancer
compound include any noted herein, including, but are not limited
to, 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 anti-cancer antibody, an anti-cancer
antibiotic, an immunotherapeutic agent, hyperthermia or
hyperthermia therapy, a bacterium and combinations thereof, such as
cisplatin, carboplatin, gemcitabine, irinotecan, an anti-EGFR
antibody and an anti-VEGF antibody.
DETAILED DESCRIPTION
Outline
[0023] A. Definitions
[0024] B. Viruses for treatment and diagnosis
[0025] C. Transporter Proteins [0026] 1. The sodium- and
chloride-dependent neurotransmitter transporter family [0027] a.
Norepinephrine Transporter [0028] i. Structure [0029] ii. Function
[0030] 2. The sodium glucose cotransporter family [0031] a. Sodium
Iodide Symporter [0032] i. Structure [0033] ii. Function
[0034] D. Methods of assessing modified viruses encoding
transporters [0035] 1. In vitro assessment [0036] 2. In vivo
assessment [0037] 3. Selection of substrates
[0038] E. Additional modifications of viruses provided [0039] 1.
Modification of viral genes [0040] 2. Expression of additional
heterologous genes [0041] a. Detectable gene product [0042] b.
Therapeutic gene product [0043] c. Superantigen [0044] d. Gene
product to be harvested [0045] e. Control of heterologous gene
expression
[0046] F. Methods for making a modified virus [0047] 1. Genetic
modifications [0048] 2. Screening of modified viruses
[0049] G. Exemplary characteristics of the viruses provided [0050]
1. Attenuated [0051] a. Reduced toxicity [0052] b. Accumulate in
tumor, not substantially in other organs [0053] c. Ability to
elicit or enhance immune response to tumor cells [0054] d. Balance
of pathogenicity and release of tumor antigens [0055] 2.
Immunogenicity [0056] 3. Replication competent [0057] 4. Genetic
variants
[0058] H. Pharmaceutical Compositions, combinations and kits [0059]
1. Pharmaceutical compositions [0060] 2. Host cells [0061] 3.
Combinations [0062] 4. Kits
[0063] I. Diagnostic and Therapeutic Methods [0064] 1.
Administration [0065] a. Steps prior to administering the virus
[0066] b. Mode of administration [0067] c. Dosages [0068] d. Number
of administrations [0069] e. Co-administrations [0070] i.
Administering a plurality of viruses [0071] ii. Therapeutic
Compounds [0072] iii. Immunotherapies and biological therapies
[0073] f. State of subject [0074] 2. Monitoring [0075] a.
Monitoring viral gene expression [0076] b. Monitoring tumor size
[0077] c. Monitoring antibody titer [0078] d. Monitoring general
health diagnostics [0079] e. Monitoring coordinated with
treatment
[0080] K. Other Microorganisms and Cells
[0081] L. Examples
A. DEFINITIONS
[0082] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of skill in the art to which the invention(s) belong. All patents,
patent applications, published applications and publications,
websites and other published materials referred to throughout the
entire disclosure herein, unless noted otherwise, are incorporated
by reference in their entirety. In the event that there are
pluralities of definitions for terms herein, those in this section
prevail. Where reference is made to a URL or other such identifier
or address, it is understood that such identifiers can change and
particular information on the internet can come and go, but
equivalent information is known and can be readily accessed, such
as by searching the internet and/or appropriate databases.
Reference thereto evidences the availability and public
dissemination of such information.
[0083] As used herein, a "transporter" is a membrane transport
protein. Transporters are involved in the movement of ions, small
molecules, or macromolecules, such as other proteins, across a
biological membrane. Transporters can be located on the outer cell
membrane or membrane-bound intracellular compartments such as the
nucleus, endoplasmic reticulum, and mitochondria. Transporters
typically show relatively high specificity for one or more
substrates and can transport solutes against their chemical or
electrochemical potential gradient. They can either function
without input of energy beyond the thermal movement (facilitated or
mediated diffusion), or be driven by electrochemical potential
gradients of H.sup.+ and Na.sup.+, or by various exergonic chemical
and photochemical reactions. Reference to transporters includes any
protein, allelic and species variants thereof and any other
variants thereof, that can be classified as a transporter using the
Transport Classification (TC) system (Saier et al., (2006) Nucleic
Acids Research 34(Database Issue):D181-D186). Such proteins are
easily identified using, for example, public databases such as the
Transport Classification Database (TCDB; www.tcdb.org).
[0084] As used herein, a "symporter" is a transporter that moves
two chemical species in the same direction, at least one of them
being ionic and driven by its electrochemical potential
gradient.
[0085] As used herein, norepinephrine transporter or NET refers to
the sodium- and chloride-dependent neurotransmitter symporter that
removes norepinephrine (NE) from the extracellular space by high
affinity reuptake into presynaptic terminals. NET also is referred
to as the "sodium-dependent noradrenaline transporter,"
"noradrenaline:Na.sup.+ symporter," "SLC6A2," "TC 2.A.22.1.2" and
"solute carrier family 6 (neurotransmitter transporter,
noradrenalin), member 2." NET is a member of the sodium- and
chloride-dependent neurotransmitter transporter family (Solute
carrier family 6; SLC6), also known as the sodium/neurotransmitter
symporter family (SNF) or the neurotransmitter/sodium symporter
family (NSS), which corresponds to TC 2.A.22 using the TC system.
Norepinephrine transporters include those of human origin (hNET)
and non-human origin. Exemplary non-human norepinephrine
transporters include, but are not limited to, bovine (SEQ ID
NO:27), mouse (SEQ ID NO:28), rat (SEQ ID NO:29), rhesus macaque
(SEQ ID NO:30), chicken (SEQ ID NO:31), ovine (fragment) (SEQ ID
NO:32) and Japanese quail (fragment) (SEQ ID NO:33) norepinephrine
transporters.
[0086] As used herein, hNET refers to the human norepinephrine
transporter. Exemplary hNETs include the wildtype hNET set forth in
SEQ ID NO:26, C-terminal variants (SEQ ID NOS:61 and 62), allelic
variants (SEQ ID NOS:45-60) and any other variants thereof,
including any variants known in the art, including polypeptides
that have at least 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the
polypeptide set forth in SEQ ID NO: 26.
[0087] Reference to norepinephrine transporters or NETs includes
wildtype polypeptides, truncated forms thereof that have activity,
and includes allelic variants and species variants, variants
encoded by splice variants, and other variants, including
polypeptides that have at least 40%, 45%, 50%, 55%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to
the polypeptide set forth in SEQ ID NO: 26 or the mature form
thereof. Norepinephrine transporters also include those that
contain chemical or posttranslational modifications and those that
do not contain chemical or posttranslational modifications. Such
modifications include, but are not limited to, pegylation,
albumination, glycosylation, farnysylation, carboxylation,
hydroxylation, phosphorylation, and other polypeptide modifications
known in the art. Reference to norepinephrine transporters also can
include fusion proteins containing a norepinephrine transporter or
portion thereof that retains activity.
[0088] As used herein, sodium-iodide symporter or NIS is an ion
pump that transports iodide (I.sup.-) into thyroid epithelial cells
and other select cells across the basolateral plasma membrane. NIS
also is referred to as the "Sodium/iodide cotransporter,"
"Na(+)/I(-) cotransporter," "SLC5A5," "TC 2.A.21.5.1" and "solute
carrier family 5 member 5." NIS is a member of the sodium glucose
cotransporter family (Solute carrier family 5; SLC5), also known as
the sodium/solute symporter family (SSSF) or TC 2.A.21 using the TC
system. Sodium-iodide symporters include those of human origin
(hNIS) and non-human origin. Exemplary non-human sodium-iodide
symporters include, but are not limited to, mouse (SEQ ID NO:65),
rat (SEQ ID NO:66), Zebrafish (SEQ ID NOS:67), and African clawed
frog mouse (SEQ ID NO:68) sodium-iodide symporters.
[0089] As used herein, hNIS refers to the human sodium-iodide
symporter. Exemplary hNETs include the wildtype hNIS set forth in
SEQ ID NO:63, allelic variants (SEQ ID NOS:87-94) and any other
variants thereof, including any variants known in the art (see e.g.
International Patent Publication WO2004000236), and includes
polypeptides that have at least 40%, 45%, 50%, 55%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to
the polypeptide set forth in SEQ ID NO: 63.
Reference to sodium-iodide symporters or NISs includes wildtype
polypeptides, truncated forms thereof that have activity, and
includes allelic variants and species variants, variants encoded by
splice variants, and other variants, including polypeptides that
have at least 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99% or more sequence identity to the
polypeptide set forth in SEQ ID NO: 63 or the mature form thereof.
Sodium-iodide symporters also include those that contain chemical
or posttranslational modifications and those that do not contain
chemical or posttranslational modifications. Such modifications
include, but are not limited to, pegylation, albumination,
glycosylation, farnysylation, carboxylation, hydroxylation,
phosphorylation, and other polypeptide modifications known in the
art. Reference to sodium-iodide symporters also can include fusion
proteins containing a sodium-iodide symporters or portion thereof
that retains activity.
[0090] As used herein, "virus" refers to any of a large group of
entities referred to as viruses. Viruses typically contain a
protein coat surrounding an RNA or DNA core of genetic material,
but no semipermeable membrane, and are capable of growth and
multiplication only in living cells. Viruses for use in the methods
provided herein include, but are not limited, to a poxvirus,
adenovirus, herpes simplex virus, Newcastle disease virus,
vesicular stomatitis virus, mumps virus, influenza virus, measles
virus, reovirus, human immunodeficiency virus (HIV), hanta virus,
myxoma virus, cytomegalovirus (CMV), lentivirus, and any plant or
insect virus.
[0091] As used herein, the term "viral vector" is used according to
its art-recognized meaning. It refers to a nucleic acid vector
construct that includes at least one element of viral origin and
can be packaged into a viral vector particle. The viral vector
particles can be used for the purpose of transferring DNA, RNA or
other nucleic acids into cells either in vitro or in vivo. Viral
vectors include, but are not limited to, retroviral vectors,
vaccinia vectors, lentiviral vectors, herpes virus vectors (e.g.,
HSV), baculoviral vectors, cytomegalovirus (CMV) vectors,
papillomavirus vectors, simian virus (SV40) vectors, semliki forest
virus vectors, phage vectors, adenoviral vectors, and
adeno-associated viral (AAV) vectors.
[0092] As used herein, the term "modified" with reference to a gene
refers to a deleted gene, a gene encoding a gene product having one
or more truncations, mutations, insertions or deletions, or a gene
that is inserted (into the chromosome or on a plasmid, phagemid,
cosmid, and phage) encoding a gene product, typically accompanied
by at least a change in function of the modified gene product or
virus.
[0093] As used herein, the term "modified virus" refers to a virus
that is altered with respect to a parental strain of the virus.
Typically modified viruses have one or more truncations, mutations,
insertions or deletions in the genome of virus. A modified virus
can have one or more endogenous viral genes modified and/or one or
more intergenic regions modified. Exemplary modified viruses can
have one or more heterologous nucleic acid sequences inserted into
the genome of the virus. Modified viruses can contain one more
heterologous nucleic acid sequences in the form of a gene
expression cassette for the expression of a heterologous gene.
[0094] As used herein, modification of a heterologous nucleic acid
molecule with respect to a virus containing a heterologous nucleic
acid molecule refers to any alteration of the heterologous nucleic
acid molecule including truncations, mutations, insertions or
deletions of the nucleic acid molecule. A deletion in a
heterologous nucleic acid molecule can include all or a portion of
the heterologous nucleic acid molecule. For example, if the
heterologous nucleic acid molecule is a double stranded DNA
molecule that is 5,000 base pairs in length, deletions of the
heterologous nucleic acid molecule can include deletions of 1, 2,
3, 4, 5 or more, 10 or more, 50 or more, 100 or more, 500 or more,
1,000 or more, or 5,000 base pairs of the heterologous nucleic acid
molecule. Deletion of all or a part of the nucleic acid molecule
can also include replacement of the heterologous nucleic acid
molecule with another nucleic acid molecule. Modification of a
heterologous nucleic acid molecule can also include alteration of
the viral genome. For example, a deletion of all or a potion
heterologous nucleic from the viral genome, for example by
homologous recombination, can also include deletion of nucleic acid
surrounding the deletion site that is part of the viral genome.
Similarly, insertion of an additional heterologous nucleic acid
molecule into the viral genome by homologous recombination, for
example, can include deletion or all, or a part of a viral gene.
When modification of a heterologous nucleic acid molecule is an
insertion, an additional nucleic acid molecule can be inserted in
the heterologous nucleic acid molecule or adjacent to the nucleic
acid molecule. Typically, insertions by homologous recombination
involve replacement of all or a part of the heterologous nucleic
acid molecule with another nucleic acid molecule.
[0095] As used herein, the term "therapeutic virus" refers to a
virus that is administered for the treatment of a disease or
disorder, such as cancer, a tumor and/or a metastasis or
inflammation or wound or diagnosis thereof and or both. A
therapeutic virus typically is modified, such as to attenuate it.
Other modifications include one or more insertions, deletions or
mutations in the genome of the virus. Therapeutic viruses all can
include modifications in one or more endogenous viral genes or one
or more intergenic regions, which attenuate the toxicity of the
virus, and can optionally express a heterologous therapeutic gene
product and/or detectable protein. Therapeutic viruses can contain
heterologous nucleic acid molecules, including one or more gene
expression cassettes for the expression of the therapeutic gene
product and/or detectable protein. Therapeutic viruses can be
replication competent viruses (e.g., oncolytic viruses) or
replication-defective viruses.
[0096] As used herein, a virus that can be detected and used for
diagnostics and is therapeutic is a theragnostic virus.
[0097] As used herein, the term, "therapeutic gene product" or
"therapeutic polypeptide" refers to any heterologous protein
expressed by the therapeutic virus that ameliorates the symptoms of
a disease or disorder or ameliorates the disease or disorder.
[0098] As used herein, the phrase "immunoprivileged cells and
tissues" refers to cells and tissues, such as solid tumors and
wounded tissues, which are sequestered from the immune system.
[0099] As used herein, preferential accumulation refers to
accumulation of a virus at a first location at a higher level than
accumulation at a second location. Thus, a virus that
preferentially accumulates in immunoprivileged tissue, such as a
tumor, relative to normal tissues or organs refers to a virus that
accumulates in immunoprivileged tissue, such as tumor, at a higher
level, or concentration, than the virus accumulates in normal
tissues or organs.
[0100] As used herein, to attenuate toxicity of a virus means to
reduce or eliminate deleterious or toxic effects to a host upon
administration of the virus compared to an un-attenuated virus. As
used herein, a virus with low toxicity means that upon
administration a virus does not accumulate in organs and tissues in
the host to an extent that results in damage or harm to organs, or
that impacts survival of the host to a greater extent than the
disease being treated does. For the purposes herein, attenuation of
toxicity is used interchangeably with attenuation of virulence and
attenuation of pathogenicity.
[0101] As used herein, the term "toxicity" with reference to a
virus refers to the ability of the virus to cause harm to the
subject to which the virus has been administered.
[0102] As used herein virulence and pathogenicity with reference to
a virus refers to the ability of the virus to cause disease or harm
in the subject to which the virus has been administered. Hence, for
the purposes herein the terms toxicity, virulence and pathogenicity
with reference to a virus are used interchangeably.
[0103] As used herein, a compound produced in a tumor or other
immunoprivileged site refers to any compound that is produced in
the tumor or tumor environment by virtue of the presence of an
introduced virus, generally a recombinant virus, expressing one or
more gene products. For example, a compound produced in a tumor can
be, for example, an encoded polypeptide, such as a recombinant
polypeptide (e.g., a transporter, a cell-surface receptor, a
cytokine, a chemokine, an apoptotic protein, a mitosis inhibitor
protein, an antimitotic oligopeptide, an antiangiogenic factor, a
single-chain antibody, a toxin, a tumor antigen, a prodrug
converting enzyme), an RNA (e.g., ribozyme, RNAi, siRNA), or a
compound that is generated by an encoded polypeptide and, in some
examples, the cellular machinery of the tumor or immunoprivileged
tissue or cells (e.g., a metabolite, a converted prodrug).
[0104] As used herein, a delivery vehicle for administration refers
to a lipid-based or other polymer-based composition, such as
liposome, micelle or reverse micelle, which associates with an
agent, such as a virus provided herein, for delivery into a host
animal.
[0105] As used herein, a disease or disorder refers to a
pathological condition in an organism resulting from, for example,
infection or genetic defect, and characterized by identifiable
symptoms.
[0106] As used herein, treatment means any manner in which the
symptoms of a condition, disorder or disease are ameliorated or
otherwise beneficially altered. Treatment also encompasses any
pharmaceutical use of the viruses described and provided
herein.
[0107] As used herein, amelioration or alleviation of the symptoms
of a particular disorder, such as by administration of a particular
pharmaceutical composition, refers to any lessening, whether
permanent or temporary, lasting or transient that can be attributed
to or associated with administration of the composition.
[0108] As used herein, an effective amount of a virus or compound
for treating a particular disease is an amount that is sufficient
to ameliorate, or in some manner reduce the symptoms associated
with the disease. Such an amount can be administered as a single
dosage or can be administered according to a regimen, whereby it is
effective. The amount can cure the disease but, typically, is
administered in order to ameliorate the symptoms of the disease.
Repeated administration can be required to achieve the desired
amelioration of symptoms.
[0109] As used herein, an in vivo method refers to a method
performed within the living body of a subject.
[0110] As used herein, a subject includes any animal for whom
diagnosis, screening, monitoring or treatment is contemplated.
Animals include mammals such as primates and domesticated animals.
An exemplary primate is human. A patient refers to a subject such
as a mammal, primate, human, or livestock subject afflicted with a
disease condition or for which a disease condition is to be
determined or risk of a disease condition is to be determined.
[0111] As used herein, the term "neoplasm" or "neoplasia" refers to
abnormal new cell growth, and thus means the same as tumor, which
can be benign or malignant. Unlike hyperplasia, neoplastic
proliferation persists even in the absence of the original
stimulus.
[0112] As used herein, neoplastic disease refers to any disorder
involving cancer, including tumor development, growth, metastasis
and progression.
[0113] As used herein, cancer is a term for diseases caused by or
characterized by any type of malignant tumor, including metastatic
cancers, lymphatic tumors, and blood cancers. Exemplary cancers
include, but are not limited to: leukemia, lymphoma, pancreatic
cancer, lung cancer, ovarian cancer, breast cancer, cervical
cancer, bladder cancer, prostate cancer, glioma tumors,
adenocarcinomas, liver cancer and skin cancer. Exemplary cancers in
humans include a bladder tumor, breast tumor, prostate tumor, basal
cell carcinoma, biliary tract cancer, bladder cancer, bone cancer,
brain and CNS cancer (e.g., glioma tumor), cervical cancer,
choriocarcinoma, colon and rectum cancer, connective tissue cancer,
cancer of the digestive system; endometrial cancer, esophageal
cancer; eye cancer; cancer of the head and neck; gastric cancer;
intra-epithelial neoplasm; kidney cancer; larynx cancer; leukemia;
liver cancer; lung cancer (e.g. small cell and non-small cell);
lymphoma including Hodgkin's and Non-Hodgkin's lymphoma; melanoma;
myeloma, neuroblastoma, oral cavity cancer (e.g., lip, tongue,
mouth, and pharynx); ovarian cancer; pancreatic cancer,
retinoblastoma; rhabdomyosarcoma; rectal cancer, renal cancer,
cancer of the respiratory system; sarcoma, skin cancer; stomach
cancer, testicular cancer, thyroid cancer; uterine cancer, cancer
of the urinary system, as well as other carcinomas and sarcomas.
Malignant disorders commonly diagnosed in dogs, cats, and other
pets include, but are not limited to, lymphosarcoma, osteosarcoma,
mammary tumors, mastocytoma, brain tumor, melanoma, adenosquamous
carcinoma, carcinoid lung tumor, bronchial gland tumor, bronchiolar
adenocarcinoma, fibroma, myxochondroma, pulmonary sarcoma,
neurosarcoma, osteoma, papilloma, retinoblastoma, Ewing's sarcoma,
Wilm's tumor, Burkitt's lymphoma, microglioma, neuroblastoma,
osteoclastoma, oral neoplasia, fibrosarcoma, osteosarcoma and
rhabdomyosarcoma, genital squamous cell carcinoma, transmissible
venereal tumor, testicular tumor, seminoma, Sertoli cell tumor,
hemangiopericytoma, histiocytoma, chloroma (e.g., granulocytic
sarcoma), corneal papilloma, corneal squamous cell carcinoma,
hemangiosarcoma, pleural mesothelioma, basal cell tumor, thymoma,
stomach tumor, adrenal gland carcinoma, oral papillomatosis,
hemangioendothelioma and cystadenoma, follicular lymphoma,
intestinal lymphosarcoma, fibrosarcoma and pulmonary squamous cell
carcinoma. In rodents, such as a ferret, exemplary cancers include
insulinoma, lymphoma, sarcoma, neuroma, pancreatic islet cell
tumor, gastric MALT lymphoma and gastric adenocarcinoma. Neoplasias
affecting agricultural livestock include leukemia,
hemangiopericytoma and bovine ocular neoplasia (in cattle);
preputial fibrosarcoma, ulcerative squamous cell carcinoma,
preputial carcinoma, connective tissue neoplasia and mastocytoma
(in horses); hepatocellular carcinoma (in swine); lymphoma and
pulmonary adenomatosis (in sheep); pulmonary sarcoma, lymphoma,
Rous sarcoma, reticulo-endotheliosis, fibrosarcoma, nephroblastoma,
B-cell lymphoma and lymphoid leukosis (in avian species);
retinoblastoma, hepatic neoplasia, lymphosarcoma (lymphoblastic
lymphoma), plasmacytoid leukemia and swimbladder sarcoma (in fish),
caseous lumphadenitis (CLA): chronic, infectious, contagious
disease of sheep and goats caused by the bacterium Corynebacterium
pseudotuberculosis, and contagious lung tumor of sheep caused by
jaagsiekte.
[0114] As used herein, the term "malignant," as it applies to
tumors, refers to primary tumors that have the capacity of
metastasis with loss of growth control and positional control.
[0115] As used herein, metastasis refers to a growth of abnormal or
neoplastic cells distant from the site primarily involved by the
morbid process.
[0116] As used herein, proliferative disorders include any
disorders involving abnormal proliferation of cells, such as, but
not limited to, neoplastic diseases.
[0117] As used herein, a method for treating or preventing
neoplastic disease means that any of the symptoms, such as the
tumor, metastasis thereof, the vascularization of the tumors or
other parameters by which the disease is characterized are reduced,
ameliorated, prevented, placed in a state of remission, or
maintained in a state of remission. It also means that the
indications of neoplastic disease and metastasis can be eliminated,
reduced or prevented by the treatment. Non-limiting examples of the
indications include uncontrolled degradation of the basement
membrane and proximal extracellular matrix, migration, division,
and organization of the endothelial cells into new functioning
capillaries, and the persistence of such functioning
capillaries.
[0118] As used herein, the term "angiogenesis" is intended to
encompass the totality of processes directly or indirectly involved
in the establishment and maintenance of new vasculature
(neovascularization), including, but not limited to,
neovascularization associated with tumors and neovascularization
associated with wounds.
[0119] As used herein, therapeutic agents are agents that
ameliorate the symptoms of a disease or disorder or ameliorate the
disease or disorder. Therapeutic agent, therapeutic compound,
therapeutic regimen, or chemotherapeutic include conventional drugs
and drug therapies, including vaccines, which are known to those
skilled in the art and described elsewhere herein. Therapeutic
agents include, but are not limited to, moieties that inhibit cell
growth or promote cell death, that can be activated to inhibit cell
growth or promote cell death, or that activate another agent to
inhibit cell growth or promote cell death. Optionally, the
therapeutic agent can exhibit or manifest additional properties,
such as, properties that permit its use as an imaging agent, as
described elsewhere herein. Therapeutic agents for the
compositions, methods and uses provided herein can be, for example,
an anti-cancer agent. Exemplary therapeutic agents include, for
example, cytokines, growth factors, photosensitizing agents,
radionuclides, toxins, anti-metabolites, signaling modulators,
anti-cancer antibiotics, anti-cancer antibodies, angiogenesis
inhibitors, radiation therapy, chemotherapeutic compounds or a
combination thereof.
[0120] As used herein, anti-cancer agents (used interchangeably
with "anti-tumor or anti-neoplastic" agent) include any anti-cancer
therapies, such as radiation therapy, surgery, hyperthermia or
hyperthermia therapy, or anti-cancer compounds useful in the
treatment of cancer. These include any agents, when used alone or
in combination with other agent, that can alleviate, reduce,
ameliorate, prevent, or place or maintain in a state of remission
of clinical symptoms or diagnostic markers associated with
neoplastic disease, tumors and cancer, and can be used in methods,
combinations and compositions provided herein. Exemplary
anti-cancer agents include, but are not limited to, the viruses
provided herein used singly or in combination and/or in combination
with other anti-cancer agents. Exemplary anti-cancer compounds
include cytokines, chemokines, growth factors, a photosensitizing
agents, toxins, anti-cancer antibiotics, chemotherapeutic
compounds, radionuclides, angiogenesis inhibitors, signaling
modulators, anti-metabolites, anti-cancer vaccines, anti-cancer
oligopeptides, mitosis inhibitor proteins, antimitotic
oligopeptides, anti-cancer antibodies (e.g., single-chain
antibodies), anti-cancer antibiotics, immunotherapeutic agents,
bacteria and any combinations thereof.
[0121] Exemplary cytokines and growth factors include, but are not
limited to, interleukins, such as, for example, interleukin-1,
interleukin-2, interleukin-6 and interleukin-12, tumor necrosis
factors, such as tumor necrosis factor alpha (TNF-.alpha.),
interferons such as interferon gamma (IFN-.gamma.), granulocyte
macrophage colony stimulating factors (GM-CSF), angiogenins, and
tissue factors.
[0122] Photosensitizing agents include, but are not limited to, for
example, indocyanine green, toluidine blue, aminolevulinic acid,
texaphyrins, benzoporphyrins, phenothiazines, phthalocyanines,
porphyrins such as sodium porfimer, chlorins such as
tetra(m-hydroxyphenyl)chlorin or tin(IV) chlorin e6, purpurins such
as tin ethyl etiopurpurin, purpurinimides, bacteriochlorins,
pheophorbides, pyropheophorbides or cationic dyes.
[0123] Radionuclides, which depending upon the radionuclide, amount
and application can be used for diagnosis and/or for treatment.
They include, but are not limited to, for example, a compound or
molecule containing .sup.11Carbon, .sup.11Fluorine, .sup.13Carbon,
.sup.15Nitrogen, .sup.18Flourine, .sup.19Flourine,
.sup.32Phosphate, .sup.60Cobalt, .sup.90Yttirum, .sup.99Technetium,
.sup.103 Palladium, .sup.106Ruthenium, .sup.111Indium,
.sup.117Lutetium, .sup.125Iodine, .sup.131Iodine, .sup.137Cesium,
.sup.153Samarium, .sup.186Rhenium, .sup.188Rhenium,
.sup.192Iridium, .sup.198Gold, .sup.211Astatine, .sup.212Bismuth or
.sup.213Bismuth.
[0124] Toxins include, but are not limited to, chemotherapeutic
compounds such as, but not limited to, 5-fluorouridine,
calicheamicin, maytansine, double-chain ricin, ricin A chain,
abrin, abrin A chain, saporin, modeccin, modeccin A chain,
Pseudomonas aeruginosa exotoxin, Cholera toxin, Shigella toxin, E.
coli heat labile toxin and Diptheria toxin. doxorubicin,
daunomycin, methotrexate, taxol, ricin A, colchicine, cytochasins,
monensin, ouabain, mitoxanthrone, vindesine, vinblastine,
vincristine and enterotoxin.
[0125] Anti-metabolites include, but are not limited to,
methotrexate, 5-fluorouracil, 6-mercaptopurine, cytosine
arabinoside, hydroxyurea and 20-chlorodeoxyadenosine.
[0126] Signaling modulators include, but are not limited to, for
example, inhibitors of macrophage inhibitory factor, toll-like
receptor agonists and stat 3 inhibitors.
[0127] Anti-cancer antibiotics include, but are not limited to,
anthracyclines such as doxorubicin hydrochloride (adriamycin),
idarubicin hydrochloride, daunorubicin hydrochloride, aclarubicin
Hydrochloride, epirubicin hydrochloride and purarubicin
hydrochloride, enomycin, phenomycin, pleomycins such as pleomycin
and peplomycin sulfate, mitomycins such as mitomycin C,
actinomycins such as actinomycin D, zinostatinstimalamer and
polypeptides such as neocarzinostatin.
[0128] Anti-cancer antibodies include, but are not limited to,
Rituximab, ADEPT, Trastuzumab (Herceptin), Tositumomab (Bexxar),
Cetuximab (Erbitux), Ibritumomab (Zevalin), Alemtuzumab
(Campath-1H), Epratuzumab (Lymphocide), Gemtuzumab ozogamicin
(Mylotarg), Bevacimab (Avastin), Tarceva (Erlotinib), SUTENT
(sunitinib malate), Panorex (Edrecolomab), RITUXAN (Rituximab),
Zevalin (90Y-ibritumomab tiuexetan), Mylotarg (Gemtuzumab
Ozogamicin) and Campath (Alemtuzumab).
[0129] Angiogenesis inhibitors include, but are not limited to,
collagenase inhibitors such as metalloproteinases and tetracyclines
such as minocycline, naturally occurring peptides such as
endostatin and angiostatin, fungal and bacterial derivatives, such
as fumagillin derivatives like TNP-470, aptamer antogonist of VEGF,
batimastat, Captopril, cartilage derived inhibitor (CDI),
genistein, interleukin 12, Lavendustin A, medroxypregesterone
acetate, recombinant human platelet factor 4(rPF4), taxol,
D-gluco-D-galactan sulfate (Tecogalan(=SP-PG, DS-4152)),
thalidomide, thrombospondin.
[0130] Radiation therapy includes, but is not limited to,
photodynamic therapy, radionuclides, radioimmunotherapy and proton
beam treatment.
[0131] Chemotherapeutic compounds include, but are not limited to
platinum; platinum analogs (e.g., platinum coordination complexes)
such as cisplatin, carboplatin, oxaliplatin, DWA2114R, NK121, IS 3
295, and 254-S; anthracenediones; vinblastine; alkylating agents
such as thiotepa and cyclosphosphamide; alkyl sulfonates such as
busulfan, improsulfan and piposulfan; aziridines such as benzodopa,
carboquone, meturedopa and uredopa; ethylenimines and
methylamelamines including altretamine, triethylenemelamine,
trietylenephosphoramide, triethylenethiophosphaoramide and
trimethylolomelamime nitrogen mustards such as chiorambucil,
chlomaphazine, cholophosphamide, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,
novembichin, phenesterine, prednimustine, trofosfamide, uracil
mustard; nitrosureas such as carmustine, chlorozotocin,
fotemustine, lomustine, nimustine, ranimustine; antibiotics such as
aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,
cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin,
chromomycins, dactinomycin, daunorubicin, detorubicin,
6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,
idarubicin, marcellomycin, mitomycins, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate and 5-fluorouracil (5-FU); folic acid analogues such
as denopterin, methotrexate, pteropterin, trimetrexate; purine
analogs such as fludarabine, 6-mercaptopurine, thiamiprine,
thioguanine; pyrimidine analogs such as ancitabine, azacitidine,
6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,
enocitabine, floxuridine; androgens such as calusterone,
dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;
demecolcine; diaziquone; elformithine; elliptinium acetate;
etoglucid; gallium nitrate; substituted ureas; hydroxyurea;
lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol;
nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid;
2-ethylhydrazide; procarbazine; anti-cancer polysaccharides;
polysaccharide-K; razoxane; sizofiran; spirogermanium; tenuazonic
acid; triaziquone; 2,2',2''-trichlorotriethylamine; urethan;
vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol;
pipobroman; gacytosine; cytosine arabinoside; cyclophosphamide;
thiotepa; taxoids, such as paclitaxel and doxetaxel; chlorambucil;
gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; etoposide
(VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine;
vinorelbine; navelbine; novantrone; teniposide; daunomycin;
aminopterin; xeloda; ibandronate; CPT11; topoisomerase inhibitor
RFS 2000; difluoromethylomithine (DMFO); retinoic acid;
esperamicins; capecitabine; methylhydrazine derivatives; and
pharmaceutically acceptable salts, acids or derivatives of any of
the above. Also included in this definition are anti-hormonal
agents that act to regulate or inhibit hormone action on tumors
such as anti-estrogens including for example tamoxifen, raloxifene,
aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen,
trioxifene, keoxifene, LY117018, onapristone and toremifene
(Fareston); adrenocortical suppressants; and antiandrogens such as
flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and
pharmaceutically acceptable salts, acids or derivatives of any of
the above. Such chemotherapeutic compounds that can be used herein
include compounds whose toxicities preclude use of the compound in
general systemic chemotherapeutic methods.
[0132] As used herein, an anti-cancer oligopeptide or an anti-tumor
oligopeptide is short polypeptide that has the ability to slow or
inhibit tumor growth and/or metastasis. Anti-cancer oligopeptide
typically have high affinity for and specificity to tumors enabling
them to target tumors. Such oligopeptides include
receptor-interacting compounds, inhibitors of protein-protein
interactions, enzyme inhibitors, and nucleic acid-interacting
compounds. As used herein an antimitotic oligopeptide is an
oligopeptide that inhibits cell division. An antimitotic
oligopeptide is an exemplary anti-cancer oligopeptide. Exemplary
antimitotic oligopeptides include, but are not limited to,
tubulysin, phomopsin, hemiasterlin, taltobulin (HTI-286, 3), and
cryptophycin.
[0133] As used herein, a prodrug is a compound that, upon in vivo
administration, is metabolized or otherwise converted to the
biologically, pharmaceutically or therapeutically active form of
the compound. To produce a prodrug, the pharmaceutically active
compound is modified such that the active compound is regenerated
by metabolic processes. The prodrug can be designed to alter the
metabolic stability or the transport characteristics of a drug, to
mask side effects or toxicity, to improve the flavor of a drug or
to alter other characteristics or properties of a drug. By virtue
of knowledge of pharmacodynamic processes and drug metabolism in
vivo, those of skill in this art, once a pharmaceutically active
compound is known, can design prodrugs of the compound (see, e.g.,
Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford
University Press, New York, pages 388-392). Prodrugs include, but
are not limited to, 5-fluorocytosine, gancyclovir, 6-methylpurine
deoxyriboside, cephalosporin-doxorubicin,
4-[(2-chloroethyl)(2-mesuloxyethyl)amino]benzoyl-L-glutamic acid,
indole-3-acetic acid,
7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycampotothecin,
bis-(2-chloroethyl)amino-4-hydroxyphenylaminomethanone 28,
1-chloromethyl-5-hydroxy-1,2-dihydro-3H-benz[e]indole,
epirubicin-glucoronide, 5'-deoxy-5-fluorouridine, cytosine
arabinoside, and linamarin.
[0134] As used herein, a compound conjugated to a moiety refers to
a complex that includes a compound bound to a moiety, where the
binding between the compound and the moiety can arise from one or
more covalent bonds or non-covalent interactions such as hydrogen
bonds, or electrostatic interactions. A conjugate also can include
a linker that connects the compound to the moiety. Exemplary
compounds include, but are not limited to, nanoparticles and
siderophores. Exemplary moieties, include, but are not limited to,
detectable moieties and therapeutic agents.
[0135] As used herein, nanoparticle refers to a microscopic
particle whose size is measured in nanometers. Often such particles
in nanoscale are used in biomedical applications acting as drug
carriers or imaging agents. Nanoparticles can be conjugated to
other agents, including, but not limited to detectable/diagnostic
agents or therapeutic agents.
[0136] As used herein, a detectable label or detectable moiety or
diagnostic moiety (also imaging label, imaging agent, or imaging
moiety) refers to an atom, molecule or composition, wherein the
presence of the atom, molecule or composition can be directly or
indirectly measured.
[0137] As used herein, a detectable moiety or an imaging moiety
refer to moieties used to image a virus in any of the methods
provided herein. Imaging (detectable) moieties include, for
example, chemiluminescent moieties, bioluminescent moieties,
fluorescent moieties, radionuclides and metals.
[0138] As used herein, a detection agent or an imaging agent refer
to any molecule, compound, or polypeptide used to image a virus in
any of the methods provided herein. Detection agents or imaging
agents can contain, for example, a detectable moiety or can be a
substrate, such as a luciferin, that produces a detectable signal
following modification, such as by chemical modification by a
luciferase.
[0139] As used herein, detect, detected and detecting refer
generally to any manner of discovering or determining the presence
of a signal, such as visual inspection, fluorescence spectroscopy,
absorption, reflectance measurement, flow cytometry, magnetic
resonance methods such as magnetic resonance imaging (MRI) and
magnetic resonance spectroscopy (MRS), ultrasound, X-rays, gamma
rays (after annihilation of a positron and an electron in PET
scanning), tomographic methods including computed tomography (CT),
computed axial tomography (CAT), electron beam computed tomography
(EBCT), high resolution computed tomography (HRCT), hypocycloidal
tomography, positron emission tomography (PET), single-photon
emission computed tomography (SPECT), spiral computed tomography
and ultrasonic tomography. Direct detection of a detectable label
refers to, for example, measurement of a physical phenomenon, such
as energy or particle emission or absorption of the moiety itself,
such as by X-ray or MRI. Indirect detection refers to measurement
of a physical phenomenon, such as energy or particle emission or
absorption, of an atom, molecule or composition that binds directly
or indirectly to the detectable moiety. In a non-limiting example
of indirect detection, a detectable label can be biotin, which can
be detected by binding to avidin. Non-labeled avidin can be
administered systemically to block non-specific binding, followed
by systemic administration of labeled avidin. Thus, included within
the scope of a detectable label or detectable moiety is a bindable
label or bindable moiety, which refers to an atom, molecule or
composition, wherein the presence of the atom, molecule or
composition can be detected as a result of the label or moiety
binding to another atom, molecule or composition. Exemplary
diagnostic agents include, for example, metals such as colloidal
gold, iron, gadolinium, and gallium-67, fluorescent moieties and
radionuclides. Exemplary fluorescent moieties and radionuclides are
provided elsewhere herein.
[0140] As used herein, magnetic resonance imaging (MRI) refers to
the use of a nuclear magnetic resonance spectrometer to produce
electronic images of specific atoms and molecular structures in
solids, especially human cells, tissues and organs. MRI is
non-invasive diagnostic technique that uses nuclear magnetic
resonance to produce cross-sectional images of organs and other
internal body structures. The subject lies inside a large, hollow
cylinder containing a strong electromagnet, which causes the nuclei
of certain atoms in the body (such as, for example, .sup.1H,
.sup.13C and .sup.19F) to align magnetically. The subject is then
subjected to radio waves, which cause the aligned nuclei to flip;
when the radio waves are withdrawn the nuclei return to their
original positions, emitting radio waves that are then detected by
a receiver and translated into a two-dimensional picture by
computer. For some MRI procedures, contrast agents such as
gadolinium are used to increase the accuracy of the images.
[0141] As used herein, an X-ray refers to a relatively high-energy
photon, or a stream of such photons, having a wavelength in the
approximate range from 0.01 to 10 nanometers. X-rays also refer to
photographs taken with x-rays.
[0142] As used herein, "optical imaging" refers to imaging of a
signal where at least some of the signal from the region of
interest is in the form of an electromagnetic radiation in the
visible light range. Non-limiting examples of optical imaging
include detection of fluorescence and luminescence signals. Such
signals can be captured by optical devices, such as a camera.
[0143] As used herein, "non-optical imaging" refers to imaging of a
signal where at least some of the signal from the region of
interest is in the form of electromagnetic radiation outside the
visible range, and can include particles, and other propagations of
energy. Non-limiting examples of non-optical imaging include
detection of gamma rays (e.g., SPECT), X-rays, RF signals (e.g.,
MRI), particles such as electrons or positrons (e.g., PET), and
other forms of energy propagations (e.g., ultrasound).
[0144] As used herein, nucleic acids include DNA, RNA and analogs
thereof, including peptide nucleic acids (PNA) and mixtures
thereof. Nucleic acids can be single or double-stranded. Nucleic
acids can encode for example gene products, such as, for example,
polypeptides, regulatory RNAs, siRNAs and functional RNAs.
[0145] As used herein, primer refers to an oligonucleotide
containing two or more deoxyribonucleotides or ribonucleotides,
typically more than three, from which synthesis of a primer
extension product can be initiated. Typically a primer contains a
free 3' hydroxy moiety. Experimental conditions conducive to
synthesis of a gene product include the presence of nucleoside
triphosphates and an agent for polymerization and extension, such
as DNA polymerase, and a suitable buffer, temperature, and pH. When
referring to probes or primers, which are optionally labeled, such
as with a detectable label, such as a fluorescent or radiolabel,
single-stranded molecules are provided. Such molecules are
typically of a length such that their target is statistically
unique or of low copy number (typically less than 5, generally less
than 3) for probing or priming a library. Generally a probe or
primer contains at least 14, 16 or 30 contiguous nucleotides of
sequence complementary to or identical to a gene of interest.
Probes and primers can be 5, 6, 7, 8, 9, 10 or more, 20 or more, 30
or more, 50 or more, 100 or more nucleic acids long.
[0146] As used herein, a sequence complementary to at least a
portion of an RNA, with reference to antisense oligonucleotides,
means a sequence of nucleotides having sufficient complementarity
to be able to hybridize with the RNA, generally under moderate or
high stringency conditions, forming a stable duplex; in the case of
double-stranded antisense nucleic acids, a single strand of the
duplex DNA (i.e., dsRNA) can thus be tested, or triplex formation
can be assayed. The ability to hybridize depends on the degree of
complementarily and the length of the antisense nucleic acid.
Generally, the longer the hybridizing nucleic acid, the more base
mismatches with an encoding RNA it can contain and still form a
stable duplex (or triplex, as the case can be). One skilled in the
art can ascertain a tolerable degree of mismatch by use of standard
procedures to determine the melting point of the hybridized
complex.
[0147] As used herein, a heterologous nucleic acid (also referred
to as exogenous nucleic acid or foreign nucleic acid) refers to a
nucleic acid that is not normally produced in vivo by an organism
or virus from which it is expressed or that is produced by an
organism or a virus but is at a different locus, expressed
differently, or that mediates or encodes mediators that alter
expression of endogenous nucleic acid, such as DNA, by affecting
transcription, translation, or other regulatable biochemical
processes. Heterologous nucleic acid is often not endogenous to a
cell or virus into which it is introduced, but has been obtained
from another cell or virus or prepared synthetically. Heterologous
nucleic acid can refer to a nucleic acid molecule from another cell
in the same organism or another organism, including the same
species or another species. Heterologous nucleic acid, however, can
be endogenous, but is nucleic acid that is expressed from a
different locus or altered in its expression or sequence (e.g., a
plasmid). Thus, heterologous nucleic acid includes a nucleic acid
molecule not present in the exact orientation or position as the
counterpart nucleic acid molecule, such as DNA, is found in a
genome. Generally, although not necessarily, such nucleic acid
encodes RNA and proteins that are not normally produced by the cell
or virus or in the same way in the cell in which it is expressed.
Any nucleic acid, such as DNA, that one of skill in the art
recognizes or considers as heterologous, exogenous or foreign to
the cell in which the nucleic acid is expressed is herein
encompassed by heterologous nucleic acid.
[0148] As used herein, a heterologous protein or heterologous
polypeptide (also referred to as exogenous protein, exogenous
polypeptide, foreign protein or foreign polypeptide) refers to a
protein that is not normally produced in vivo by an organism.
[0149] As used herein, operative linkage of heterologous nucleic
acids to regulatory and effector sequences of nucleotides, such as
promoters, enhancers, transcriptional and translational stop sites,
and other signal sequences refers to the relationship between such
nucleic acid, such as DNA, and such sequences of nucleotides. For
example, operative linkage of heterologous DNA to a promoter refers
to the physical relationship between the DNA and the promoter such
that the transcription of such DNA is initiated from the promoter
by an RNA polymerase that specifically recognizes, binds to and
transcribes the DNA. Thus, operatively linked or operationally
associated refers to the functional relationship of a nucleic acid,
such as DNA, with regulatory and effector sequences of nucleotides,
such as promoters, enhancers, transcriptional and translational
stop sites, and other signal sequences. For example, operative
linkage of DNA to a promoter refers to the physical and functional
relationship between the DNA and the promoter such that the
transcription of such DNA is initiated from the promoter by an RNA
polymerase that specifically recognizes, binds to and transcribes
the DNA. In order to optimize expression and/or transcription, it
can be necessary to remove, add or alter 5' untranslated portions
of the clones to eliminate extra, potentially inappropriate,
alternative translation initiation (i.e., start) codons or other
sequences that can interfere with or reduce expression, either at
the level of transcription or translation. In addition, consensus
ribosome binding sites can be inserted immediately 5' of the start
codon and can enhance expression (see, e.g., Kozak J. Biol. Chem.
266: 19867-19870 (1991); Shine and Delgarno Nature 254(5495): 34-38
(1975)). The desirability of (or need for) such modification can be
empirically determined.
[0150] As used herein, a promoter, a promoter region or a promoter
element or regulatory region or regulatory element refers to a
segment of DNA or RNA that controls transcription of the DNA or RNA
to which it is operatively linked. The promoter region includes
specific sequences that are involved in RNA polymerase recognition,
binding and transcription initiation. In addition, the promoter
includes sequences that modulate recognition, binding and
transcription initiation activity of RNA polymerase (i.e., binding
of one or more transcription factors). These sequences can be cis
acting or can be responsive to trans acting factors. Promoters,
depending upon the nature of the regulation, can be constitutive or
regulated. Regulated promoters can be inducible or environmentally
responsive (e.g. respond to cues such as pH, anaerobic conditions,
osmoticum, temperature, light, or cell density). Many such promoter
sequences are known in the art. See, for example, U.S. Pat. Nos.
4,980,285; 5,631,150; 5,707,928; 5,759,828; 5,888,783; 5,919,670,
and, Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd
Ed., Cold Spring Harbor Press (1989).
[0151] As used herein, a native promoter is a promoter that is
endogenous to the organism or virus and is unmodified with respect
to its nucleotide sequence and its position in the viral genome as
compared to a wild-type organism or virus.
[0152] As used herein, a heterologous promoter refers to a promoter
that is not normally found in the wild-type organism or virus or
that is at a different locus as compared to a wild-type organism or
virus. A heterologous promoter is often not endogenous to a cell or
virus into which it is introduced, but has been obtained from
another cell or virus or prepared synthetically. A heterologous
promoter can refer to a promoter from another cell in the same
organism or another organism, including the same species or another
species. A heterologous promoter, however, can be endogenous, but
is a promoter that is altered in its sequence or occurs at a
different locus (e.g., at a different location in the genome or on
a plasmid). Thus, a heterologous promoter includes a promoter not
present in the exact orientation or position as the counterpart
promoter is found in a genome.
[0153] A synthetic promoter is a heterologous promoter that has a
nucleotide sequence that is not found in nature. A synthetic
promoter can be a nucleic acid molecule that has a synthetic
sequence or a sequence derived from a native promoter or portion
thereof. A synthetic promoter can also be a hybrid promoter
composed of different elements derived from different native
promoters.
[0154] As used herein a "gene expression cassette" or "expression
cassette" is a nucleic acid construct, containing nucleic acid
elements that are capable of effecting expression of a gene in
hosts that are compatible with such sequences. Expression cassettes
include at least promoters and optionally, transcription
termination signals. Typically, the expression cassette includes a
nucleic acid to be transcribed operably linked to a promoter.
Additional factors helpful in effecting expression can also be used
as described herein. Expression cassettes can contain genes that
encode, for example, a therapeutic gene product or a detectable
protein or a selectable marker gene,
[0155] As used herein, replacement of a promoter with a stronger
promoter refers to removing a promoter from a genome and replacing
it with a promoter that effects an increased the level of
transcription initiation relative to the promoter that is replaced.
Typically, a stronger promoter has an improved ability to bind
polymerase complexes relative to the promoter that is replaced. As
a result, an open reading frame that is operably linked to the
stronger promoter has a higher level of gene expression. Similarly,
replacement of a promoter with a weaker promoter refers to removing
a promoter from a genome and replacing it with a promoter that
decreases the level of transcription initiation relative to the
promoter that is replaced. Typically, a weaker promoter has a
lessened ability to bind polymerase complexes relative to the
promoter that is replaced. As a result, an open reading frame that
is operably linked to the weaker promoter has a lower level of gene
expression.
[0156] As used herein, production by recombinant means by using
recombinant DNA methods means the use of the well known methods of
molecular biology for expressing proteins encoded by cloned
DNA.
[0157] As used herein, vector (or plasmid) refers to discrete
elements that are used to introduce heterologous nucleic acid into
cells for either expression or replication thereof. The vectors
typically remain episomal, but can be designed to effect
integration of a gene or portion thereof into a chromosome of the
genome. Selection and use of such vectors are well known to those
of skill in the art. An expression vector includes vectors capable
of expressing DNA that is operatively linked with regulatory
sequences, such as promoter regions, that are capable of effecting
expression of such DNA fragments. Thus, an expression vector refers
to a recombinant DNA or RNA construct, such as a plasmid, a phage,
recombinant virus or other vector that, upon introduction into an
appropriate host cell, results in expression of the cloned DNA.
Appropriate expression vectors are well known to those of skill in
the art and include those that are replicable in eukaryotic cells
and/or prokaryotic cells and those that remain episomal or those
which integrate into the host cell genome. Vectors can be used in
the generation of a recombinant genome by integration or homologous
recombination, such as in the generation of a recombinant virus as
described elsewhere herein.
[0158] As used herein, genetic therapy or gene therapy involves the
transfer of heterologous nucleic acid, such as DNA or RNA, into
certain cells, target cells, of a mammal, particularly a human,
with a disorder or conditions for which such therapy is sought. As
used herein, genetic therapy or gene therapy can involve the
transfer of heterologous nucleic acid, such as DNA, into a virus,
which can be transferred to a mammal, particularly a human, with a
disorder or conditions for which such therapy is sought. The
nucleic acid, such as DNA, is introduced into the selected target
cells, such as directly or indirectly, in a manner such that the
heterologous nucleic acid, such as DNA, is expressed and a
therapeutic product encoded thereby is produced. Alternatively, the
heterologous nucleic acid, such as DNA, can in some manner mediate
expression of DNA that encodes the therapeutic product, or it can
encode a product, such as a peptide or RNA that is in some manner a
therapeutic product, or which mediates, directly or indirectly,
expression of a therapeutic product. Genetic therapy also can be
used to deliver nucleic acid encoding a gene product that replaces
a defective gene or supplements a gene product produced by the
mammal or the cell in which it is introduced. The introduced
nucleic acid can encode a therapeutic compound. The heterologous
nucleic acid, such as DNA, encoding the therapeutic product can be
modified prior to introduction into the cells of the afflicted host
in order to enhance or otherwise alter the product or expression
thereof. Genetic therapy also can involve delivery of an inhibitor
or repressor or other modulator of gene expression.
[0159] As used herein, a therapeutically effective product for gene
therapy is a product that is encoded by heterologous nucleic acid,
typically DNA, or an RNA product such as dsRNA, RNAi, including
siRNA, that upon introduction of the nucleic acid into a host, a
product is expressed that ameliorates or eliminates the symptoms,
manifestations of an inherited or acquired disease or that cures
the disease. Also included are biologically active nucleic acid
molecules, such as RNAi and antisense nucleic acids.
[0160] As used herein, an agent or compound that modulates the
activity of a protein or expression of a gene or nucleic acid
either decreases or increases or otherwise alters the activity of
the protein or, in some manner, up- or down-regulates or otherwise
alters expression of the nucleic acid in a cell.
As used herein, recitation that amino acids of a polypeptide
"correspond to" amino acids in a disclosed sequence, such as amino
acids set forth in the Sequence listing, refers to amino acids
identified upon alignment of the polypeptide with the disclosed
sequence to maximize identity or homology (where conserved amino
acids are aligned) using a standard alignment algorithm, such as
the GAP algorithm. By aligning the sequences of polypeptides, one
skilled in the art can identify corresponding residues, using
conserved and identical amino acid residues as guides.
[0161] As used herein, "amino acids" are represented by their full
name or by their known, three-letter or one-letter abbreviations
(Table 1). The nucleotides which occur in the various nucleic acid
fragments are designated with the standard single-letter
designations used routinely in the art.
TABLE-US-00001 TABLE 1 Table of Correspondence SYMBOL 1-Letter
3-Letter AMINO ACID Y Tyr tyrosine G Gly glycine F Phe
phenylalanine M Met methionine A Ala alanine S Ser serine I Ile
isoleucine L Leu leucine T Thr threonine V Val valine P Pro proline
K Lys lysine H His histidine Q Gln glutamine E Glu glutamic acid Z
Glx Glu and/or Gln w Trp tryptophan R Arg arginine D Asp aspartic
acid N Asn asparagine B Asx Asn and/or Asp C Cys cysteine X Xaa
Unknown or other
[0162] As used herein, the terms "homology" and "identity" are used
interchangeably, but homology for proteins can include conservative
amino acid changes. In general, to identify corresponding
positions, the sequences of amino acids are aligned so that the
highest order match is obtained (see, e.g., Computational Molecular
Biology, Lesk, A.M.; ed., Oxford University Press, New York, 1988;
Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,
Academic Press, New York, 1993; Computer Analysis of Sequence Data,
Part I, Griffin, A.M., and Griffin, H. G., eds., Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje,
G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov,
M. and Devereux, J., eds., M Stockton Press, New York, 1991;
Carillo et al. (1988) SIAM J Applied Math 48:1073).
[0163] As use herein, "sequence identity" refers to the number of
identical amino acids (or nucleotide bases) in a comparison between
a test and a reference polypeptide or polynucleotide. Homologous
polypeptides refer to a pre-determined number of identical or
homologous amino acid residues. Homology includes conservative
amino acid substitutions as well identical residues. Sequence
identity can be determined by standard alignment algorithm programs
used with default gap penalties established by each supplier.
Homologous nucleic acid molecules refer to a pre-determined number
of identical or homologous nucleotides. Homology includes
substitutions that do not change the encoded amino acid (i.e.,
"silent substitutions") as well identical residues. Substantially
homologous nucleic acid molecules hybridize typically at moderate
stringency or at high stringency all along the length of the
nucleic acid or along at least about 70%, 80% or 90% of the
full-length nucleic acid molecule of interest. Also contemplated
are nucleic acid molecules that contain degenerate codons in place
of codons in the hybridizing nucleic acid molecule. (For
determination of homology of proteins, conservative amino acids can
be aligned as well as identical amino acids; in this case,
percentage of identity and percentage homology vary). Whether any
two nucleic acid molecules have nucleotide sequences (or any two
polypeptides have amino acid sequences) that are at least 80%, 85%,
90%, 95%, 96%, 97%, 98% or 99% "identical" can be determined using
known computer algorithms such as the "FAST A" program, using for
example, the default parameters as in Pearson et al. Proc. Natl.
Acad. Sci. USA 85: 2444 (1988) (other programs include the GCG
program package (Devereux, J., et al., Nucleic Acids Research
12(I): 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S. F., et al,
J. Molec. Biol. 215:403 (1990); Guide to Huge Computers, Martin J.
Bishop, ed., Academic Press, San Diego (1994), and Carillo et al.
SIAM J Applied Math 48: 1073 (1988)). For example, the BLAST
function of the National Center for Biotechnology Information
database can be used to determine identity. Other commercially or
publicly available programs include DNAStar "MegAlign" program
(Madison, Wis.) and the University of Wisconsin Genetics Computer
Group (UWG) "Gap" program (Madison Wis.)). Percent homology or
identity of proteins and/or nucleic acid molecules can be
determined, for example, by comparing sequence information using a
GAP computer program (e.g., Needleman et al. J. Mol. Biol. 48: 443
(1970), as revised by Smith and Waterman (Adv. Appl. Math. 2: 482
(1981)). Briefly, a GAP program defines similarity as the number of
aligned symbols (i.e., nucleotides or amino acids) which are
similar, divided by the total number of symbols in the shorter of
the two sequences. Default parameters for the GAP program can
include: (1) a unary comparison matrix (containing a value of 1 for
identities and 0 for non identities) and the weighted comparison
matrix of Gribskov et al. Nucl. Acids Res. 14: 6745 (1986), as
described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence
and Structure, National Biomedical Research Foundation, pp. 353-358
(1979); (2) a penalty of 3.0 for each gap and an additional 0.10
penalty for each symbol in each gap; and (3) no penalty for end
gaps. Therefore, as used herein, the term "identity" represents a
comparison between a test and a reference polypeptide or
polynucleotide. In one non-limiting example, "at least 90%
identical to" refers to percent identities from 90 to 100% relative
to the reference polypeptides. Identity at a level of 90% or more
is indicative of the fact that, assuming for exemplification
purposes a test and reference polynucleotide length of 100 amino
acids are compared, no more than 10% (i.e., 10 out of 100) of amino
acids in the test polypeptide differs from that of the reference
polypeptides. Similar comparisons can be made between a test and
reference polynucleotides. 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.,
10/100 amino acid difference (approximately 90% identity).
Differences are defined as nucleic acid or amino acid
substitutions, insertions or deletions. At the level of homologies
or identities above about 85-90%, the result should be independent
of the program and gap parameters set; such high levels of identity
can be assessed readily, often without relying on software.
[0164] The term substantially identical or homologous or similar
varies with the context as understood by those skilled in the
relevant art and generally means at least 60% or 70%, preferably
means at least 80%, more preferably at least 90%, and most
preferably at least 95%, 96%, 97%, 98%, 99% or greater identity. As
used herein, substantially identical to a product means
sufficiently similar so that the property of interest is
sufficiently unchanged so that the substantially identical product
can be used in place of the product.
[0165] As used herein, substantially pure means sufficiently
homogeneous to appear free of readily detectable impurities as
determined by standard methods of analysis, such as thin layer
chromatography (TLC), gel electrophoresis and high performance
liquid chromatography (HPLC), used by those of skill in the art to
assess such purity, or sufficiently pure such that further
purification would not detectably alter the physical and chemical
properties, such as enzymatic and biological activities, of the
substance. Methods for purification of the compounds to produce
substantially chemically pure compounds are known to those of skill
in the art. A substantially chemically pure compound can, however,
be a mixture of stereoisomers or isomers. In such instances,
further purification might increase the specific activity of the
compound.
[0166] As used herein equivalent, when referring to two sequences
of nucleic acids, means that the two sequences in question encode
the same sequence of amino acids or equivalent proteins. When
equivalent is used in referring to two proteins or peptides or
other molecules, it means that the two proteins or peptides have
substantially the same amino acid sequence with only amino acid
substitutions (such as, but not limited to, conservative changes)
or structure and the any changes do not substantially alter the
activity or function of the protein or peptide. When equivalent
refers to a property, the property does not need to be present to
the same extent (e.g., two peptides can exhibit different rates of
the same type of enzymatic activity), but the activities are
usually substantially the same. Complementary, when referring to
two nucleotide sequences, means that the two sequences of
nucleotides are capable of hybridizing, typically with less than
25%, 15% or 5% mismatches between opposed nucleotides. If
necessary, the percentage of complementarity will be specified.
Typically the two molecules are selected such that they will
hybridize under conditions of high stringency.
[0167] As used herein, a receptor refers to a molecule that has an
affinity for a ligand. Receptors can be naturally-occurring or
synthetic molecules. Receptors also can be referred to in the art
as anti-ligands. As used herein, the receptor and anti-ligand are
interchangeable. Receptors can be used in their unaltered state or
bound to other polypeptides, including as homodimers. Receptors can
be attached to, covalently or noncovalently, or in physical contact
with, a binding member, either directly or indirectly via a
specific binding substance or linker. Examples of receptors,
include, but are not limited to: antibodies, cell membrane
receptors surface receptors and internalizing receptors, monoclonal
antibodies and antisera reactive with specific antigenic
determinants (such as on viruses, cells, or other materials),
drugs, polynucleotides, nucleic acids, peptides, cofactors,
lectins, sugars, polysaccharides, cells, cellular membranes, and
organelles.
[0168] As used herein, bind, bound and binding refer to the binding
between atoms or molecules with a K.sub.d in the range of 10.sup.-2
to 10.sup.-15 mole/L, generally, 10.sup.-6 to 10.sup.-15, 10.sup.-7
to 10.sup.-15 and typically 10.sup.-8 to 10.sup.-15 (and/or a
K.sub.a of 10.sup.5-10.sup.12, 10.sup.7-10.sup.12,
10.sup.8-10.sup.12 L/mole).
[0169] As used herein, luminescence refers to the detectable
electromagnetic (EM) radiation, generally, ultraviolet (UV),
infrared (IR) or visible EM radiation that is produced when the
excited product of an exergonic chemical process reverts to its
ground state with the emission of light. Chemiluminescence is
luminescence that results from a chemical reaction. Bioluminescence
is chemiluminescence that results from a chemical reaction using
biological molecules (or synthetic versions or analogs thereof) as
substrates and/or enzymes. Fluorescence is luminescence in which
light of a visible color is emitted from a substance under
stimulation or excitation by light or other forms radiation such as
ultraviolet (UV), infrared (1R) or visible EM radiation.
[0170] As used herein, chemiluminescence refers to a chemical
reaction in which energy is specifically channeled to a molecule
causing it to become electronically excited and subsequently to
release a photon thereby emitting visible light. Temperature does
not contribute to this channeled energy. Thus, chemiluminescence
involves the direct conversion of chemical energy to light
energy.
[0171] As used herein, bioluminescence, which is a type of
chemiluminescence, refers to the emission of light by biological
molecules, particularly proteins. The essential condition for
bioluminescence is molecular oxygen, either bound or free in the
presence of an oxygenase, a luciferase, which acts on a substrate,
a luciferin. Bioluminescence is generated by an enzyme or other
protein (luciferase) that is an oxygenase that acts on a substrate
luciferin (a bioluminescence substrate) in the presence of
molecular oxygen and transforms the substrate to an excited state,
which, upon return to a lower energy level releases the energy in
the form of light.
[0172] As used herein, the substrates and enzymes for producing
bioluminescence are generically referred to as luciferin and
luciferase, respectively. When reference is made to a particular
species thereof, for clarity, each generic term is used with the
name of the organism from which it derives such as, for example,
click beetle luciferase or firefly luciferase.
[0173] As used herein, luciferase refers to oxygenases that
catalyze a light emitting reaction. For instance, bacterial
luciferases catalyze the oxidation of flavin mononucleotide (FMN)
and aliphatic aldehydes, which reaction produces light. Another
class of luciferases, found among marine arthropods, catalyzes the
oxidation of Cypridina (Vargula) luciferin and another class of
luciferases catalyzes the oxidation of Coleoptera luciferin.
[0174] Thus, luciferase refers to an enzyme or photoprotein that
catalyzes a bioluminescent reaction (a reaction that produces
bioluminescence). The luciferases, such as firefly and Gaussia and
Renilla luciferases are enzymes which act catalytically and are
unchanged during the bioluminescence generating reaction. The
luciferase photoproteins, such as the aequorin photoprotein to
which luciferin is non-covalently bound, are changed, such as by
release of the luciferin, during bioluminescence generating
reaction. The luciferase is a protein, or a mixture of proteins
(e.g., bacterial luciferase), that occurs naturally in an organism
or a variant or mutant thereof, such as a variant produced by
mutagenesis that has one or more properties, such as thermal
stability, that differ from the naturally-occurring protein.
Luciferases and modified mutant or variant forms thereof are well
known. For purposes herein, reference to luciferase refers to
either the photoproteins or luciferases.
[0175] Thus, reference, for example, to Renilla luciferase refers
to an enzyme isolated from member of the genus Renilla or an
equivalent molecule obtained from any other source, such as from
another related copepod, or that has been prepared synthetically.
It is intended to encompass Renilla luciferases with conservative
amino acid substitutions that do not substantially alter activity.
Conservative substitutions of amino acids are known to those of
skill in this art and can be made generally without altering the
biological activity of the resulting molecule. Those of skill in
this art recognize that, in general, single amino acid
substitutions in non-essential regions of a polypeptide do not
substantially alter biological activity (see, e.g., Watson et al.
Molecular Biology of the Gene, 4th Edition, 1987, The
Benjamin/Cummings Pub. co., p. 224).
[0176] As used herein, bioluminescence substrate refers to the
compound that is oxidized in the presence of a luciferase and any
necessary activators and generates light. These substrates are
referred to as luciferins herein, are substrates that undergo
oxidation in a bioluminescence reaction. These bioluminescence
substrates include any luciferin or analog thereof or any synthetic
compound with which a luciferase interacts to generate light.
Typical substrates include those that are oxidized in the presence
of a luciferase or protein in a light-generating reaction.
Bioluminescence substrates, thus, include those compounds that
those of skill in the art recognize as luciferins. Luciferins, for
example, include firefly luciferin, Cypridina (also known as
Vargula) luciferin (coelenterazine), bacterial luciferin as well as
synthetic analogs of these substrates or other compounds that are
oxidized in the presence of a luciferase in a reaction the produces
bioluminescence.
[0177] As used herein, capable of conversion into a bioluminescence
substrate refers to being susceptible to chemical reaction, such as
oxidation or reduction, which yields a bioluminescence substrate.
For example, the luminescence producing reaction of bioluminescent
bacteria involves the reduction of a flavin mononucleotide group
(FMN) to reduced flavin mononucleotide (FMNH.sub.2) by a flavin
reductase enzyme. The reduced flavin mononucleotide (substrate)
then reacts with oxygen (an activator) and bacterial luciferase to
form an intermediate peroxy flavin that undergoes further reaction,
in the presence of a long-chain aldehyde, to generate light. With
respect to this reaction, the reduced flavin and the long chain
aldehyde are bioluminescence substrates.
[0178] As used herein, a bioluminescence generating system refers
to the set of reagents required to conduct a bioluminescent
reaction. Thus, the specific luciferase, luciferin and other
substrates, solvents and other reagents that can be required to
complete a bioluminescent reaction form a bioluminescence system.
Thus a bioluminescence generating system refers to any set of
reagents that, under appropriate reaction conditions, yield
bioluminescence. Appropriate reaction conditions refer to the
conditions necessary for a bioluminescence reaction to occur, such
as pH, salt concentrations and temperature. In general,
bioluminescence systems include a bioluminescence substrate,
luciferin, a luciferase, which includes enzymes luciferases and
photoproteins and one or more activators. A specific
bioluminescence system can be identified by reference to the
specific organism from which the luciferase derives; for example,
the Renilla bioluminescence system includes a Renilla luciferase,
such as a luciferase isolated from Renilla or produced using
recombinant methods or modifications of these luciferases. This
system also includes the particular activators necessary to
complete the bioluminescence reaction, such as oxygen and a
substrate with which the luciferase reacts in the presence of the
oxygen to produce light.
[0179] As used herein, a fluorescent protein (FP) refers to a
protein that possesses the ability to fluoresce (i.e., to absorb
energy at one wavelength and emit it at another wavelength). For
example, a green fluorescent protein (GFP) refers to a polypeptide
that has a peak in the emission spectrum at 510 nm or about 510 nm.
A variety of FPs that emit at various wavelengths are known in the
art. Exemplary FPs include, but are not limited to, a green
fluorescent protein (GFP), yellow fluorescent protein (YFP), orange
fluorescent protein (OFP), cyan fluorescent protein (CFP), blue
fluorescent protein (BFP), red fluorescent protein (RFP), far-red
fluorescent protein, or near-infrared fluorescent protein.
Extending the spectrum of available colors of fluorescent proteins
to blue, cyan, orange yellow and red variants, provides a method
for multicolor tracking of fusion proteins.
[0180] As used herein, Aequorea GFP refers to GFPs from the genus
Aequorea and to mutants or variants thereof. Such variants and GFPs
from other species, such as Anthozoa reef coral, Anemonia sea
anemone, Renilla sea pansy, Galaxea coral, Acropora brown coral,
Trachyphyllia and Pectimidae stony coral and other species are well
known and are available and known to those of skill in the art.
Exemplary GFP variants include, but are not limited to BFP, CFP,
YFP and OFP. Examples of florescent proteins and their variants
include GFP proteins, such as Emerald (InVitrogen, Carlsbad,
Calif.), EGFP (Clontech, Palo Alto, Calif.), Azami-Green (MBL
International, Woburn, Mass.), Kaede (MBL International, Woburn,
Mass.), ZsGreen1 (Clontech, Palo Alto, Calif.) and CopGFP
(Evrogen/Axxora, LLC, San Diego, Calif.); CFP proteins, such as
Cerulean (Rizzo, Nat. Biotechnol. 22(4):445-9 (2004)), mCFP (Wang
et al., PNAS US A. 101 (48):16745-9 (2004)), AmCyan1 (Clontech,
Palo Alto, Calif.), MiCy (MBL International, Woburn, Mass.), and
CyPet (Nguyen and Daugherty, Nat. Biotechnol. 23(3):355-60 (2005));
BFP proteins such as EBFP (Clontech, Palo Alto, Calif.); YFP
proteins such as EYFP (Clontech, Palo Alto, Calif.), YPet (Nguyen
and Daugherty, Nat. Biotechnol. 23(3):355-60 (2005)), Venus (Nagai
et al., Nat. Biotechnol. 20(1):87-90 (2002)), ZsYellow (Clontech,
Palo Alto, Calif.), and mCitrine (Wang et al., PNAS USA.
101(48):16745-9 (2004)); OFP proteins such as cOFP (Strategene, La
Jolla, Calif.), mKO (MBL International, Woburn, Mass.), and
mOrange; and others (Shaner N C, Steinbach P A, and Tsien R Y.,
Nat. Methods. 2(12):905-9 (2005)).
[0181] As used herein, red fluorescent protein, or RFP, refers to
the Discosoma RFP (DsRed) that has been isolated from the
corallimorph Discosoma (Matz et al., Nature Biotechnology 17:
969-973 (1999)), and red or far-red fluorescent proteins from any
other species, such as Heteractis reef coral and Actinia or
Entacmaea sea anemone, as well as variants thereof. RFPs include,
for example, Discosoma variants, such as mRFP1, mCherry, tdTomato,
mStrawberry, mTangerine (Wang et al., PNAS US A. 101 (48):16745-9
(2004)), DsRed2 (Clontech, Palo Alto, Calif.), and DsRed-T1 (Bevis
and Glick, Nat. Biotechnol., 20: 83-87 (2002)), Anthomedusa J-Red
(Evrogen) and Anemonia AsRed2 (Clontech, Palo Alto, Calif.).
Far-red fluorescent proteins include, for example, Actinia AQ143
(Shkrob et al., Biochem J. 392(Pt 3):649-54 (2005)), Entacmaea
eqFP611 (Wiedenmann et al. Proc Natl Acad Sci USA. 99(18):11646-51
(2002)), Discosoma variants such as mPlum and mRasberry (Wang et
al., PNAS US A. 101(48):16745-9 (2004)), and Heteractis HcRed1 and
t-HcRed (Clontech, Palo Alto, Calif.).
[0182] As used herein the term assessing or determining is intended
to include quantitative and qualitative determination in the sense
of obtaining an absolute value for the activity of a product, and
also of obtaining an index, ratio, percentage, visual or other
value indicative of the level of the activity. Assessment can be
direct or indirect.
[0183] As used herein, activity refers to the in vivo activities of
a compound or viruses on physiological responses that result
following in vivo administration thereof. (or of a composition or
other mixture). Activity, thus, encompasses resulting therapeutic
effects and pharmaceutical activity of such compounds, compositions
and mixtures. Activities can be observed in in vitro and/or in vivo
systems designed to test or use such activities.
[0184] As used herein, a vaccine refers to a composition which,
upon administration to a subject, elicits an immune response in a
subject to which it is administered and which protects the
immunized subject against subsequent challenge by the immunizing
agent or an immunologically cross-reactive agent. A vaccine can be
used to enhance the immune response against a pathogen, such as a
virus, that expresses the immunological agent and/or has already
infected the subject. Protection can be complete or partial (i.e.,
a reduction in symptoms or infection as compared with an
unvaccinated subject). Typically a vaccine is administered to a
subject that is a mammal. An immunologically cross-reactive agent
can be, for example, the whole protein (e.g., tumor antigen) from
which a subunit peptide used as the immunogen is derived.
Alternatively, an immunologically cross-reactive agent can be a
different protein which is recognized in whole or in part by the
antibodies elicited by the immunizing agent. Exemplary vaccines can
be modified vaccinia viruses that express an immunologically
cross-reactive agent.
[0185] As used herein, a "pharmaceutically acceptable carrier"
refers to any carrier, diluent, excipient, wetting agent, buffering
agent, suspending agent, lubricating agent, adjuvant, solid binder,
vehicle, delivery system, emulsifier, disintegrant, absorbent,
preservative, surfactant, colorant, flavorant, or sweetener,
preferably non-toxic, that are suitable for use in a pharmaceutical
composition.
[0186] As used herein, complex refers generally to an association
between two or more species regardless of the nature of the
interaction between the species (i.e., ionic, covalent, or
electrostatic).
[0187] As used herein, "a combination" refers to any association
between two or among more items or elements. Exemplary combinations
include, but are not limited to, two or more pharmaceutical
compositions, a composition containing two or more active
ingredients, such as two viruses, or a virus and a chemotherapeutic
compound, two or more viruses, a virus and a therapeutic agent, a
virus and an imaging agent, a virus and a plurality therapeutic
and/or imaging agents, or any association thereof. Such
combinations can be packaged as kits.
[0188] As used herein, a composition refers to any mixture. It can
be a solution, a suspension, an emulsion, liquid, powder, a paste,
aqueous, non-aqueous or any combination of such ingredients.
[0189] As used herein, fluid refers to any composition that can
flow. Fluids thus encompass compositions that are in the form of
semi-solids, pastes, solutions, aqueous mixtures, gels, lotions,
creams and other such compositions.
[0190] As used herein, a kit is a packaged combination, optionally,
including instructions for use of the combination and/or other
reactions and components for such use.
[0191] For clarity of disclosure, and not by way of limitation, the
detailed description is divided into the subsections that
follow.
B. VIRUSES FOR TREATMENT AND DIAGNOSIS
[0192] Provided herein are viruses for therapeutic and diagnostic
use. Also provided elsewhere herein are methods for making and
using such viruses for therapeutic and diagnostic uses. The viruses
provided herein encode gene products that can enhance the uptake or
retention of compounds useful for diagnostic and/or therapeutic
purposes. Exemplary of such viruses are viruses that encode a
transporter protein. The viruses provided herein that encode a
transporter protein can localize to and infect target cells, such
as tumor cells, resulting expression of the transporter protein by
the infected cells. Transporter proteins expressed by an infected
cell can result in increased uptake diagnostic and therapeutic
moieties across the cell membrane. Diagnostic moieties include
those that can emit a signal that is detectable by optical or
non-optical imaging methods. Detection of the signal by imaging
modalities such as, for example, by positron emission tomography
(PET) and, thereby allows visualization of the infected tissues,
such a tumor or an inflammation.
[0193] Exemplary diagnostic moieties are provided elsewhere herein
and include radiolabeled compounds that can be imaged in a subject.
The uptake of such compounds by the expressed transporter protein
allows amplification of signal emitted by the compounds due to the
increased accumulation of the labeled compound in the infected
cells. Hence, the transporter-based imaging methods using the
viruses provided herein can result in greater signal strength at
the target tissue as compared to receptor-based imaging methods
because a higher number of diagnostic moieties can be taken up by
the infected cells as compared to the limited binding of one moiety
to one receptor. Thus such methods can provide high signal to noise
ratios in vivo for infected versus non-infected tissues.
[0194] Exemplary transporter proteins that can be encoded by the
viruses provided herein are listed elsewhere herein and include,
for example, the human norepinephrine transporter (hNET) and the
human sodium iodide symporter (hNIS). Exemplary viruses provided
herein that encode the human norepinephrine transporter (hNET)
include, but are not limited to, GLV-1h99, GLV-1h100, GLV-1h101,
GLV-1h139, GLV-1h146 and GLV-1h150. Exemplary viruses provided
herein the encode the human norepinephrine transporter (hNET)
include, but are not limited to, GLV-1h151, GLV-1h152 and
GLV-1h153.
[0195] The viruses provided herein that encode a transporter
protein are typically attenuated. Attenuated viruses have a
decreased capacity to cause disease in a host. The decreased
capacity can result from any of a variety of different
modifications to the ability of a virus to be pathogenic. For
example, a virus can have reduced toxicity, reduced ability to
accumulate in non-tumorous organs or tissue, reduced ability to
cause cell lysis or cell death, or reduced ability to replicate
compared to the non-attenuated form thereof. The attenuated viruses
provided herein, however, retain at least some capacity to
replicate and to cause immunoprivileged cells and tissues, such as
tumor cells to leak or lyse, undergo cell death, or otherwise cause
or enhance an immune response to immunoprivileged cells and
tissues, such as tumor cells. Such characteristics of the viruses
provided are described in detail elsewhere herein.
[0196] The viruses provided herein that encode a transporter
protein can accumulate in and infect immunoprivileged cells or
immunoprivileged tissues, including tumors and/or metastases, and
also including inflamed or wounded tissues and cells. While the
viruses provided herein can typically be cleared from the subject
to whom the viruses are administered by activity of the subject's
immune system, viruses can nevertheless accumulate, survive and
proliferate in immunoprivileged cells and tissues such as tumors
because such immunoprivileged areas are sequestered from the host's
immune system. Accordingly, the methods provided herein, as applied
to tumors and/or metastases, and diagnostic and therapeutic methods
relating thereto, can readily be applied to other immunoprivileged
cells and tissues, including inflamed and wounded cells and
tissues.
[0197] Among the viruses provided herein that encode a transporter
protein are cytoplasmic viruses, which do not require entry of
viral nucleic acid molecules in to the nucleus of the host cell
during the viral life cycle. Exemplary cytoplasmic viruses provided
herein are viruses of the poxvirus family, including
orthopoxviruses. Exemplary of poxviruses provided herein are
vaccinia viruses. Vaccinia virus possesses a variety of features
for use in cancer gene therapy and vaccination, including broad
host and cell type range, a large carrying capacity for foreign
genes and high sequence homology among different strains for
designing and generating modified viruses in other strains.
Techniques for production of modified vaccinia strains by genetic
engineering are well established (Moss (1993) Curr. Opin. Genet.
Dev. 3: 86-90; Broder and Earl (1999) Mol. Biotechnol. 13: 223-245;
Timiryasova et al. (2001) Biotechniques 31: 534-540). A variety of
vaccinia virus strains are available, including Western Reserve
(WR), Copenhagen, Tashkent, Tian Tan, Lister, Wyeth, IHD-J, and
IHD-W, Brighton, Ankara, MVA, Dairen I, LIPV, LC1 6M8, LC16MO,
LIVP, WR 65-16, Connaught, New York City Board of Health. Exemplary
of vaccinia viruses provided herein are Lister strain or LIVP
vaccinia viruses.
[0198] The modifications of the Lister strain provided herein also
can be adapted to other vaccinia viruses (e.g., Western Reserve
(WR), Copenhagen, Tashkent, Tian Tan, Lister, Wyeth, 1HD-J, and
IHD-W, Brighton, Ankara, MVA, Dairen I, LIPV, LC16M8, LC16MO, LIVP,
WR 65-16, Connaught, New York City Board of Health). The
modifications of the Lister strain provided herein also can be
adapted to other viruses, including, but not limited to, viruses of
the poxvirus family, adenoviruses, herpes viruses and
retroviruses.
[0199] Typically, DNA encoding the transporter protein is inserted
into a nonessential gene of the virus genome that does not
significantly affecting viral replication and infection in the
target tissue. For example, in a vaccinia virus, non essential gene
loci include, but are not limited to, TK, HA, F14.5L, E2L/E3L, K1
L/K2L, superoxide dismutase locus, 7.5K, C7-K1L, J2R, B13R+B14R,
A56R, A26L or 14L gene loci. The heterologous nucleic acid encoding
the transporter is typically operably linked to a promoter for
expression of the transporter protein in the infected cells.
Suitable promoter include viral promoters, such as a vaccinia virus
natural and synthetic promoters. Exemplary vaccinia viral promoters
include, but are not limited to, P11k, P7.5k early/late, P7.5k
early, P28 late, synthetic early P.sub.SE, synthetic early/late
P.sub.SEL and synthetic late P.sub.SL promoters.
[0200] Exemplary vaccinia viruses provided herein that encode a
transporter protein were derived from vaccinia virus strain
GLV-1h68 (also named RVGL21, SEQ ID NO: 1). GLV-1h68, which has
been described in U.S. Pat. Pub. No. 2005-0031643, contains DNA
insertions gene loci of the vaccinia virus LIVP strain (a vaccinia
virus strain, originally derived by adapting the Lister strain
(ATCC Catalog No. VR-1549) to calf skin (Institute of Viral
Preparations, Moscow, Russia, Al'tshtein et al., (1983) Dokl. Akad.
Nauk USSR 285:696-699)). GLV-1h68 contains expression cassettes
encoding detectable marker proteins in the F14.5L (also designated
in LIVP as F3) gene locus, thymidine kinase (TK) gene locus, and
hemagglutinin (HA) gene locus. An expression cassette containing a
Ruc-GFP cDNA molecule (a fusion of DNA encoding Renilla luciferase
and DNA encoding GFP) under the control of a vaccinia synthetic
early/late promoter P.sub.SEL ((P.sub.SEL)Ruc-GFP) was inserted
into the F14.5L gene locus; an expression cassette containing a DNA
molecule encoding beta-galactosidase under the control of the
vaccinia early/late promoter P.sub.7.5k ((P.sub.7.5k)LacZ) and DNA
encoding a rat transferrin receptor positioned in the reverse
orientation for transcription relative to the vaccinia synthetic
early/late promoter P.sub.SEL ((P.sub.SEL)rTrfR) was inserted into
the TK gene locus (the resulting virus does not express transferrin
receptor protein since the DNA molecule encoding the protein is
positioned in the reverse orientation for transcription relative to
the promoter in the cassette); and an expression cassette
containing a DNA molecule encoding .beta.-glucuronidase under the
control of the vaccinia late promoter P.sub.11k ((P.sub.11k)gusA)
was inserted into the HA gene locus. The GLV-1h68 virus exhibited a
strong preference for accumulation in tumor tissues as compared to
non-tumorous tissues following systemic administration of the virus
to tumor bearing subjects. This preference was significantly higher
than the tumor selective accumulation of other vaccinia viral
strains, such as WR (see, e.g. U.S. Pat. Pub. No. 2005-0031643 and
Zhang et al. (2007) Cancer Res. 67(20):10038-46). The viruses
provided herein that encode a transporter protein were generated by
replacement of one or more expression cassettes of the GLV-1h68
strain with heterologous DNA encoding a transporter protein.
[0201] In some examples, nucleic acid encoding the transporter
protein can be introduced into a viral genome that contains one or
more additional heterologous genes or is modified by removal or
replacement of one or more viral genes to attenuate the virus. The
heterologous genes can encode additional diagnostic or therapeutic
proteins as described elsewhere herein. Non-limiting examples
viruses that can be modified by insertion of a nucleic acid
encoding a transporter protein include attenuated Lister strain
LIVP viruses including, but not limited to, LIVP viruses described
in U.S. Patent Publication Nos. 2005/0031643, 2004/0234455 and
2004/0213741 and U.S. patent application Ser. No. 11/975,088.
Exemplary viruses contained therein that can be modified as
described here include viruses which have one or more expression
cassettes removed from GLV-1h68 and replaced with a heterologous
non-coding DNA molecule (e.g., GLV-1h70, GLV-1h71, GLV-1h72,
GLV-1h73, GLV-1h74, GLV-1h85, and GLV-1h86). GLV-1h70 contains
(P.sub.SEL)Ruc-GFP inserted into the F14.5L gene locus,
(P.sub.SEL)rTrfR and (P.sub.7.5k)LacZ inserted into the TK gene
locus, and a non-coding DNA molecule inserted into the HA gene
locus in place of (P.sub.11k)gusA. GLV-1h71 contains a non-coding
DNA molecule inserted into the F14.5L gene locus in place of
(P.sub.SEL)Ruc-GFP, (P.sub.SEL)rTrfR and (P.sub.7.5k)LacZ inserted
into the TK gene locus, and (P.sub.11k)gusA inserted into the HA
gene locus. GLV-1h72 contains (P.sub.SEL)Ruc-GFP inserted into the
F14.5L gene locus, a non-coding DNA molecule inserted into the TK
gene locus in place of (P.sub.SEL)rTrfR and (P.sub.7.5k)LacZ, and
P.sub.11kgusA inserted into the HA gene locus. GLV-1h73 contains a
non-coding DNA molecule inserted into the F14.5L gene locus in
place of (P.sub.SEL)Ruc-GFP, (P.sub.SEL)rTrfR and (P.sub.7.5k)LacZ
inserted into the TK gene locus, and a non-coding DNA molecule
inserted into the HA gene locus in place of (P.sub.11k)gusA.
GLV-1h74 contains a non-coding DNA molecule inserted into the
F14.5L gene locus in place of (P.sub.SEL)Ruc-GFP, a non-coding DNA
molecule inserted into the TK gene locus in place of
(P.sub.SEL)rTrfR and (P.sub.7.5k)LacZ, and a non-coding DNA
molecule inserted into the HA gene locus in place
of(P.sub.11k)gusA. GLV-1h85 contains a non-coding DNA molecule
inserted into the F14.5L gene locus in place of (P.sub.SEL)Ruc-GFP,
a non-coding DNA molecule inserted into the TK gene locus in place
of (P.sub.SEL)rTrfR and (P.sub.7.5k)LacZ, and (P.sub.11k)gusA
inserted into the HA gene locus. GLV-1h86 contains
(P.sub.SEL)Ruc-GFP inserted into the F14.5L gene locus, a
non-coding DNA molecule inserted into the TK gene locus in place of
(P.sub.SEL)rTrfR and (P.sub.7.5k)LacZ, and a non-coding DNA
molecule inserted into the HA gene locus in place of
(P.sub.11k)gusA. Other exemplary viruses include, but are not
limited to, LIVP viruses that express one or more therapeutic gene
products, such as angiogenesis inhibitors (e.g., GLV-1h81, which
contains DNA encoding the plasminogen K.sub.5 domain (SEQ ID NO:
109) under the control of the vaccinia synthetic early-late
promoter in place of the gusA expression cassette in GLV-1h68;
GLV-1h104, GLV-1h105 and GLV-1h106, which contain DNA encoding a
truncated human tissue factor fused to the
.alpha..sub.v.beta..sub.3-integrin RGD binding motif (tTF-RGD) (SEQ
ID NO: 105) under the control of a vaccinia synthetic early
promoter, vaccinia synthetic early/late promoter or vaccinia
synthetic late promoter, respectively, in place of the LacZ/rTFr
expression cassette at the TK locus of GLV-1h68; GLV-1h107,
GLV-1h108 and GLV-1h109, which contain DNA encoding an anti-VEGF
single chain antibody G6 (SEQ ID NO: 106) under the control of a
vaccinia synthetic early promoter, vaccinia synthetic early/late
promoter or vaccinia synthetic late promoter, respectively, in
place of the LacZ/rTFr expression cassette at the TK locus of
GLV-1h68) and proteins for tumor growth suppression (e.g.,
GLV-1h90, GLV-1h91 and GLV-1h92, which express a fusion protein
containing an IL-6 fused to an IL-6 receptor (sIL-6R/IL-6) (SEQ ID
NO: 108) under the control of a vaccinia synthetic early promoter,
vaccinia synthetic early/late promoter or vaccinia synthetic late
promoter, respectively, in place of the gusA expression cassette in
GLV-1h68; and GLV-1h96, GLV-1h97 and GLV-1h98, which express IL-24
(melanoma differentiation gene, mda-7; SEQ ID NO: 107) under the
control of a vaccinia synthetic early promoter, vaccinia synthetic
early/late promoter or vaccinia synthetic late promoter,
respectively, in place of the Ruc-GFP fusion gene expression
cassette at the F14.5L locus of GLV-1h68). Additional therapeutic
gene products that can be engineered in the viruses provided herein
also are described elsewhere herein.
C. TRANSPORTER PROTEINS
[0202] Membrane transport proteins, also known as transporters, are
proteins involved in the movement of ions, small molecules, or
macromolecules, such as other proteins, across a membrane.
Transport proteins are integral membrane proteins that span the
membrane across which they transport substances. These proteins can
assist in the movement of substances by facilitated diffusion or
active transport. Transporters can be located on the outer cell
membrane, mitochondria or other intracellular organelles. When
expressed in viruses using the methods described herein, these
transporters can function to transport and accumulate detectable
and/or therapeutic substrates in cells, such as tumor cells. For
example, transporters can provide signal amplification through
transport-mediated concentrative intracellular accumulation of
radiolabeled substrates for use in imaging, and can provide a means
to deliver therapeutic substances to virally-targeted tumors.
[0203] Transporters can be classified and identified using various
systems and databases well known in the art. Such systems can be
used to help identify transporters that can be expressed in the
viruses using the methods described herein, and to identify the
substrates for each transporter. For example, the Transporter
Classification database (TCDB; www.tcdb.org/) is an IUBMB
(International Union of Biochemistry and Molecular
Biology)-approved classification system for membrane transport
proteins, including ion channels (Saier et al., (2006) Nucl. Acids.
Res. 34: D181-D186). This was designed to be analogous to the EC
number system for classifying enzymes, but it also uses
phylogenetic information. The TC system classifies approximately
3000 proteins into over 550 transporter families. Included Another
system is the Solute Carrier (SLC) gene nomenclature system, which
is the basis for the Human Genome Organization (HUGO) names of the
genes that encode this group of transporters, and includes over 300
members organized into 47 families. Members within an individual
SLC family have greater than 20% sequence homology to each other.
The criteria for inclusion of a family into the SLC group is
functional (i.e., an integral membrane protein which transports a
solute) rather than evolutionary. The SLC group include
transporters that are facilitative transporters (allow solutes to
flow downhill with their electrochemical gradients) and secondary
active transporters (allow solutes to flow uphill against their
electrochemical gradient by coupling to transport of a second
solute that flows downhill with its gradient such that the overall
free energy change is still favorable). The SLC group does not
include ATP-driven transporters, ion channels or aquaporins. Most
members of the SLC group are located in the outer cell membrane,
although some members are located in mitochondria (most notably SLC
family 25) or other intracellular organelles. Table 2 provides the
SLC families (e.g. SLC1), the subfamiles (e.g. SLC1A) and the
member of the family (e.g. SLC1A1, corresponding to "Solute carrier
family 1, member 1").
TABLE-US-00002 TABLE 2 Solute Carrier (SLC) Transporter families
Family Members SLC1: The high affinity SLC1A1, SLC1A2, SLC1A3,
SLC1A4, SLC1A5, SLC1A6, glutamate and neutral SLC1A7 amino acid
transporter family SLC2: The facilitative SLC2A1, SLC2A2, SLC2A3,
SLC2A4, SLC2A5, SLC2A6, GLUT transporter SLC2A7, SLC2A8, SLC2A9,
SLC2A10, SLC2A11, family SLC2A12, SLC2A13, SLC2A14 SLC3: The heavy
SLC4A1, SLC4A1, SLC4A2, SLC4A3, SLC4A4, SLC4A5, subunits of the
SLC4A6, SLC4A7, SLC4A8, SLC4A9, SLC4A10, SLC4A11 heteromeric amino
acid transporters SLC4: The bicarbonate SLC4A1, SLC4A1, SLC4A2,
SLC4A3, SLC4A4, SLC4A5, transporter family SLC4A6, SLC4A7, SLC4A8,
SLC4A9, SLC4A10, SLC4A11 SLC5: The sodium SLC5A1, SLC5A2, SLC5A3,
SLC5A4, SLC5A5, SLC5A6, glucose cotransporter SLC5A7, SLC5A8,
SLC5A9, SLC5A10, SLC5A11, SLC5A12 family SLC6: The sodium- and
SLC6A1, SLC6A2, SLC6A3, SLC6A4, SLC6A5, SLC6A6, chloride-dependent
SLC6A7, SLC6A8, SLC6A9, SLC6A10, SLC6A11, neurotransmitter SLC6A12,
SLC6A13, SLC6A14, SLC6A15, SLC6A16, transporter family SLC6A17,
SLC6A18, SLC6A19, SLC6A20 SLC7: The cationic SLC7A1, SLC7A2,
SLC7A3, SLC7A4, SLC7A5, SLC7A6, amino acid SLC7A7, SLC7A8, SLC7A9,
SLC7A10, SLC7A11, transporter/glycoprotein- SLC7A13, SLC7A14
associated family SLC8: The Na+/Ca2+ SLC8A1, SLC8A2, SLC8A3
exchanger family SLC9: The Na+/H+ SLC9A1, SLC9A2, SLC9A3, SLC9A4,
SLC9A5, SLC9A6, exchanger family SLC9A7, SLC9A8, SLC9A9, SLC9A10,
SLC9A11 SLC10: The sodium bile SLC10A1, SLC10A2, SLC10A3, SLC10A4,
SLC10A5, salt cotransport family SLC10A6, SLC10A7 SLC11: The proton
SLC11A1, SLC11A2 coupled metal ion transporter family SLC12: The
SLC12A1, SLC12A1, SLC12A2, SLC12A3, SLC12A4, electroneutral
cation-Cl SLC12A5, SLC12A6, SLC12A7, SLC12A8, SLC12A9 cotransporter
family SLC13: The human SLC13A1, SLC13A2, SLC13A3, SLC13A4, SLC13A5
Na+-sulfate/carboxylate cotransporter family SLC14: The urea
SLC14A1, SLC14A2 transporter family SLC15: The proton SLC15A1,
SLC15A2, SLC15A3, SLC15A4 oligopeptide cotransporter family SLC16:
The SLC16A1, SLC16A2, SLC16A3, SLC16A4, SLC16A5, monocarboxylate
SLC16A6, SLC16A7, SLC16A8, SLC16A9, SLC16A10, transporter family
SLC16A11, SLC16A12, SLC16A13, SLC16A14 SLC17: The vesicular
SLC17A1, SLC17A2, SLC17A3, SLC17A4, SLC17A5, glutamate transporter
SLC17A6, SLC17A7, SLC17A8 family SLC18: The vesicular SLC18A1,
SLC18A2, SLC18A3 amine transporter family SLC19: The SLC19A1,
SLC19A2, SLC19A3 folate/thiamine transporter family SLC20: The type
III SLC20A1, SLC20A2 Na+-phosphate cotransporter family SLC21/SLCO:
The subfamily 1; SLCO1A2, SLCO1B1, SLCO1B3, SLCO1B4, organic anion
SLCO1C1 transporting family subfamily 2; SLCO2A1, SLCO2B1 subfamily
3; SLCO3A1 subfamily 4; SLCO4A1, SLCO4C1 subfamily 5; SLCO5A1
SLC22: The organic SLC22A1, SLC22A2, SLC22A3, SLC22A4, SLC22A5,
cation/anion/zwitterion SLC22A6, SLC22A7, SLC22A8, SLC22A9,
SLC22A10, transporter family SLC22A11, SLC22A12, SLC22A13,
SLC22A14, SLC22A15, SLC22A16, SLC22A17, SLC22A18, SLC22A19,
SLC22A20 SLC23: The Na+- SLC23A1, SLC23A2, SLC23A3, SLC23A4
dependent ascorbic acid transporter family SLC24: The SLC24A1,
SLC24A2, SLC24A3, SLC24A4, SLC24A5, Na+/(Ca2+-K+) SLC24A6 exchanger
family SLC25: The SLC25A1, SLC25A2, SLC25A3, SLC25A4, SLC25A5,
mitochondrial carrier SLC25A6, SLC25A7, SLC25A8, SLC25A9, SLC25A10,
family SLC25A11, SLC25A12, SLC25A13, SLC25A14, SLC25A15, SLC25A16,
SLC25A17, SLC25A18, SLC25A19, SLC25A20, SLC25A21, SLC25A22,
SLC25A23, SLC25A24, SLC25A25, SLC25A26, SLC25A27, SLC25A28,
SLC25A29, SLC25A30, SLC25A31, SLC25A32, SLC25A33, SLC25A34,
SLC25A35, SLC25A36, SLC25A37, SLC25A38, SLC25A39, SLC25A40,
SLC25A41, SLC25A42, SLC25A43, SLC25A44, SLC25A45, SLC25A46 SLC26:
The SLC26A1, SLC26A2, SLC26A3, SLC26A4, SLC26A5, multifunctional
anion SLC26A6, SLC26A7, SLC26A8, SLC26A9, SLC26A10, exchanger
family SLC26A11 SLC27: The fatty acid SLC27A1, SLC27A2, SLC27A3,
SLC27A4, SLC27A5, transport protein family SLC27A6 SLC28: The Na+-
SLC28A1, SLC28A2, SLC28A3 coupled nucleoside transport family
SLC29: The facilitative SLC29A1, SLC29A2, SLC29A3, SLC29A4
nucleoside transporter family SLC30: The zinc efflux SLC30A1,
SLC30A2, SLC30A3, SLC30A4, SLC30A5, family SLC30A6, SLC30A7,
SLC30A8, SLC30A9, SLC30A10 SLC31: The copper SLC31A1 transporter
family SLC32: The vesicular SLC32A1 inhibitory amino acid
transporter family SLC33: The Acety-CoA SLC33A1 transporter family
SLC34: The type II SLC34A1, SLC34A2, SLC34A3 Na+-phosphate
cotransporter family SLC35: The nucleoside- subfamily A; SLC35A1,
SLC35A2, SLC35A3, SLC35A4, sugar transporter family SLC35A5
subfamily B; SLC35B1, SLC35B2, SLC35B3, SLC35B4 subfamily C;
SLC35C1, SLC35C2 subfamily D; SLC35D1, SLC35D2, SLC35D3 subfamily
E; SLC35E1, SLC35E2, SLC35E3, SLC35E4 SLC36: The proton- SLC36A1,
SLC36A2, SLC36A3, SLC36A4 coupled amino acid transporter family
SLC37: The sugar- SLC37A1, SLC37A2, SLC37A3, SLC37A4
phosphate/phosphate exchanger family SLC38: The System A SLC38A1,
SLC38A2, SLC38A3, SLC38A4, SLC38A5, &N, sodium-coupled SLC38A6
neutral amino acid transporter family SLC3 9: The metal ion
SLC39A1, SLC39A2, SLC39A3, SLC39A4, SLC39A5, transporter family
SLC39A6, SLC39A7, SLC39A8, SLC39A9, SLC39A10, SLC39A11, SLC39A12,
SLC39A13, SLC39A14 SLC40: The basolateral SLC40A1 iron transporter
family SLC41: The MgtE-like SLC41A1, SLC41A2, SLC41A3 magnesium
transporter family SLC42: The Rh RHAG, RhBG, RhCG ammonium
transporter family (pending) SLC43: Na+- SLC43A1, SLC43A2, SLC43A3
independent, system-L like amino acid transporter family SLC44:
Choline-like SLC44A1, SLC44A2, SLC44A3, SLC44A4, SLC44A5
transporter family SLC45: Putative sugar SLC45A1, SLC45A2, SLC54A3,
SLC45A4 transporter family SLC46: Heme SLC46A1, SLC46A2 transporter
family SLC47: Multidrug and SLC47A1, SLC47A2 toxin extrusion
[0204] 1. The Sodium- and Chloride-Dependent Neurotransmitter
Transporter Family
[0205] Exemplary of transporters for use in the viruses and methods
described herein are members of the sodium- and chloride-dependent
neurotransmitter transporter family (Solute carrier family 6;
SLC6), also known as the sodium/neurotransmitter symporter family
(SNF) or the neurotransmitter/sodium symporter family (NSS) of
neurotransmitter transporters, which corresponds to TC 2.A.22 using
the TCDB system). Transporters in this family derive energy from
the co-transport of Na.sup.+ and Cl.sup.- in order to transport
neurotransmitter molecules into the cell against their
concentration gradient. The family has a common structure of 12
conserved transmembrane helices and includes transporters for
gamma-aminobutyric acid (GABA), noradrenaline/adrenaline, dopamine,
serotonin, proline, glycine, choline, betaine and taurine. Sequence
analysis of the Na.sup.+/Cl.sup.- neurotransmitter superfamily
reveals that it can be divided into four subfamilies, these being
transporters for monoamines, the amino acids proline and glycine,
GABA, and a group of orphan transporters (Nelson et al. (1998)
Methods Enzymol. 296:425-436). Table 3 sets forth the gene name of
exemplary members of this family, the corresponding protein name
and the sequence identifier of the corresponding human protein.
TABLE-US-00003 TABLE 3 SLC6 family members SLC6 gene Protein name
SEQ ID NO: SLC6A1 sodium- and chloride-dependent GABA transporter 1
SEQ ID NO: 36 SLC6A2 norepinephrine transporter (sodium-dependent
SEQ ID NO: 26 noradrenaline transporter) SLC6A3 sodium-dependent
dopamine transporter SEQ ID NO: 35 SLC6A4 sodium-dependent
serotonin transporter SEQ ID NO: 34 SLC6A5 sodium- and
chloride-dependent glycine transporter 1 SEQ ID NO: 40 SLC6A6
sodium- and chloride-dependent taurine transporter SEQ ID NO: 42
SLC6A7 sodium-dependent proline transporter SEQ ID NO: 41 SLC6A8
sodium- and chloride-dependent creatine transporter SEQ ID NO: 44
SLC6A9 sodium- and chloride-dependent glycine transporter SEQ ID
NO: 40 1, isoform 1 sodium- and chloride-dependent glycine
transporter SEQ ID NO: 95 1, isoform 2 sodium- and
chloride-dependent glycine transporter SEQ ID NO: 96 1, isoform 3
SLC6A10 sodium- and chloride-dependent creatine transporter SEQ ID
NO: 97 2 SLC6A11 sodium- and chloride-dependent GABA transporter 3
SEQ ID NO: 38 SLC6A12 sodium- and chloride-dependent betaine
transporter SEQ ID NO: 43 SLC6A13 sodium- and chloride-dependent
GABA transporter 2 SEQ ID NO: 37 SLC6A14 Sodium- and
chloride-dependent neutral and basic SEQ ID NO: 98 amino acid
transporter B(0+) SLC6A15 Orphan sodium- and chloride-dependent SEQ
ID NO: 99 neurotransmitter transporter NTT73 SLC6A16 Orphan sodium-
and chloride-dependent SEQ ID NO: 100 neurotransmitter transporter
NTT5 SLC6A17 Orphan sodium- and chloride-dependent SEQ ID NO: 101
neurotransmitter transporter NTT4 SLC6A18 Sodium- and
chloride-dependent transporter XTRP2 SEQ ID NO: 102 SLC6A19
Sodium-dependent neutral amino acid transporter SEQ ID NO: 103 B(0)
SLC6A20 Sodium- and chloride-dependent transporter XTRP3 SEQ ID NO:
104
[0206] a. Norepinephrine Transporter
[0207] The norepinephrine transporter (NET) is a sodium
chloride-dependent neurotransmitter symporter located primarily on
the plasma membrane of noradrenergic neurons that removes
norepinephrine (NE) from the extracellular space by high affinity
reuptake into presynaptic terminals. NET also is referred to as the
"sodium-dependent noradrenaline transporter,"
"noradrenaline:Na.sup.+ symporter," "SLC6A2," "TC 2.A.22.1.2" and
"solute carrier family 6 (neurotransmitter transporter,
noradrenalin), member 2." The NET not only regulates the longevity
of norepinephrine in the synapse but also plays an important role
in presynaptic and postsynaptic homeostasis. The norepinephrine
transporter belongs to NET is a monoamine transporter which, like
other monoamine transporters such as the dopamine transporter (DAT)
and the serotonin transporter (SAT), transfers monoamine
neurottransmitters in and out of cells.
[0208] i. Structure
[0209] The norepinephrine transporter (NET) is encoded by the
SLC6A2 gene. The cloned human NET cDNA (Pacholcznyk et al. (1991)
Nature 350:350-354) is an 1857 base pair open reading frame (SEQ ID
NO:25) encoding a 617 amino acid polypeptide (SEQ ID NO:26) with 12
transmembrane domains. The human NET shows sequence and structural
similarity to norepinephrine transporters from other species
including, but not limited to, bovine (SEQ ID NO:27, mouse (SEQ ID
NO:28), rat (SEQ ID NO:29), rhesus macaque (SEQ ID NO:30), chicken
(SEQ ID NO:31), ovine (fragment) (SEQ ID NO:32) and Japanese quail
(SEQ ID NO:33). In addition to multiple single amino acid variants,
such as those set forth in SEQ ID NOS:45-60, there are three known
C-terminal isoforms of human NET resulting from alternative
splicing. Typically, exon 14 of the human NET gene encodes the last
7 amino acids of the 617 amino acid polypeptide (SEQ ID NO:26). In
the two additional variants, alternative splicing joins exon 13 to
a new exon 15 (skipping exon 14). The alternatively spliced exon 15
encodes for either 3 or 18 amino acids, resulting in polypeptides
with a sequence set forth in SEQ ID NOS: 61 and 62, respectively
(Distelmaier et al., (2004) J. Neurochem. 91: 537-546). It has been
observed that while there was no difference in the binding
abilities of these isoforms, expression of the long C-terminal NET
splice variant (SEQ ID NO:62) exerted a dominant negative effect on
cell surface expression of the wild-type 617 amino acid NET. It has
been postulated, therefore, that this isoform may be involved in a
mechanism for a regulating noradrenergic neurotransmission
(Distelmaier et al., (2004) J. Neurochem. 91: 537-546).
[0210] Like other sodium/neurotransmitter symporter family members,
the human NET protein contains 12 hydrophobic segments, each of
18-25 amino acids in length, forming the transmembrane domains (TM1
to TM12). The amino and carboxy termini are located in the
cytoplasmic space. There are three possible N-glycosylation sites
located on the extracellular loop between TM3 and TM4 at amino acid
positions 184, 192 and 198 of SEQ ID NO:26). N-linked glycosylation
of NET results in increased cellular expression of the transporter
(Nguyen et al., (1996) J. Neurochem. 67:645-655, Melikian et al.,
(1996) Mol. Pharmacol. 50:266-276).
[0211] ii. Function
[0212] Human NET is expressed almost exclusively in the central and
peripheral sympathetic nervous system on the plasma membrane of
noradrenergic neurons, where it functions to take up synaptically
released norepinephrine. NET mRNA expression is localized to
monoaminergic cell bodies rather than to nerve terminals and is
generally restricted to cells that synthesize the corresponding NE.
As such, NET mRNA expression in the brain is an indicator for
noradrenergic pathways with cell bodies primarily located in the
brain stem, and in the locus coeruleus complex in the dorsal pons,
particularly in the nucleus locus coeruleus proper. NET mRNA also
is expressed in the lateral tegmentum of the medulla and pons. All
these regions encompass most of the noradrenergic cell bodies in
the CNS. In the periphery, NET mRNA is expressed in sympathetic
ganglia, in the adrenal medulla and the placenta (Bonisch H and
Bruss M. (2006) Handb Exp Pharmacol. 175:485-524).
[0213] NET acts as the primary mechanism for the inactivation of
noradrenergic signaling. Norepinephrine, also known as
noradrenaline, is a neurotransmitter found in the sympathetic
nervous system and is an important neurochemical messenger in the
central nervous system, playing a crucial role in physiology and
pathology. NE is involved in mood regulation, sleep regulation,
expression of behavior, memory, and the extent of alertness and
arousal. NE also exerts control over the endocrine system and
autonomic nervous system, and is involved in the regulation of
heart rate and blood pressure. Reuptake of NE by NET is the primary
mechanism by which the biological effects of NE in the synapse are
terminated. Thus, NET is critical in preventing excessive increases
of NE concentrations in the synaptic cleft, which regulate
adrenergic neurotransmission in the brain as well as the removal of
NE from the heart and other peripheral organs (Tellioglu et al.
(2001) Expert Rev. Mol. Med. 19 November). Approximately 70-90% of
the NE released into the synapses is cleared by this mechanism,
while the remaining 10-30% leaks out into the circulation or
extraneuronal tissue (Esler et al. (1990) Physiol. Rev.
70:963-985).
[0214] In addition to the transport of NE, the norepinephrine
transporter also has been shown in vitro using human
NET-transfected cells to transport other substrates, including, but
not limited to, dopamine, epinephrine, metaraminol, tyramine,
phenylethylamine, d-amphetamine, ephedrine and serotonin, in
addition to other substances including, but not limited to, the
drugs tranylcypromine, selegiline and amezinium, the adrenergic
neuron blocking agents bretylium and guanethidine,
meta-iodobenzylguanadine (MIBG), the covalently binding NET suicide
substrates and noradrenergic neurotoxins xylamine, and DSP-4, the
dopaminergic neurotoxin 1-methyl-4-tetrahydropyridinium) MPP.sup.+
and the fluorescent model substrate ASP.sup.+ (Bonisch H and Bruss
M. (2006) Handb Exp Pharmacol. 175:485-524). In addition to binding
substrates, NET also binds various molecules that act as
inhibitors, including, but not limited to, antidepressants such as
desipramine, nortriptyline, reboxetine, maprotiline and
nomeifensine, and other substances such as nisoxetine, sibutramine,
atomoxetine, RTI-55 and cocaine (Bonisch H and Bruss M. (2006)
Handb Exp Pharmacol. 175:485-524).
[0215] Substrate transport by the NET is dependent on Na.sup.+ and
Cl.sup.- ions and the Na.sup.+ gradient across the plasma membrane
that drives the intracelleular accumulation of the substrate.
Studies indicate that sequential binding of Na.sup.+ and then
Cl.sup.- is required for substrate binding to the transporter
(Bonisch H and Bruss M. (1994) Ann. N.Y. Acad. Sci. 733:193-202).
Following binding of the substrate, both Na.sup.+ and Cl.sup.- are
co-transported with the substrate into the cytoplasm. The ion pump
Na.sup.+/-K.sup.+-ATPase is the driving force for this transport by
maintaining a Na.sup.+ concentration gradient across the plasma
membrane. Na.sup.+ and Cl.sup.- dependent transport of NE and other
substrates into the cytoplasm also requires a balance of K.sup.+
ions across the cell membrane.
[0216] 2. The Sodium Glucose Cotransporter Family
[0217] Exemplary of transporters for use in the viruses and methods
described herein are members of the sodium glucose cotransporter
family (Solute carrier family 5; SLC5), also known as the
sodium/solute symporter family (SSSF) or TC 2.A.21. This family has
over a hundred members of prokaryotic and eukaryotic origin. The
family members typically have 11 to 15 transmembrane domains (TMs)
in alpha-helical conformation. Proteins of this family utilize a
sodium motive force (the energy stored in an inwardly directed
electrochemical sodium gradient) to drive uphill transport of
substrates such as sugars, amino acids, vitamins, ions,
myo-inositol, phenyl acetate, urea, and water. Most of the
transporters are part of catabolic pathways and are used by the
cells to acquire the corresponding substrate. Among the best
characterized members of the SSSF are the human sodium/glucose
transporter (SGLT1), the sodium/iodide transporter (NIS), and PutP
of E. coli. Table 4 sets forth the gene name of exemplary members
of the SLC5 family, the corresponding protein name and the sequence
identifier of the corresponding human protein.
TABLE-US-00004 TABLE 4 SLC5 family members SLC5 gene Protein name
SEQ ID NO: SLC5A1 sodium/glucose cotransporter 1 SEQ ID NO: 69
SLC5A2 sodium/glucose cotransporter 2 SEQ ID NO: 70 SLC5A3
sodium/myo-inositol cotransporter SEQ ID NO: 71 SLC5A4 low affinity
sodium-glucose SEQ ID NO: 72 cotransporter SLC5A5 sodium/iodide
cotransporter SEQ ID NO: 63 SLC5A6 sodium-dependent multivitamin
SEQ ID NO: 73 transporter SLC5A7 high affinity choline transporter
1 SEQ ID NO: 74 SLC5A8 sodium-coupled monocarboxylate SEQ ID NO: 75
transporter 1 SLC5A9 sodium/glucose cotransporter 4 SEQ ID NO: 76
SLC5A10 sodium/glucose cotransporter 5, SEQ ID NO: 77 isoform 1
sodium/glucose cotransporter 5, SEQ ID NO: 78 isoform 2
sodium/glucose cotransporter 5, SEQ ID NO: 79 isoform 3
sodium/glucose cotransporter 5, SEQ ID NO: 80 isoform 4 SLC5A11
sodium/myo-inositol cotransporter 2, SEQ ID NO: 81 isoform 1
sodium/myo-inositol cotransporter 2, SEQ ID NO: 82 isoform 2
sodium/myo-inositol cotransporter 2, SEQ ID NO: 83 isoform 3
sodium/myo-inositol cotransporter 2, SEQ ID NO: 84 isoform 4
SLC5A12 sodium-coupled monocarboxylate SEQ ID NO: 85 transporter 2,
isoform 1 sodium-coupled monocarboxylate SEQ ID NO: 86 transporter
2, isoform 2
[0218] a. Sodium Iodide Symporter
[0219] The sodium-iodide symporter (NIS) is an ion pump that
transports iodide (I.sup.-) into thyroid epithelial cells across
the basolateral plasma membrane, an important step in the process
of iodide organification and the formation of triiodothyronine
(T.sub.3) and thyroxine (T.sub.4). NIS also is referred to as the
"Sodium/iodide cotransporter," "Na(+)/I(-) cotransporter,"
"SLC5A5," "TC 2.A.21.5.1" and "solute carrier family 5 member
5."
[0220] i. Structure
[0221] The human sodium-iodide symporter gene (SLC5A5) encodes a
glycoprotein of 643 amino acids (SEQ ID NO:63) with a molecular
mass of approximately 70-90 kDa. The gene has 15 exons and has an
open reading frame of 1929 nucleotides (SEQ ID NO:64). The NIS is
predicted to have a 13-transmembrane segment pattern with the
N-terminus facing the extracellular milieu, and the COOH-terminus
facing the cytosol (Levy et al. (1997) Proc. Nat. Acad. Sci. 94:
5568-5573.). The human NIS shares sequence and structural
similarity to NIS proteins from other species, including but not
limited to, the NIS from mouse (SEQ ID NO:65), rat (SEQ ID NO:66),
Zebrafish (SEQ ID NO:67), and African clawed frog mouse (SEQ ID
NO:68). Multiple natural variants of human NIS also exist,
including, but not limited to, those set forth in SEQ ID
NOS:87-94). Synthetic variants of hNIS also are available and
include hNIS transporters with multiple lysine residues inserted at
that C-terminus of the protein (see, e.g., PCT Patent publication
2004/000236), which increases the net positive electrostatic charge
of the protein, allowing increased transport of NIS substrates.
[0222] There are at least two potential sites of glycosylation in
the human NIS at amino acid positions 489, 502 of SEQ ID NO:63.
However, studies using rat NIS indicate that glycosylation does not
play an important role in the stability, activity, or targeting to
the membrane of the symporter (Levy et al., (1998) J. Biol. Chem.
273: 22657-22663). NIS also a phosphoprotein (Riedel et al. (2001)
J. Biol. Chem. 276: 21458-21463). Amino acid position 556 of the
human NIS polypeptide set forth in SEQ ID NO:63 is a potential site
for phosphorylation by phosphokinase A (PKA).
[0223] ii. Function
[0224] The NIS is an intrinsic transmembrane protein that is
expressed on the basolateral membrane of thyroid follicular cells
and mediates the accumulation of iodide from the bloodstream to
thyroid follicles as the crucial first step for thyroid hormone
biosynthesis. The thyroid hormones T.sub.3 and T.sub.4 are the only
iodine-containing hormones in vertebrates. Because I.sup.- is an
essential constituent of T.sub.3 and T.sub.4, thyroid function
depends on an adequate supply of I.sup.- to the gland. In normal
thyroid tissue, NIS transports Na.sup.+ I.sup.- ions at a 2:1
stoichiometry down the Na.sup.+ ion gradient that generated from
the activity of Na.sup.+/K.sup.+ ATPase. NIS actively transports
iodide producing an iodine concentration gradient from the thyroid
cell to extracellular fluid greater than 30:1. This process is
stimulated by thyrotropin (TSH) and inhibitable by the competitive
inhibitors thiocyanate (SCN.sup.-) and perchlorate
(ClO.sub.4.sup.-). I.sup.- is then translocated from the cytoplasm
across the apical plasma membrane toward the colloid in a process
called I.sup.- efflux. I.sup.- is oxidized and incorporated into
tyrosyl residues within the thyroglobulin (Tg) molecule, leading to
the subsequent coupling of iodotyrosine residues at the
cell-colloid interface. This process is called organification of
I.sup.-. Iodinated Tg is stored extracellularly in the colloid. In
response to demand for thyroid hormones, phagolysosomal hydrolysis
of endocytosed iodinated Tg is effected. T.sub.3 and T.sub.4 are
secreted into the bloodstream, and nonsecreted iodotyrosines are
metabolized to tyrosine and I.sup.- (Dohan et al. (2003) Endocrine
Reviews 24:48-77).
[0225] The human NIS also is expressed in extrathyroidal tissues,
including the salivary gland, gastric mucosa, mammary gland,
ciliary body of the eye, and the choroid plexus. Thus, NIS also
mediates active I.sup.- transport in other tissues, including
salivary glands, gastric mucosa, and lactating mammary gland,
although this process is not stimulated by TSH in these tissues
(Dohan et al. (2003) Endocrine Reviews 24:48-77). Whereas the
functional significance of NIS in the gastric mucosa and salivary
glands is unknown, in the lactating mammary gland NIS mediates the
translocation of I.sup.- into the milk, making this anion available
for the nursing newborn to biosynthesize his/her own thyroid
hormones. Studies using whole body imaging with radioiodide
indicate that lactating breast tissue concentrates a significant
amount of iodide as a result of stimulation of NIS expression. The
trapped iodide is secreted in milk and provides iodine for thyroid
hormone synthesis to the developing infant (Kogai et al., (2006)
Endocrine-Related Cancer 13:797-826).
[0226] The ability of the thyroid to accumulate I.sup.- via NIS has
enabled diagnostic scintigraphic imaging of the thyroid with
radioiodide and has served as an effective means for therapeutic
doses of radioiodide to target and destroy hyperfunctioning thyroid
tissue, such as in Graves' disease and I.sup.--transporting thyroid
cancer and its metastases. The expression of NIS on various thyroid
tumors can differ. For example, in some studies, reduced expression
of NIS mRNA and protein was been observed in papillary and
follicular thyroid cancer, while other studies indicated increased
NIS expression in papillary (Kogai et al., (2006) Endocrine-Related
Cancer 13:797-826). In addition to being expressed on thyroid
tumors, NIS expression has been demonstrated in more than 80% of
breast cancer tissue (Wapnir et al. (2003) J. Clin. End. Met. 88:
1880-1888). As a result, agents that stimulate NIS expression in
breast cancer sufficient to concentrate radioiodide have been
considered as a source of potential therapy for some differentiated
breast cancer (Boelaert et al. (2003) Lancet 361: 796-797).
[0227] In addition to transporting I.sup.- ions, studies have
indicated that NIS also can transport additional anions, including,
but not limited to, ClO.sub.3.sup.-, SCN.sup.-, SeCN.sup.-,
NO.sub.3.sup.-, BR.sup.-, BF.sub.4.sup.-, IO.sub.4.sup.- and
BrO.sub.3.sup.-, CIO.sub.3.sup.-, ReO.sub.4.sup.-, At.sup.-,
TcO.sub.4.sup.- (Van Sande et al., (2003) Endocrinology 144:10,
International Patent Publication WO2004000236). Such substrates can
be administered to patients in chemical forms well known in the art
for therapeutic or imaging purposes. For example, I.sup.- can be
administered as NaI, TcO.sub.4.sup.- -can be administered as
Na.sup.+(TcO.sub.4.sup.-). In some instances, the NIS substrates
used in the methods herein and other methods for imaging of therapy
are isotopic and include radionuclides. For example, .sup.123I,
.sup.124I, .sup.125I and .sup.131I are forms of isotopic
radioactive iodide, .sup.99mTcO.sub.4 is a form of isotopic
pertechnate, .sup.188ReO.sub.4 is an isotopic form of perrhenate
and .sup.211At is an isotopic form of astatide.
D. METHODS OF ASSESSING MODIFIED VIRUSES ENCODING TRANSPORTERS
[0228] The activity of transporters used in the viruses and methods
herein, and the ability of the modified viruses to deliver and
express these transporters on infected cells, can be assayed using
methods well known in the art. The ability of transporters to
transport one or more substrates can be assessed prior to
expression in a modified virus, such as by transfection of cell
lines with nucleic acid encoding the transporter of interest, to
assist in determining suitable transporters for inclusion in the
modified viruses. The activity of the transporters also can be
assessed in the context of a modified virus, such as any of those
provided herein. The transporters can be assessed, for example, to
determine the expression profile, including the cellular
localization and level of expression, on a cell following infection
with the modified virus that expresses the transporter. In
addition, the ability of virus to produce high levels of expression
of the transporter in tumor tissue can be assessed to determine
whether the expressed transporter can concentrate the signals
emitted by the transporter substrates at the tumor tissue. The
specificity of the transporter, such as the ability of the
transporter to transport one or more substrates, and level of
activity, such as how much substrate is transported, can be
assessed. Such assays are available and know by those skill in the
art. Exemplary assays are provided herein.
[0229] 1. In Vitro Assessment
[0230] The expression of transporters on cells infected by modified
viruses carrying those transporters, such as those viruses
described herein, can be assessed using assays well known in the
art. In some instances, immunoassays can be used to assess the
expression of transporters on cells following infection with the
modified viruses. For example, modified viruses encoding a
transporter can be used to infect cells and the level of expression
determined by immunoblot with a transporter-specific antibody
following cell lysis (see e.g. Example 2.C, below). Alternatively,
the cell lysates could be analyzed by enzyme-linked immunosorbant
assays (ELISA) to assess transporter expression. In another
example, infected cells are analyzed by, for example,
immunohistochemistry, flow cytometry analysis or
immunoprecipitation using transporter-specific antibodies.
[0231] Assays to determine the cellular localization of a
transporter following infection with a modified virus encoding the
transporter also are known in the art and can be used herein. For
example, techniques such as immunocytochemistry and cell surface
biotinylation can be used to determine the localization of
transporters such as NET, NIS and GABA transporters (Gu et al.,
(2001) Mol. Cell. Biol. 12:3797-3807, Ahn et al., (1996) J. Biol.
Chem. 27: 6917-6924, Gu et al. (1996) J. Biol. Chem. 271,
18100-18106, Dayem et al., (2008) J. Endocrinol. 197:95-109).
[0232] The ability of transporters to bind and transport substrates
across a membrane can be assessed in vitro using any one or more
assays known in the art. In some instances, radiotracer assays are
used to assess transporter activity (such as those described in
Example 2.D, below. Such assays can be performed prior to
introducing the nucleic acid encoding the transporter into the
modified viruses. This can be done, for example, to screen
transporters for substrate specificity to assist in determining
which transporters are suitable for use in the viruses and methods
herein. For example, nucleic acid encoding the transporter can be
stably or transiently transfected, or can be introduced using viral
vectors, into an appropriate cell line (see e.g. Moroz et al.,
(2007) J Nucl. Med. 48:827-836) and a radiotracer assay performed.
Alternatively, the modified virus expressing the transporter can be
used to infect an appropriate cell line, such a tumor cell line,
such as described in Example 2.D. The amount of radiolabeled
substrate that is transported into the cell is then assessed, such
as by using a gamma counter, to determine whether the transporter
is specific for the substrate and, if so, how efficient the
transporter is in transporting the substrate. Many radiolabeled
substrates are readily available, and methods for radiolabeling
substrates also are well known in the art and can be used in the
methods herein to label a desired substrate for use in a
radiotracer assay. Isotopic labels can be incorporated at specific
positions of a compound and can be incorporated into peptides and
polypeptides and other substances (see e.g. U.S. Pat. No.
5,102,651, 5,277,893, U.S. Patent Publication No. 20070280883,
Sandell et al., (2002) Bior. Med. Clin. Lett. 14:3611-3613). Such
assays can be performed using cells infected with modified viruses
encoding the transporter and cells infected with modified viruses
that do not encode the transporter. The level by which substrate
uptake is increased when the transporter is expressed can then be
determined.
[0233] Other assays to detect transport of substrates are known in
the art and can be used herein. In some instances, these assays
utilize fluorescent substrates. For example, the fluorescent
substrate 4-(4-dimethylaminostyryl)-N-methylpyridinium (ASP.sup.+)
can be used to detect transporting activity of NET (see e.g. U.S.
Patent Publication No. 2004115703, International Pat. Pub.
WO2008043851, Hauns (2007) J. Biomol. Screen. 12:378-384). Other
fluorescent substrates can be used to detect the activity or
neurotransmitter transporters (International Pat. Pub.
WO2008021870). Fluorescent-based assays also are known for other
transporters, including, but not limited to, NIS (see e.g. Rhoden
et al., (2007) Am. J. Physiol. 61:C814-C823). Other methods for
identifying substrates specific for transporters are known in the
art and can be used herein (see e.g. U.S. Pat. No. 7,309,576,
European Patent No EP1212616).
[0234] 2. In Vivo Assessment
[0235] The modified viruses encoding transporters also can be
assessed in vivo, both in human and animal models. Animal models,
such as mouse models, of different types of human and non-human
animal cancers can be employed to assess the transporter activity
imparted by infection with the modified viruses. Tumors can be
established by implantation of different tumor cell types.
Exemplary human tumor xenograft models in mice, such as nude or
SCID mice, include, but are not limited to, human lung carcinoma
(A549 cells, ATCC No. CCL-185); human breast tumor (GI-101A cells,
Rathinavelu et al., Cancer Biochem. Biophys., 17:133-146 (1999));
human ovarian carcinoma (OVCAR-3 cells, ATCC No. HTB-161); human
pancreatic carcinoma (PANC-1cells, ATCC No. CRL-1469 and MIA PaCa-2
cells, ATCC No. CRL-1420); DU145 cells (human prostate cancer
cells, ATCC No. HTB-81); human prostate cancer (PC-3 cells, ATCC#
CRL-1435); colon carcinoma (HT-29 cells); human melanoma (888-MEL
cells, 1858-MEL cells or 1936-MEL cells; see e.g. Wang et al.,
(2006) J. Invest. Dermatol. 126:1372-1377); and human fibrosarcoma
(HT-1080 cells, ATCC No. CCL-121,) and human mesothelioma
(MSTO-211H cells).
[0236] The ability of the virus to deliver the transporter to the
tumor and for the transporter to be subsequently expressed and be
active can be assessed using such models (see e.g. Example 3,
below). For example, transporter expression can be detected using
methods such as immunohistochemistry, immunoblot and flow cytometry
analysis of tumor and non-tumor tissues using transporter-specific
antibodies. The activity of the transporter also be assessed using,
for example, radiolabeled or fluorescently labeled substrate. Whole
animal imaging can be then performed, such as PET or gamma imaging
(see e.g. Example 3.B and C, below) to detect the transport and
accumulation of the labeled substrate in the tumor cells. In other
examples, tissues from the mouse can be extracted and the
radiolabeled or fluorescently labeled substrate can be detected by,
for example, imaging, flow cytometry, gamma counting or fluorescent
microscopy.
[0237] In some instances, the radiolabeled substrate also can be a
chemotherapeutic agent or can be linked to a therapeutic agent.
Thus, the therapeutic effect of transport of the agent into the
tumor cell also can be assessed in animal models. For example, the
change in tumor growth can be assessed following injection with a
modified virus encoding a transporter/chemotherapeutic agent and
compared with tumor growth following injection with a modified
virus that does not encode the transporter.
[0238] 3. Selection of Substrates
[0239] Substrates that can be transported into cells by particular
transporter proteins are known in the art and can be selected for
use as imaging and/or therapeutic agents. The substrates can be
radiolabeled for detection in vivo by imaging methods, including
PET and SPECT imaging and other imaging methods as described
elsewhere herein. The radiolabeled substrates can be employed for
either or both imaging and therapy of tumors. For example, the
accumulation of radioactive substrates within infected tumor cells
that express the corresponding transporter proteins can provide
radiotherapy treatment of the tumor.
[0240] Exemplary detectable substrates that can be selected for
transport by norepinephrine, dopamine and serotonin transporters
include, but are not limited to, radiolabeled
metaiodobenzylguanidine (MIBG), such as .sup.124I-MIBG and
.sup.123I-MIBG .sup.131I-MIBG and .sup.11C-labeled hydroxyephedrine
(HED) (Shulkin et al. (1986) J Nucl Med 27:1138-42 and Glowniak et
al. (1993) J Nucl Med 34:1140-6). Exemplary detectable substrates
that can be selected for transport by hNIS include, but are not
limited to radioactive iodide (.sup.123I.sup.-, .sup.124I.sup.- and
.sup.131I.sup.-), other radiolabeled anions
(.sup.99mTcO.sub.4.sup.- (pertechnetate), .sup.76Br.sup.-), the
.beta.-emitter .sup.188rhenium (.sup.188 ReO.sub.4.sup.-) and the
.alpha.-emitter .sup.211astatine (.sup.211At.sup.-) (Van Sande et
al. (2003) Endocrinology 144:247-52; Barton et al. (2003) Mol Ther
8:508-18; Kang et al. (2004) J Nucl Med 45:1571-6; Cho et al.
(2002) Gene Ther 9:1139-45; Groot-Wassink et al. (2004) Mol Ther
9:436-42; Niu et al. (2004) J Nucl Med 45:445-9; Groot-Wassink et
al. (2002) Hum Gene Ther 13:1723-35; Shimura et al. (1997)
Endocrinology 138:4493-6; Mandell et al. (1999) Cancer Res
59:661-8; Boland et al. (2000) Cancer Res 60:3484-92; Carlin et al.
(2002) Nucl Med Biol 29:729-39; Haberkorn et al. (2003) Gene Ther
10:774-80). hNIS can also transport anions such as ClO.sub.3.sup.-,
SCN.sup.-, SeCN.sup.-, NO.sub.3.sup.-, Br.sup.-, BF.sub.4.sup.-,
IO.sub.4.sup.- and BrO.sub.3.sup.- that can be labeled according to
methods known in the art (Van Sande et al. (2003) Endocrinology
144:247-52).
[0241] Selected substrates can also be conjugated to cytotoxic
agents for therapy tumors. Uptake of the transporter substrates
thus permits accumulation of the cytotoxic agent in the infected
tumors that express the transporter. The accumulation also results
in high levels of the cytotoxic agents in the surrounding tumorous
tissue, thus promoting uptake of the cytotoxic agents by
neighboring tumor cells. Such cytotoxic agents for the therapy of
tumor are know in the art and include, but are not limited to,
double-chain ricin, ricin A chain, abrin, abrin A chain, saporin,
modeccin, modeccin A chain, Pseudomonas aeruginosa exotoxin,
Cholera toxin, Shigella toxin, E. coli heat labile toxin and
Diptheria toxin. doxorubicin, daunomycin, 5-fluorouracil,
methotrexate, taxol, ricin A, colchicine, cytochasins, monensin,
ouabain, mitoxanthrone, vindesine, vinblastine, vincristine or
enterotoxin
E. ADDITIONAL MODIFICATIONS OF VIRUSES PROVIDED
[0242] Viruses provided herein that encode a transporter protein
can contain additional modifications. Such modifications can be
generated using any known method for modifying a virus. The
additional modifications of the virus can be introduced prior to,
simultaneously with or following modification of the viral genome
to introduce DNA encoding the transporter protein. Furthermore,
viruses provided herein also can be further modified to attenuate
the virus. Hence, the methods provided herein can be combined with
any known method for modifying a virus. Furthermore, the methods
provided herein can be combined with any known method for
modulating the attenuation of a virus. For example, such methods
include modification of one or more viral genes, such as by a point
mutation, a deletion mutation, an interruption by an insertion, a
substitution or a mutation of the viral gene promoter or enhancer
regions.
[0243] Viruses provided herein can contain one or more additional
heterologous nucleic acid molecules inserted into the genome of the
virus. A heterologous nucleic acid molecule can contain an open
reading frame or can be a non-coding sequence. In some cases, the
heterologous nucleic acid replaces all or a portion of a viral
gene.
[0244] Further modifications of the viruses provided can enhance
one or more characteristics of the virus. Such characteristics can
include, but are not limited to, attenuated pathogenicity, reduced
toxicity, preferential accumulation in tumor, increased ability to
activate an immune response against tumor cells, increased
immunogenicity, increased or decreased replication competence, and
ability to express additional exogenous proteins, and combinations
thereof. In some examples, the modified viruses have an ability to
activate an immune response against tumor cells without
aggressively killing the tumor cells. In other examples, the
viruses can be modified to express one or more detectable gene
products, including additional gene products that can be used for
imaging. In other examples, the viruses can be modified to express
one or more genes for the therapy of a tumor or an inflamed or
wounded tissue. In other examples, the viruses can be modified to
express one or more genes for harvesting the gene products and/or
for harvesting antibodies against the gene products.
[0245] 1. Modification of Viral Genes
[0246] Methods for modifying a virus include modifications in one
or more viral genes. Modification can include those that inactivate
viral gene or abolish or decrease the activity of a viral gene
product. In some example the viral gene is replaced with non-coding
nucleic acid. Such modifications in a viral gene can alter the
viral processes, such as, for example, viral infectivity, viral DNA
replication, viral protein synthesis, virus particle assembly and
maturation, and viral particle release. Exemplary viral genes for
modification include, but are not limited to, viral surface
antigens (e.g. proteins that mediate viral attachment to host cell
receptors), viral proteases, and viral enzymes involved in viral
replication and transcription of viral genes (e.g., polymerases,
replicases and helicases). Modifications in such genes can decrease
the overall replication of the virus and production of viral
particles thus resulting in a more attenuated virus.
[0247] In some examples, the viral gene can be replaced with
homologous gene from another virus or a different gene. For
example, vaccinia viruses provided herein can be modified by
replacement of the A34R gene with another A34R gene from a
different strain in order to increase the EEV form of the virus. In
one example, the A34R gene from the Lister strain of vaccinia can
be replaced with A34R gene from the IHD-J strain of vaccinia
virus
[0248] In another embodiment, a viral surface antigen gene can be
modified to produce a chimeric protein such that the heterologous
epitope is expressed on the surface of the virus. Viruses
expressing such chimeric proteins are thus useful as vaccines for
use in generating an immune response in the host subject. Exemplary
epitopes include but are not limited to tumor antigens, viral and
bacterial antigens. Many exemplary antigens are known in the art,
and include, for example, those listed and/or described in
Novellino et al. (2005) Cancer Immunol Immunother. 54(3): 187-207;
Eisenberger et al. (2006) Hematol Oncol Clin North Am.
20(3):661-87. In one embodiment, insertion of a heterologous
epitope into the viral gene can affect the level of attenuation of
the virus. In an alternative embodiment, the level of attenuation
of the virus is unaffected by insertion of a heterologous epitope
into the viral gene.
[0249] 2. Expression of Additional Heterologous Genes
[0250] Viruses provided herein and viruses generated using the
methods provided herein can be further modified to express one or
more additional heterologous genes. Gene expression can include
expression of a protein encoded by a gene and/or expression of an
RNA molecule encoded by a gene. In some embodiments, the viruses
can express heterologous genes at levels high enough that permit
harvesting products of the heterologous gene from the tumor.
[0251] The heterologous nucleic acid can be operably linked to a
promoter for expression of an open reading frame encoding the
heterologous protein. Expression of heterologous genes can be
controlled by a constitutive promoter, or by an inducible promoter.
Expression can also be influenced by one or more proteins or RNA
molecules expressed by the virus. Gene regulatory elements, such as
promoters and enhancers, possess cell type specific activities and
can be activated by certain induction factors (e.g., hormones,
growth factors, cytokines, cytostatics, irradiation, heat shock)
via responsive elements. A controlled and restricted expression of
these genes can be achieved using such regulatory elements as
internal promoters to drive the expression of therapeutic genes in
viral vector constructs. Heterologous genes expressed can include
genes encoding a therapeutic gene product, genes encoding a
detectable gene product, such as a gene product that can be used
for imaging, genes encoding a gene product to be harvested, genes
encoding an antigen of an antibody to be harvested or to elicit an
immune response. The viruses provided herein can be used for
expressing genes in vivo and in vitro. Exemplary proteins include
reporter proteins (E. coli .beta.-galactosidase,
.beta.-glucuronidase, xanthineguanine phosphoribosyltransferase),
proteins facilitating detection, such as a detectable protein or a
protein capable of inducing a detectable signal, (luciferase,
fluorescent proteins, transferrin receptor, for example), gene
products (i.e., proteins and RNAs) useful for tumor therapy (e.g.,
a transporter, a cell-surface receptor, a cytokine, a chemokine, an
apoptotic protein, a mitosis inhibitor protein, an antimitotic
oligopeptide, an antiangiogenic factor, anti-cancer antibodies,
such as a single-chain antibody, a toxin, a tumor antigen, a
prodrug converting enzyme, a ribozyme, RNAi, siRNA, pseudomonas A
endotoxin, diphtheria toxin, p53, Arf, Bax, tumor necrosis
factor-alpha, HSV TK, E. coli purine nucleoside phosphorylase,
angiostatin and endostatin) and other anticancer or therapeutic
gene products.
[0252] a. Detectable Gene Product
[0253] Viruses provided herein and viruses generated using the
methods provided herein can express one or more genes whose
products are detectable or whose products can provide a detectable
signal. A variety of detectable gene products, such as detectable
proteins are known in the art, and can be used with the viruses
provided herein. Detectable proteins include receptors or other
proteins that can specifically bind a detectable compound, proteins
that can emit a detectable signal such as a fluorescence signal,
and enzymes that can catalyze a detectable reaction or catalyze
formation of a detectable product.
[0254] In some embodiments, the virus expresses a gene encoding a
protein that can emit a detectable signal or that can catalyze a
detectable reaction. A variety of DNA sequences encoding proteins
that can emit a detectable signal or that can catalyze a detectable
reaction, such as luminescent or fluorescent proteins, are known
and can be used in the viruses and methods provided herein.
Exemplary genes encoding light-emitting proteins include genes from
bacterial luciferase from Vibrio harveyi (Belas et al., Science 218
(1982), 791-793), bacterial luciferase from Vibrio fischerii (Foran
and Brown, Nucleic acids Res. 16 (1988), 177), firefly luciferase
(de Wet et al., Mol. Cell. Biol. 7 (1987), 725-737), aequorin from
Aequorea victoria (Prasher et al., Biochem. 26 (1987), 1326-1332),
Renilla luciferase from Renilla renformis (Lorenz et al, PNAS USA
88 (1991), 4438-4442) and green fluorescent protein from Aequorea
victoria (Prasher et al., Gene 111: 229-233 (1987)). The luxA and
luxB genes of bacterial luciferase can be fused to produce the
fusion gene (Fab.sub.2), which can be expressed to produce a fully
functional luciferase protein (Escher et al., PNAS 86: 6528-6532
(1989)). Transformation and expression of these genes in viruses
can permit detection of viral infection, for example, using a low
light and/or fluorescence imaging camera. In some embodiments,
luciferases expressed by viruses can require exogenously added
substrates such as decanal or coelenterazine for light emission. In
other embodiments, viruses can express a complete lux operon, which
can include proteins that can provide luciferase substrates such as
decanal. For example, viruses containing the complete lux operon
sequence, when injected intraperitoneally, intramuscularly, or
intravenously, allowed the visualization and localization of
microorganisms in live mice indicating that the luciferase light
emission can penetrate the tissues and can be detected externally
(Contag et al. (995) Mol. Microbiol. 18: 593-603).
[0255] In other embodiments, the virus can express a gene that can
bind a detectable compound or that can form a product that can bind
a detectable compound. A variety of gene products, such as
proteins, that can specifically bind a detectable compound are
known in the art, including receptors, metal binding proteins
(e.g., siderophores, ferritins, transferrin receptors), ligand
binding proteins, and antibodies. Any of a variety of detectable
compounds can be used, and can be imaged by any of a variety of
known imaging methods. Exemplary compounds include receptor ligands
and antigens for antibodies. The ligand can be labeled according to
the imaging method to be used. Exemplary imaging methods include
any of X-rays, a variety magnetic resonance methods such as
magnetic resonance imaging (MRI) and magnetic resonance
spectroscopy (MRS), and also include any of a variety of
tomographic methods including computed tomography (CT), computed
axial tomography (CAT), electron beam computed tomography (EBCT),
high resolution computed tomography (HRCT), hypocycloidal
tomography, positron emission tomography (PET), single-photon
emission computed tomography (SPECT), spiral computed tomography
and ultrasonic tomography.
[0256] Labels appropriate for X-ray imaging are known in the art,
and include, for example, Bismuth (III), Gold (III), Lanthanum
(III) or Lead (II); a radioactive ion, such as .sup.67Copper,
.sup.67Gallium, .sup.68Gallium, .sup.111Indium, .sup.113Indium,
.sup.123Iodine, .sup.125Iodine, .sup.131Iodine, .sup.97Mercury,
.sup.203Mercury, .sup.186Rhenium, .sup.188Rhenium, .sup.97Rubidium,
.sup.103Rubidium, .sup.99Technetium or .sup.90Yttrium; a nuclear
magnetic spin-resonance isotope, such as Cobalt (II), Copper (II),
Chromium (III), Dysprosium (III), Erbium (III), Gadolinium (III),
Holmium (III), Iron (II), Iron (III), Manganese (II), Neodymium
(III), Nickel (II), Samarium (III), Terbium (III), Vanadium (II) or
Ytterbium (III); or rhodamine or fluorescein.
[0257] Labels appropriate for magnetic resonance imaging are known
in the art, and include, for example, gadolinium chelates and iron
oxides. Use of chelates in contrast agents is known in the art.
Labels appropriate for tomographic imaging methods are known in the
art, and include, for example, .beta.-emitters such as .sup.11C,
.sup.13N, .sup.150 or .sup.64Cu or (b) .gamma.-emitters such as
.sup.123I. Other exemplary radionuclides that can, be used, for
example, as tracers for PET include .sup.55Co, .sup.67Ga,
.sup.68Ga, .sup.60Cu(II), .sup.67Cu(II), .sup.57Ni, .sup.52Fe and
.sup.18F (e.g., .sup.18F-fluorodeoxyglucose (FDG)). Examples of
useful radionuclide-labeled agents are .sup.64Cu-labeled engineered
antibody fragment (Wu et al. (2002) PNAS USA 97: 8495-8500),
.sup.64Cu-labeled somatostatin (Lewis et al. (1999) J. Med. Chem.
42: 1341-1347),
.sup.64Cu-pyruvaldehyde-bis(N4methylthiosemicarbazone)(64Cu-PTSM)
(Adonai et al. (2002) PNAS USA 99: 3030-3035), .sup.52Fe-citrate
(Leenders et al.(1994) J. Neural. Transm. Suppl. 43: 123-132),
.sup.52Fe/.sup.52mMn-citrate (Calonder et al.(1999) J. Neurochem.
73" 2047-2055) and .sup.52Fe-labeled iron (III) hydroxide-sucrose
complex (Beshara et al. (1999) Br. J. Haematol. 104: 288-295,
296-302).
[0258] In some examples dual imaging in vitro and/or in vivo can be
used to detect two or more detectable gene products, gene products
that produce a detectable signal, gene products that can bind a
detectable compound, or gene products that can bind other molecules
to form a detectable product. In some examples, the two or more
gene products are expressed by different viruses, whereas in other
examples the two or more gene products are produced by the same
virus. For example, a virus can express a gene product that emits a
detectable signal and also express a gene product that catalyzes a
detectable reaction. In other examples, a virus can express one or
more gene products that emit a detectable signal, one or more gene
products that catalyze a detectable reaction, one or more gene
products that can bind a detectable compound or that can form a
detectable product, or any combination thereof. Any combination of
such gene products can be expressed by the viruses provided herein
and can be used in combination with any of the methods provided
herein. Imaging of such gene products can be performed, for
example, by various imaging methods as described herein and known
in the art (e.g., fluorescence imaging, MRI, PET, among many other
methods of detection). Imaging of gene products can also be
performed using the same method, whereby gene products are
distinguished by their properties, such as by differences in
wavelengths of light emitted. For example, a virus can express more
than one fluorescent protein that differs in the wavelength of
light emitted (e.g., a GFP and an RFP). In another non-limiting
example, an RFP can be expressed with a luciferase. In yet other
non-limiting examples, a fluorescent gene product can be expressed
with a gene product, such as a ferritin or a transferrin receptor,
used for magnetic resonance imaging. A virus expressing two or more
detectable gene products or two or more viruses expressing two or
more detectable gene products can be imaged in vitro or in vivo
using such methods. In some embodiments the two or more gene
products are expressed as a single polypeptide, such as a fusion
protein. For example a fluorescent protein can be expressed as a
fusion protein with a luciferase protein.
[0259] b. Therapeutic Gene Product
[0260] Viruses provided herein can be modified to express one or
more genes whose products cause cell death or whose products cause
an anti-tumor immune response; such genes can be considered
therapeutic genes. A variety of therapeutic gene products, such as
toxic or apoptotic proteins, or siRNA, are known in the art, and
can be used with the viruses provided herein. The therapeutic genes
can act by directly killing the host cell, for example, as a
channel-forming or other lytic protein, or by triggering apoptosis,
or by inhibiting essential cellular processes, or by triggering an
immune response against the cell, or by interacting with a compound
that has a similar effect, for example, by converting a less active
compound to a cytotoxic compound. A large number of therapeutic
proteins that can be expressed for tumor treatment are known in the
art, including, but not limited to, a transporter, a cell-surface
receptor, a cytokine, a chemokine, an apoptotic protein, a mitosis
inhibitor protein, an antimitotic oligopeptide, an antiangiogenic
factor (e.g., hk5), anti-cancer antibodies, such as a single-chain
antibody (e.g., anti-VEGF), a toxin, a tumor antigen, a prodrug
converting enzyme, a ribozyme, RNAi, and siRNA. Costimulatory
molecules for the methods provided herein include any molecules
which are capable of enhancing immune responses to an
antigen/pathogen in vivo and/or in vitro. Costimulatory molecules
also encompass any molecules which promote the activation,
proliferation, differentiation, maturation or maintenance of
lymphocytes and/or other cells whose function is important or
essential for immune responses. An exemplary, non-limiting list of
therapeutic proteins includes IL-24, WT1, p53, pseudomonas A
endotoxin, diphtheria toxin, Arf, Bax, HSV TK, E. coli purine
nucleoside phosphorylase, angiostatin and endostatin, p16, Rb,
BRCA1, cystic fibrosis transmembrane regulator (CFTR), Factor VIII,
low density lipoprotein receptor, beta-galactosidase,
alpha-galactosidase, beta-glucocerebrosidase, insulin, parathyroid
hormone, alpha-1-antitrypsin, rsCD40L, Fas-ligand, TRAIL, TNF,
antibodies, microcin E492, diphtheria toxin, Pseudomonas exotoxin,
Escherichia coli Shiga toxin, Escherichia coli Verotoxin 1, and
hyperforin. Exemplary cytokines include, but are not limited to,
chemokines and classical cytokines, such as the interleukins,
including for example, interleukin-1, interleukin-2, interleukin-6
and interleukin-12, tumor necrosis factors, such as tumor necrosis
factor alpha (TNF-.alpha.), interferons such as interferon gamma
(IFN-.gamma.), granulocyte macrophage colony stimulating factor
(GM-CSF) and exemplary chemokines including, but not limited to CXC
chemokines such as IL-8 GRO.alpha., GRO.beta., GRO.gamma., ENA-78,
LDGF-PBP, GCP-2, PF4, Mig, IP-10, SDF-1.alpha./.beta.,
BUNZO/STRC33, I-TAC, BLC/BCA-1; CC chemokines such as MIP-1.alpha.,
MIP-1.beta., MDC, TECK, TARC, RANTES, HCC-1, HCC-4, DC-CK1,
MIP-3.alpha., MIP-3.beta., MCP-1, MCP-2, MCP-3, MCP-4, Eotaxin,
Eotaxin-2/MPIF-2, I-309, MIP-5/HCC-2, MPIF-1, 6Ckine, CTACK, MEC;
lymphotactin; and fractalkine. Exemplary other costimulatory
molecules include immunoglobulin superfamily of cytokines, such as
B7.1, B7.2. Other therapeutic proteins that can be expressed by the
viruses include but are not limited to an anti-VEGF single chain
antibody (e.g., SEQ ID NO: 106), a plasminogen K.sub.5 domain
(e.g., SEQ ID NO: 109), a human tissue
factor-.alpha.v.beta.3-integrin RGD fusion protein (e.g., SEQ ID
NO: 105), interleukin-24 (e.g., SEQ ID NO: 107) or an IL-6-IL-6
receptor fusion protein (e.g., SEQ ID NO: 108). Exemplary viruses
are provided herein that encode a transporter protein, such as
hNET, and a therapeutic protein, IL-24 (see description of
GLV-1h146 and GLV-1h150 provided in the Examples below).
[0261] In other embodiments, the viruses can express a protein that
converts a less active compound into a compound that causes tumor
cell death. Exemplary methods of conversion of such a prodrug
compound include enzymatic conversion and photolytic conversion. A
large variety of protein/compound pairs are known in the art, and
include, but are not limited to, Herpes simplex virus thymidine
kinase/ganciclovir, Herpes simplex virus thymidine
kinase/(E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), varicella
zoster thymidine kinase/ganciclovir, varicella zoster thymidine
kinase/BVDU, varicella zoster thymidine
kinase/(E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil
(BVaraU), cytosine deaminase/5-fluorouracil, cytosine
deaminase/5-fluorocytosine, purine nucleoside
phosphorylase/6-methylpurine deoxyriboside, beta
lactamase/cephalosporin-doxorubicin, carboxypeptidase
G2/4-[(2-chloroethyl)(2-mesuloxyethyl)amino]benzoyl-L-glutamic acid
(CMDA), carboxypeptidase A/methotrexate-phenylamine, cytochrome
P450/acetominophen, cytochrome P450-2B1/cyclophosphamide,
cytochrome P450-4B1/2-aminoanthracene, 4-ipomeanol, horseradish
peroxidase/indole-3-acetic acid, nitroreductase/CB1954, rabbit
carboxylesterase/7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxy-ca-
mptothecin (CPT-11), mushroom
tyrosinase/bis-(2-chloroethyl)amino-4-hydroxyphenylaminomethanone
28, beta
galactosidase/1-chloromethyl-5-hydroxy-1,2-dihydro-3H-benz[e]indole,
beta glucuronidase/epirubicin glucuronide, thymidine
phosphorylase/5'-deoxy-5-fluorouridine, deoxycytidine
kinase/cytosine arabinoside, and linamerase/linamarin.
[0262] In another embodiment, the therapeutic gene product can be
an siRNA molecule. The siRNA molecule can be directed against
expression of a tumor-promoting gene, such as, but not limited to,
an oncogene, growth factor, angiogenesis promoting gene, or a
receptor. The siRNA molecule also can be directed against
expression of any gene essential for cell growth, cell replication
or cell survival. The siRNA molecule also can be directed against
expression of any gene that stabilizes the cell membrane or
otherwise limits the number of tumor cell antigens released from
the tumor cell. Design of an siRNA can be readily determined
according to the selected target of the siRNA; methods of siRNA
design and down-regulation of genes are known in the art, as
exemplified in U.S. Pat. Pub. No. 2003-0198627.
[0263] In another embodiment, the therapeutic gene product can be a
viral attenuation factor. Antiviral proteins or peptides can be
expressed by the viruses provided herein. Expression of antiviral
proteins or peptides can control viral pathogenicity. Exemplary
viral attenuation factors include, but are not limited to,
virus-specific antibodies, mucins, thrombospondin, and soluble
proteins such as cytokines, including, but not limited to
TNF.alpha., interferons (for example IFN.alpha., IFN.beta., or
IFN.gamma.) and interleukins (for example IL-1, IL-12 or
IL-18).
[0264] In another embodiment, the therapeutic gene product can be a
protein ligand, such as antitumor oligopeptide. Antitumor
oligopeptides are short protein peptides with high affinity and
specificity to tumors. Such oligopeptides could be enriched and
identified using tumor-associated phage libraries (Akita et
al.(2006) Cancer Sci. 97(10):1075-1081). These oligopeptides have
been shown to enhance chemotherapy (U.S. Pat. No. 4,912,199). The
oligopeptides can be expressed by the viruses provided herein.
Expression of the oligopeptides can elicit anticancer activities on
their own or in combination with other chemotherapeutic agents. An
exemplary group of antitumor oligopeptides is antimitotic peptides,
including, but not limited to, tubulysin (Khalil et al. (2006)
Chembiochem. 7(4):678-683), phomopsin, hemiasterlin, taltobulin
(HTI-286, 3), and cryptophycin. Tubulysin is from myxobacteria and
can induce depletion of cell microtubules and trigger the apoptotic
process. The antimitotic peptides can be expressed by the viruses
provide herein and elicit anticancer activities on their own or in
combination with other therapeutic modalities.
[0265] In another embodiment, the therapeutic gene product can be a
protein that sequesters molecules or nutrients needed for tumor
growth. For example, the virus can express one or more proteins
that bind iron, transport iron, or store iron, or a combination
thereof. Increased iron uptake and/or storage by expression of such
proteins not only, increases contrast for visualization and
detection of a tumor or tissue in which the virus accumulates, but
also depletes iron from the tumor environment. Iron depletion from
the tumor environment removes a vital nutrient from the tumors,
thereby deregulating iron hemostasis in tumor cells and delaying
tumor progression and/or killing the tumor.
[0266] Additionally, iron, or other labeled metals, can be
administered to a tumor-bearing subject, either alone, or in a
conjugated form. An iron conjugate can include, for example, iron
conjugated to an imaging moiety or a therapeutic agent. In some
cases, the imaging moiety and therapeutic agent are the same, e.g.,
a radionuclide. Internalization of iron in the tumor, wound, area
of inflammation or infection allows the internalization of iron
alone, a supplemental imaging moiety, or a therapeutic agent (which
can deliver cytotoxicity specifically to tumor cells or deliver the
therapeutic agent for treatment of the wound, area of inflammation
or infection). These methods can be combined with any of the other
methods provided herein.
[0267] c. Superantigen
[0268] The viruses provided herein can be modified to express one
or more superantigens. Superantigens are antigens that can activate
a large immune response, often brought about by a large response of
T cells. A variety of superantigens are known in the art including,
but not limited to, diphtheria toxin, staphylococcal enterotoxins
(SEA, SEB, SEC1, SEC2, SED, SEE and SEH), Toxic Shock Syndrome
Toxin 1, Exfoliating Toxins (EXft), Streptococcal Pyrogenic
Exotoxin A, B and C (SPE A, B and C), Mouse Mammary Tumor Virus
proteins (MMTV), Streptococcal M proteins, Clostridial Perfringens
Enterotoxin (CPET), Listeria monocytogenes antigen p60, and
mycoplasma arthritis superantigens.
[0269] Since many superantigens also are toxins, if expression of a
virus of reduced toxicity is desired, the superantigen can be
modified to retain at least some of its superantigenicity while
reducing its toxicity, resulting in a compound such as a toxoid. A
variety of recombinant superantigens and toxoids of superantigens
are known in the art, and can readily be expressed in the viruses
provided herein. Exemplary toxoids include toxoids of diphtheria
toxin, as exemplified in U.S. Pat. No. 6,455,673 and toxoids of
Staphylococcal enterotoxins, as exemplified in U.S. Pat. Pub. No.
20030009015.
[0270] d. Gene Product to be Harvested
[0271] Exemplary genes expressible by a virus provided herein for
the purpose of harvesting include human genes. An exemplary list of
genes includes the list of human genes and genetic disorders
authored and edited by Dr. Victor A. McKusick and his colleagues at
Johns Hopkins University and elsewhere, and developed for the World
Wide Web by NCBI, the National Center for Biotechnology
Information. Online Mendelian Inheritance in Man, OMIM.TM., Center
for Medical Genetics, Johns Hopkins University (Baltimore, Md.) and
National Center for Biotechnology Information, National Library of
Medicine (Bethesda, Md.), and those available in public databases,
such as PubMed and GenBank (see, for example, genes provided in the
website ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM).
[0272] e. Control of Heterologous Gene Expression
[0273] In one embodiment, expression the therapeutic compound can
be controlled by a regulatory sequence. Suitable regulatory
sequences which, for example, are functional in a mammalian host
cell are well known in the art. In one example, the regulatory
sequence contains a poxvirus promoter. In another embodiment, the
regulatory sequence can contain a natural or synthetic vaccinia
virus promoter. Strong late promoters can be used to achieve high
levels of expression of the foreign genes. Early and
intermediate-stage promoters can also be used. In one embodiment,
the promoters contain early and late promoter elements, for
example, the vaccinia virus early/late promoter P7.5k, vaccinia
late promoter P11k, a synthetic early/late vaccinia P.sub.SEL
promoter (Patel et al., (1988) Proc. Natl. Acad. Sci. USA 85:
9431-9435; Davison and Moss, (1989) J Mol Biol 210: 749-769;
Davison et al. (1990) Nucleic Acids Res. 18: 4285-4286; Chakrabarti
et al. (1997), BioTechniques 23: 1094-1097). As described in the
Examples and elsewhere herein, the viruses provided can exhibit
differences in characteristics, such as attenuation, as a result of
using a stronger promoter versus a weaker promoter. For example, in
vaccinia, synthetic early/late and late promoters are relatively
strong promoters, whereas vaccinia synthetic early, P7.5k
early/late, P7.5k early, and P28 late promoters are relatively
weaker promoters (see e.g., Chakrabarti et al. (1997) BioTechniques
23(6) 1094-1097). Combinations of different promoters can be used
to express different gene products in the same virus or two
different viruses. In one embodiment, different therapeutic or
detectable gene products are expressed from different promoters,
such as two different vaccinia synthetic promoters.
F. METHODS FOR MAKING A MODIFIED VIRUS
[0274] The viruses provided herein can be formed by standard
methodologies well known in the art for modifying viruses. Briefly,
the methods include introducing into viruses one or more genetic
modifications, followed by screening the viruses for properties
reflective of the modification or for other desired properties.
[0275] 1. Genetic Modifications
[0276] Standard techniques in molecular biology can be used to
generate the modified viruses provided herein. Such techniques
include various nucleic acid manipulation techniques, nucleic acid
transfer protocols, nucleic acid amplification protocols, and other
molecular biology techniques known in the art. For example, point
mutations can be introduced into a gene of interest through the use
of oligonucleotide mediated site-directed mutagenesis.
Alternatively, homologous recombination can be used to introduce a
mutation or exogenous sequence into a target sequence of interest.
In an alternative mutagenesis protocol, point mutations in a
particular gene can also be selected for using a positive selection
pressure. See, e.g., Current Techniques in Molecular Biology, (Ed.
Ausubel, et al.). Nucleic acid amplification protocols include but
are not limited to the polymerase chain reaction (PCR). Use of
nucleic acid tools such as plasmids, vectors, promoters and other
regulating sequences, are well known in the art for a large variety
of viruses and cellular organisms. Nucleic acid transfer protocols
include calcium chloride transformation/transfection,
electroporation, liposome mediated nucleic acid transfer,
N-[1-(2,3-Dioloyloxy)propyl]-N,N,N-trimethylammonium methylsulfate
meditated transformation, and others. Further a large variety of
nucleic acid tools are available from many different sources
including ATCC, and various commercial sources. One skilled in the
art will be readily able to select the appropriate tools and
methods for genetic modifications of any particular virus according
to the knowledge in the art and design choice.
[0277] Any of a variety of modifications can be readily
accomplished using standard molecular biological methods known in
the art. The modifications will typically be one or more
truncations, deletions, mutations or insertions of the viral
genome. In one embodiment, the modification can be specifically
directed to a particular sequence. The modifications can be
directed to any of a variety of regions of the viral genome,
including, but not limited to, a regulatory sequence, to a
gene-encoding sequence, or to a sequence without a known role. Any
of a variety of regions of viral genomes that are available for
modification are readily known in the art for many viruses,
including the viruses specifically listed herein. As a non-limiting
example, the loci of a variety of vaccinia genes provided herein
and elsewhere exemplify the number of different regions that can be
targeted for modification in the viruses provided herein. In
another embodiment, the modification can be fully or partially
random, whereupon selection of any particular modified virus can be
determined according to the desired properties of the modified the
virus. These methods include, for example, in vitro recombination
techniques, synthetic methods and in vivo recombination methods as
described, for example, in Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory
Press, cold Spring Harbor N.Y. (1989), and in the Examples
disclosed herein.
[0278] In some embodiments, the virus can be modified to express an
exogenous gene. Exemplary exogenous gene products include proteins
and RNA molecules. The modified viruses can express a detectable
gene product, a therapeutic gene product, a gene product for
manufacturing or harvesting, or an antigenic gene product for
antibody harvesting. The characteristics of such gene products are
described herein and elsewhere. In some embodiments of modifying an
organism to express an exogenous gene, the modification can also
contain one or more regulatory sequences to regulate expression of
the exogenous gene. As is known in the art, regulatory sequences
can permit constitutive expression of the exogenous gene or can
permit inducible expression of the exogenous gene. Further, the
regulatory sequence can permit control of the level of expression
of the exogenous gene. In some examples, inducible expression can
be under the control of cellular or other factors present in a
tumor cell or present in a virus-infected tumor cell. In other
examples, inducible expression can be under the control of an
administrable substance, including IPTG, RU486 or other known
induction compounds. Any of a variety of regulatory sequences are
available to one skilled in the art according to known factors and
design preferences. In some embodiments, such as gene product
manufacture and harvesting, the regulatory sequence can result in
constitutive, high levels of gene expression. In some embodiments,
such as anti-(gene product) antibody harvesting, the regulatory
sequence can result in constitutive, lower levels of gene
expression. In tumor therapy embodiments, a therapeutic protein can
be under the control of an internally inducible promoter or an
externally inducible promoter.
[0279] In other embodiments, organ or tissue-specific expression
can be controlled by regulatory sequences. In order to achieve
expression only in the target organ, for example, a tumor to be
treated, the foreign nucleotide sequence can be linked to a tissue
specific promoter and used for gene therapy. Such promoters are
well known to those skilled in the art (see e.g., Zimmermann et
al., Neuron 12: 11-24 (1994); Vidal et al., EMBO J. 9: 833-840
(1990); Mayford et al., Cell 81: 891-904 (1995); and Pinkert et
al., Genes & Dev. 1: 268-76 (1987)).
[0280] In some embodiments, the viruses can be modified to express
two or more proteins, where any combination of the two or more
proteins can be one or more detectable gene products, therapeutic
gene products, gene products for manufacturing or harvesting or
antigenic gene products for antibody harvesting. In one embodiment,
a virus can be modified to express a detectable protein and a
therapeutic protein. In another embodiment, a virus can be modified
to express two or more gene products for detection or two or more
therapeutic gene products. For example, one or more proteins
involved in biosynthesis of a luciferase substrate can be expressed
along with luciferase. When two or more exogenous genes are
introduced, the genes can be regulated under the same or different
regulatory sequences, and the genes can be inserted in the same or
different regions of the viral genome, in a single or a plurality
of genetic manipulation steps. In some embodiments, one gene, such
as a gene encoding a detectable gene product, can be under the
control of a constitutive promoter, while a second gene, such as a
gene encoding a therapeutic gene product, can be under the control
of an inducible promoter. Methods for inserting two or more genes
in to a virus are known in the art and can be readily performed for
a wide variety of viruses using a wide variety of exogenous genes,
regulatory sequences, and/or other nucleic acid sequences.
[0281] Methods of producing recombinant viruses are known in the
art. Provided herein for exemplary purposes are methods of
producing a recombinant vaccinia virus. A recombinant vaccinia
virus with an insertion in the F14.5L gene (NotI site of LIVP) can
be prepared by the following steps: (a) generating (i) a vaccinia
shuttle plasmid containing the modified F14.5L gene inserted at
restriction site X and (ii) a dephosphorylated wt VV (VGL) DNA
digested at restriction site X; (b) transfecting host cells
infected with PUV-inactivated helper VV (VGL) with a mixture of the
constructs of (i) and (ii) of step a; and (c) isolating the
recombinant vaccinia viruses from the transfectants. One skilled in
the art knows how to perform such methods, for example by following
the instructions given in co-pending U.S. application Ser. Nos.
10/872,156 and 11/238,025; see also Timiryasova et al.
(Biotechniques 31: 534-540 (2001)). In one embodiment, restriction
site X is a unique restriction site. A variety of suitable host
cells also are known to the person skilled in the art and include
many mammalian, avian and insect cells and tissues which are
susceptible for vaccinia virus infection, including chicken embryo,
rabbit, hamster and monkey kidney cells, for example, HeLa cells,
RK.sub.13, CV-1, Vero, BSC40 and BSC-1 monkey kidney cells.
[0282] 2. Screening of Modified Viruses
[0283] Modified viruses can be screened for any desired
characteristics, including the characteristics described herein
such as attenuated pathogenicity, reduced toxicity, preferential
accumulation in tumor, increased ability to activate an immune
response against tumor cells, increased immunogenicity, increased
or decreased replication competence, and are able to express
exogenous proteins, and combinations thereof. For example, the
modified viruses can be screened for the ability to activate an
immune response against tumor cells without aggressively killing
the tumor cells. In another example, the viruses can be screened
for expression of one or more detectable genes, including genes
that can be used for imaging, or for expression of one or more
genes for manufacture or harvest of the gene products and/or for
harvest of antibodies against the gene products.
[0284] Any of a variety of known methods for screening for such
characteristics can be performed, as demonstrated in the Examples
provided herein. One exemplary method for screening for desired
characteristics includes, but is not limited to, monitoring growth,
replication and/or gene expression (including expression of an
exogenous gene) in cell culture or other in vitro medium. The cell
culture can be from any organism, and from any tissue source, and
can include tumorous tissues. Other exemplary methods for screening
for desired characteristics include, but are not limited to,
administering a virus to animal, including non-human animals such
as a mouse, monkey or ape, and optionally also including humans,
and monitoring the virus, the tumor, and or the animal; monitoring
can be performed by in vivo imaging of the virus and/or the tumor
(e.g., low light imaging of viral gene expression or ultrasonic
tumor imaging), external monitoring of the tumor (e.g., external
measurement of tumor size), monitoring the animal (e.g., monitoring
animal weight, blood panel, antibody titer, spleen size, or liver
size). Other exemplary methods for screening for desired
characteristics include, but are not limited to, harvesting a
non-human animal for the effects and location of the virus and
expression by the virus, including methods such as harvesting a
variety of organs including a tumor to determine presence of the
virus and/or gene expression by the virus in the organs or tumor,
harvesting of organs associated with an immune response or viral
clearance such as the spleen or liver, harvesting the tumor to
determine tumor size and viability of tumor cells, harvesting
antibodies or antibody producing cells. Such screening and
monitoring methods can be used in any of a variety of combinations,
as is known in art. In one embodiment, a virus can be screened by
administering the virus to an animal such as a non-human animal or
a human, followed by monitoring by in vivo imaging. In another
embodiment, a virus can be screened by administering the virus to
an animal such as a non-human animal, monitoring by in vivo
imaging, and then harvesting the animal. Thus, provided herein are
methods for screening a virus for desired characteristics by
administering the virus to an animal such as an animal with a
tumor, and monitoring the animal, tumor (if present), and/or virus
in the animal for one or more characteristics. Also provided herein
are methods for screening a virus for desired characteristics by
administering the virus to a non-human animal such as a non-human
animal with a tumor, harvesting the animal, and assaying the
animal's organs, antibody titer, and/or tumor (if present) for one
or more characteristics.
[0285] Provided herein are methods for screening a virus for
attenuated pathogenicity or reduced toxicity, where the
pathogenicity or toxicity can be determined by a variety of
techniques, including, but not limited to, assessing the health
state of the subject, measuring the body weight of a subject, blood
or urine analysis of a subject, and monitoring tissue distribution
of the virus within the subject; such techniques can be performed
on a living subject in vivo, or can be performed post mortem.
Methods also can include the ability of the viruses to lyse cells
or cause cell death, which can be determined in vivo or in
vitro.
[0286] When a subject drops below a threshold body weight, the
virus can be considered pathogenic to the subject. Exemplary
thresholds can be a drop of about 5% or more, a drop of about 10%
or more, or a drop of about 15% or more in body weight relative to
a reference. A body weight reference can be selected from any of a
variety of references used in the art; for example, a body weight
reference can be the weight of the subject prior to administration
of the virus, the body weight reference can be a control subject
having the same condition as the test subject (e.g., normal or
tumor-injected), where the change in weight of the control is
compared to the change in weight of the test subject for the time
period after administration of the virus.
[0287] Blood or urine analysis of the subject can indicate level of
immune response, level of toxins in the subject, or other levels of
stress to cells, tissues or organs of the subject such as kidneys,
pancreas, liver and spleen. Levels increased above established
threshold levels can indicate pathogenicity of the virus to the
subject. Threshold levels of components of blood or urine for
indicating viral pathogenicity are well known in the art, and any
such thresholds can be selected herein according to the desired
tolerance of pathogenicity or toxicity of the virus.
[0288] Tissue distribution of a virus in a subject can indicate
pathogenicity or toxicity of the virus. In one embodiment, tissue
distribution of a virus that is not pathogenic or toxic can be
mostly in tumor relative to other tissues or organs. Microorganisms
located mostly in tumor can accumulate, for example, at least about
2-fold greater, at least about 5-fold greater, at least about
10-fold greater, at least about 100-fold greater, at least about
1,000-fold greater, at least about 10,000-fold greater, at least
about 100,000-fold greater, or at least about 1,000,000-fold
greater, than the viruses accumulate in any other particular organ
or tissue.
[0289] Provided herein are methods for screening a virus for tissue
distribution or accumulation, where the tissue distribution can be
determined by a variety of techniques, including, but not limited
to, harvesting a non-human subject, in vivo imaging a detectable
gene product in subject. Harvesting can be accomplished by
euthanizing the non-human subject, and determining the accumulation
of viruses in tumor and, optionally, the accumulation in one or
more additional tissues or organs. The accumulation can be
determined by any of a variety of methods, including, but not
limited to, detecting gene products such as detectable gene
products (e.g., GFP or beta galactosidase), histological or
microscopic evaluation of tissue, organ or tumor samples, or
measuring the number of plaque or colony forming units present in a
tissue, organ or tumor sample. In one embodiment, the desired
amount of tissue distribution of a virus can be mostly in tumor
relative to other tissues or organs. Microorganisms located mostly
in tumor can accumulate, for example, at least about 2-fold
greater, at least about 5-fold greater, at least about 10-fold
greater, at least about 100-fold greater, at least about 1,000-fold
greater, at least about 10,000-fold greater, at least about
100,000-fold greater, or at least about 1,000,000-fold greater,
than the viruses accumulate in any other particular organ or
tissue.
[0290] Also provided herein are methods of screening for viruses
that can elicit an immune response, where the immune response can
be against the tumor cells or against the viruses. A variety of
methods for measuring the ability to elicit an immune response are
known in the art, and include measuring an overall increase in
immune activity in a subject, measuring an increase in anti-virus
or anti-tumor antibodies in a subject, testing the ability of a
virus-treated (typically a non-human) subject to prevent later
infection/tumor formation or to rapidly eliminate viruses or tumor
cells. Methods also can include the ability of the viruses to lyse
cells or cause cell death, which can be determined in vivo or in
vitro.
[0291] Also provided herein are methods for determining increased
or decreased replication competence, by monitoring the speed of
replication of the viruses. Such measurements can be performed in
vivo or in vitro. For example, the speed of replication in a cell
culture can be used to determine replication competence of a virus.
In another example, the speed of replication in a tissue, organ or
tumor in a subject can be used to measure replication competence.
In some embodiments, decreased replication competence in non-tumor
tissues and organs can be the characteristic to be selected in a
screen. In other embodiments, increased replication competence in
tumors can be the characteristic to be selected in a screen.
[0292] Also provided herein are methods for determining the ability
of a virus to express genes, such as exogenous gene. Such methods
can be performed in vivo or in vitro. For example, the viruses can
be screened on selective plates for the ability to express a gene
that permits survival of the virus or permits the virus to provide
a detectable signal, such as turning X-gal blue. Such methods also
can be performed in vivo, where expression can be determined, for
example, by harvesting tissues, organs or tumors a non-human
subject or by in vivo imaging of a subject.
[0293] Also provided herein are methods for determining the ability
of a virus to express genes toward which the subject can develop
antibodies, including exogenous genes toward which the subject can
develop antibodies. Such methods can be performed in vivo using any
of a variety of non-human subjects. For example, gene expression
can be determined, for example, by bleeding a non-human subject to
which a virus has been administered, and assaying the blood (or
serum) for the presence of antibodies against the virus-expressed
gene, or by any other method generally used for polyclonal antibody
harvesting, such as production bleeds and terminal bleeds.
[0294] Also provided herein are methods for screening a virus that
has two or more characteristics provided herein, including
screening for attenuated pathogenicity, reduced toxicity,
preferential accumulation in tumor, increased ability to activate
an immune response against tumor cells, increased immunogenicity,
increased or decreased replication competence, ability to express
exogenous proteins, and ability to elicit antibody production
against a virally expressed gene product. A single monitoring
technique, such as in vivo imaging, can be used to verify two or
more characteristics, or a variety of different monitoring
techniques can be used, as can be determined by one skilled in the
art according to the selected characteristics and according to the
monitoring techniques used.
[0295] Mouse models of different types of human and non-human
animal cancers can be employed to assess the properties of the
modified viruses. Tumors can be established by implantation of
different tumor cell types. Exemplary human tumor xenograft models
in mice include, but are not limited to, human lung carcinoma (A549
cells, ATCC No. CCL-185); human breast tumor (GI-101A cells,
Rathinavelu et al., Cancer Biochem. Biophys., 17:133-146 (1999));
human ovarian carcinoma (OVCAR-3 cells, ATCC No. HTB-161); human
pancreatic carcinoma (PANC-1cells, ATCC No. CRL-1469 and MIA PaCa-2
cells, ATCC No. CRL-1420); DU145 cells (human prostate cancer
cells, ATCC No. HTB-81); human prostate cancer (PC-3 cells, ATCC#
CRL-1435); colon carcinoma (HT-29 cells); human melanoma (888-MEL
cells, 1858-MEL cells or 1936-MEL cells; see e.g. Wang et al.,
(2006) J. Invest. Dermatol. 126:1372-1377); and human fibrosarcoma
(HT-1080 cells, ATCC No. CCL-121,). Exemplary rat tumor xenograft
models in mice include, but are not limited to, glioma tumor (C6
cells; ATCC No. CCL-107). Exemplary mouse tumor homograft models
include, but are not limited to, mouse melanoma (B16-F10 cells;
ATCC No. CRL-6475). Exemplary cat tumor xenograft models in mice
include, but are not limited to, feline fibrosarcoma (FC77.T cells;
ATCC No. CRL-6105). Exemplary dog tumor xenograft models in mice
include, but are not limited to, canine osteosarcoma (D17 cells;
ATCC No. CCL-183).
G. EXEMPLARY CHARACTERISTICS OF THE VIRUSES PROVIDED
[0296] The viruses provided herein can accumulate in
immunoprivileged cells or immunoprivileged tissues, including
tumors and/or metastases, and also including wounded tissues and
cells. While the viruses provided herein can typically be cleared
from the subject to whom the viruses are administered by activity
of the subject's immune system, viruses can nevertheless
accumulate, survive and proliferate in immunoprivileged cells and
tissues such as tumors because such immunoprivileged areas are
sequestered from the host's immune system. Accordingly, the methods
provided herein, as applied to tumors and/or metastases, and
therapeutic methods relating thereto, can readily be applied to
other immunoprivileged cells and tissues, including wounded cells
and tissues.
[0297] 1. Attenuated
[0298] The viruses provided herein and viruses provided for use in
the methods are typically attenuated. Attenuated viruses have a
decreased capacity to cause disease in a host. The decreased
capacity can result from any of a variety of different
modifications to the ability of a virus to be pathogenic. For
example, a virus can have reduced toxicity, reduced ability to
accumulate in non-tumorous organs or tissue, reduced ability to
cause cell lysis or cell death, or reduced ability to replicate
compared to the non-attenuated form thereof. The attenuated viruses
provided herein, however, retain at least some capacity to
replicate and to cause immunoprivileged cells and tissues, such as
tumor cells to leak or lyse, undergo cell death, or otherwise cause
or enhance an immune response to immunoprivileged cells and
tissues, such as tumor cells.
[0299] a. Reduced Toxicity
[0300] Viruses can be toxic to their hosts by manufacturing one or
more compounds that worsen the health condition of the host.
Toxicity to the host can be manifested in any of a variety of
manners, including septic shock, neurological effects or muscular
effects. The viruses provided herein can have a reduced toxicity to
the host. The reduced toxicity of a virus of the present methods
and compositions can range from a toxicity in which the host
experiences no toxic effects, to a toxicity in which the host does
not typically die from the toxic effects of the microbes. In some
embodiments, the viruses are of a reduced toxicity such that a host
typically has no significant long-term effect from the presence of
the viruses in the host, beyond any effect on tumorous, metastatic
or necrotic organs or tissues. For example, the reduced toxicity
can be a minor fever or minor infection, which lasts for less than
about a month, and following the fever or infection, the host
experiences no adverse effects resultant from the fever or
infection. In another example, the reduced toxicity can be measured
as an unintentional decline in body weight of about 5% or less for
the host after administration of the microbes. In other examples,
the virus has no toxicity to the host.
[0301] b. Accumulate in Tumor, not Substantially in Other
Organs
[0302] Viruses can accumulate in any of a variety of tissues and
organs of the host. Accumulation can be evenly distributed over the
entire host organism, or can be concentrated in one or a few organs
or tissues. The viruses provided herein can accumulate in targeted
tissues, such as immunoprivileged cells and tissues, such as tumors
and also metastases. In some embodiments, the viruses provided
herein exhibit accumulation in immunoprivileged cells and tissues,
such as tumor cells relative to normal organs or tissues that is
equal to or greater than the accumulation that occurs with
wild-type viruses. In other embodiments, the viruses provided
herein exhibit accumulation in immunoprivileged cells and tissues,
such as tumor cells that is equal to or greater than the
accumulation in any other particular organ or tissue. For example,
the viruses provided herein can demonstrate an accumulation in
immunoprivileged cells and tissues, such as tumor cells that is at
least about 2-fold greater, at least about 5-fold greater, at least
about 10-fold greater, at least about 100-fold greater, at least
about 1,000-fold greater, at least about 10,000-fold greater, at
least about 100,000-fold greater, or at least about 1,000,000-fold
greater, than the accumulation in any other particular organ or
tissue.
[0303] In some embodiments, a virus can accumulate in targeted
tissues and cells, such as immunoprivileged cells and tissues, such
as tumor cells, without accumulating in one or more selected
tissues or organs. For example, a virus can accumulate in tumor
cells without accumulating in the brain. In another example, a
virus can accumulate in tumor cells without accumulating in neural
cells. In another example, a virus can accumulate in tumor cells
without accumulating in ovaries. In another example, a virus can
accumulate in tumor cells without accumulating in the blood. In
another example, a virus can accumulate in tumor cells without
accumulating in the heart. In another example, a virus can
accumulate in tumor cells without accumulating in the bladder. In
another example, a virus can accumulate in tumor cells without
accumulating in testes. In another example, a virus can accumulate
in tumor cells without accumulating in the spleen. In another
example, a virus can accumulate in tumor cells without accumulating
in the lungs.
[0304] One skilled in the art can determine the desired capability
for the viruses to selectively accumulate in targeted tissue or
cells, such as in an immunoprivileged cells and tissues, such as
tumor rather than non-target organs or tissues, according to a
variety of factors known in the art, including, but not limited to,
toxicity of the viruses, dosage, tumor to be treated,
immunocompetence of host, and disease state of the host.
[0305] c. Ability to Elicit or Enhance Immune Response to Tumor
Cells
[0306] Viruses herein can cause or enhance an immune response to
antigens in the targeted tissues or cells, such as immunoprivileged
cells and tissues, such as tumor cells. The immune response can be
triggered by any of a variety of mechanisms, including the presence
or expression of immunostimulatory cytokines and the expression or
release antigenic compounds that can cause an immune response.
[0307] Cells, in response to an infection such as a viral
infection, can send out signals to stimulate an immune response
against the cells. Exemplary signals sent from such cells include
antigens, cytokines and chemokines such as interferon-gamma and
interleukin-15. The viruses provided herein can cause targeted
cells to send out such signals in response to infection by the
microbes, resulting in a stimulation of the host's immune system
against the targeted cells or tissues, such as tumor cells.
[0308] In another embodiment, targeted cells or tissues, such as
tumor cells, can contain one or more compounds that can be
recognized by the host's immune system in mounting an immune
response against a tumor. Such antigenic compounds can be compounds
on the cell surface or the tumor cell, and can be protein,
carbohydrate, lipid, nucleic acid or combinations thereof.
Viral-mediated release of antigenic compounds can result in
triggering the host's immune system to mount an immune response
against the tumor. The amount of antigenic compound released by the
tumor cells is any amount sufficient to trigger an immune response
in a subject; for example, the antigenic compounds released from
one or more tumor cells can trigger a host immune response in the
organism that is known to be accessible to leukocytes.
[0309] The time duration of antigen release is an amount of time
sufficient for the host to establish an immune response to one or
more tumor antigens. In some embodiments, the duration is an amount
of time sufficient for the host to establish a sustained immune
response to one or more tumor antigens. One skilled in the art can
determine such a time duration based on a variety of factors
affecting the time duration for a subject to develop an immune
response, including the level of the tumor antigen in the subject,
the number of different tumor antigens, the antigenicity of the
antigen, the immunocompetence of the host, and the access of the
antigenic material to the vasculature of the host. Typically, the
duration of antigen release can be at least about a week, at least
about 10 days, at least about two weeks, or at least about a
month.
[0310] The viruses provided herein can have any of a variety of
properties that can cause target cells and tissues, such as tumor
cells, to release antigenic compounds. Exemplary properties are the
ability to lyse cells and the ability to elicit apoptosis in tumor
cells. Viruses that are unable to lyse tumor cells or cause tumor
cell death can nevertheless be used in the methods provided herein
when such viruses can cause some release or display of antigenic
compounds from tumor cells. A variety of mechanisms for antigen
release or display without lysis or cell death are known in the
art, and any such mechanism can be used by the viruses provided
herein, including, but not limited to, secretion of antigenic
compounds, enhanced cell membrane permeability, expression of
immunostimulatory proteins or altered cell surface expression or
altered MHC presentation in tumor cells when the tumor cells can be
accessed by the host's immune system. Regardless of the mechanism
by which the host's immune system is activated, the net result of
the presence of the viruses in the tumor is a stimulation of the
host's immune system, at least in part, against the tumor cells. In
one example, the viruses can cause an immune response against tumor
cells not infected by the viruses.
[0311] In one embodiment, the viruses provided herein can cause
tumor cells to release an antigen that is not present on the tumor
cell surface. Tumor cells can produce compounds such as proteins
that can cause an immune response; however, in circumstances in
which the antigenic compound is not on the tumor cell surface, the
tumor can proliferate, and even metastasize, without the antigenic
compound causing an immune response. Within the scope of the
present methods, the viruses provided herein can cause antigenic
compounds within the cell to release away from the cell and away
from the tumor, which can result in triggering an immune response
to such an antigen. Even if not all cells of a tumor are releasing
antigens, the immune response can initially be targeted toward the
"leaky" tumor cells, and the bystander effect of the immune
response can result in further tumor cell death around the "leaky"
tumor cells.
[0312] d. Balance of Pathogenicity and Release of Tumor
Antigens
[0313] Typical methods of involving treatment of targeted cells and
tissues, such as immunoprivileged cells and tissues, such as
tumors, are designed to cause rapid and complete removal thereof.
For example, many viruses can cause lysis and/or apoptosis in a
variety of cells, including tumor cells. Viruses that can
vigorously lyse or cause cell death can be highly pathogenic, and
can even kill the host. Furthermore, therapeutic methods based upon
such rapid and complete lysis are typically therapeutically
ineffective.
[0314] In contrast, the viruses provided herein are not aggressive
in causing cell death or lysis. They can have a limited or no
ability to cause cell death as long as they accumulate in the
target cells or tissues and result in alteration of cell membranes
to cause leakage of antigens against which an immune response is
mounted. It is desirable that their apoptotic or lytic effect is
sufficiently slow or ineffective to permit sufficient antigenic
leakage for a sufficient time for the host to mount an effective
immune response against the target tissues. Such immune response
alone or in combination with the lytic/apoptotic effect of the
virus results in elimination of the target tissue and also
elimination of future development, such as metastases and
reoccurrence, of such tissues or cells. While the viruses provided
herein can have a limited ability to cause cell death, the viruses
provided herein can nevertheless stimulate the host's immune system
to attack tumor cells. As a result, such viruses also are typically
unlikely to have substantial toxicity to the host.
[0315] In one embodiment, the viruses have a limited, or no ability
to cause tumor cell death, while still causing or enhancing an
immune response against tumor cells. In one example, the rate of
viral-mediated tumor cell death is less than the rate of tumor cell
growth or replication. In another example, the rate of
viral-mediated tumor cell death is slow enough for the host to
establish a sustained immune response to one or more tumor
antigens. Typically, the time for cell death is sufficient to
establish an anti-tumor immune response and can be at least about a
week, at least about 10 days, at least about two weeks, or at least
about a month, depending upon the host and the targeted cells or
tissues.
[0316] In another embodiment, the viruses provided herein can cause
cell death in tumor cells, without causing substantial cell death
in non-tumor tissues. In such an embodiment, the viruses can
aggressively kill tumor cells, as long as no substantial cell death
occurs in non-tumor cells, and optionally, so long as the host has
sufficient capability to mount an immune response against the tumor
cells.
[0317] In one embodiment, the ability of the viruses to cause cell
death is slower than the host's immune response against the
viruses. The ability for the host to control infection by the
viruses can be determined by the immune response (e.g., antibody
titer) against viral antigens. Typically, after the host has
mounted immune response against the viruses, the viruses can have
reduced pathogenicity in the host. Thus, when the ability of the
viruses to cause cell death is slower than the host's immune
response against the microbes, viral-mediated cell death can occur
without risk of serious disease or death to the host. In one
example, the ability of the viruses to cause tumor cell death is
slower than the host's immune response against the microbes.
[0318] 2. Immunogenicity
[0319] The viruses provided herein also can be immunogenic. An
immunogenic virus can create a host immune response against the
virus. In one embodiment, the viruses can be sufficiently
immunogenic to result in a large anti-viral antibody titer. The
viruses provided herein can have the ability to elicit an immune
response. The immune response can be activated in response to viral
antigens or can be activated as a result of viral-infection induced
cytokine or chemokine production. Immune response against the
viruses can decrease the likelihood of pathogenicity toward the
host organism.
[0320] Immune response against the viruses also can result in
target tissue or cell, such as tumor cell, killing. In one
embodiment, the immune response against viral infection can result
in an immune response against tumor cells, including developing
antibodies against tumor antigens. In one example, an immune
response mounted against the virus can result in tumor cell killing
by the "bystander effect," where uninfected tumor cells nearby
infected tumor cells are killed at the same time as infected cells,
or alternatively, where uninfected tumor cells nearby extracellular
viruses are killed at the same time as the viruses. As a result of
bystander effect tumor cell death, tumor cell antigens can be
released from cells, and the host organism's immune system can
mount an immune response against tumor cell antigens, resulting in
an immune response against the tumor itself.
[0321] In one embodiment, the virus can be selected or modified to
express one or more antigenic compounds, including superantigenic
compounds. The antigenic compounds such as superantigens can be
endogenous gene products or can be exogenous gene products.
Superantigens, including toxoids, are known in the art and
described elsewhere herein.
[0322] 3. Replication Competent
[0323] The viruses provided herein can be replication competent. In
a variety of viral systems, the administered virus is rendered
replication incompetent to limit pathogenicity risk to the host.
While replication incompetence can protect the host from the virus,
it also limits the ability of the virus to infect and kill tumor
cells, and typically results in only a short-lived effect. In
contrast, the viruses provided herein can be attenuated but
replication competent, resulting in low toxicity to the host and
accumulation mainly or solely in tumors. Thus, the viruses provided
herein can be replication competent without creating a
pathogenicity risk to the host.
[0324] Attenuation of the viruses provided herein can include, but
is not limited to, reducing the replication competence of the
virus. For example, a virus can be modified to decrease or
eliminate an activity related to replication, such as a
transcriptional activator that regulates replication in the virus.
In an example, a virus, can have the viral thymidine kinase (TK)
gene modified, which decreases replication of the virus.
[0325] 4. Genetic Variants
[0326] The viruses provided herein can be modified from their wild
type form. Modifications can include any of a variety of changes,
and typically include changes to the genome or nucleic acid
molecules of the viruses. Exemplary nucleic acid molecular
modifications include truncations, insertions, deletions and
mutations. In an exemplary modification, a viral gene can be
modified by truncation, insertion, deletion or mutation. In an
exemplary insertion, an exogenous gene can be inserted into the
genome of the virus.
[0327] Modifications of the viruses provided herein can result in a
modification of viral characteristics, including those provided
herein such as pathogenicity, toxicity, ability to preferentially
accumulate in tumor, ability to lyse cells or cause cell death,
ability to elicit an immune response against tumor cells,
immunogenicity and replication competence. Variants can be obtained
by general methods such as mutagenesis and passage in cell or
tissue culture and selection of desired properties, as is known in
the art, as exemplified for respiratory syncytial virus in Murphy
et al., Virus Res. 1994, 32:13-26.
[0328] Variants also can be obtained by mutagenic methods in which
nucleic acid residues of the virus are added, removed or modified
relative to the wild type. Any of a variety of known mutagenic
methods can be used, including recombination-based methods,
restriction endonuclease-based methods, and PCR-based methods.
Mutagenic methods can be directed against particular nucleotide
sequences such as genes, or can be random, where selection methods
based on desired characteristics can be used to select mutated
viruses. Any of a variety of viral modifications can be made,
according to the selected virus and the particular known
modifications of the selected virus.
H. PHARMACEUTICAL COMPOSITIONS, COMBINATIONS AND KITS
[0329] Provided herein are pharmaceutical compositions,
combinations and kits containing a virus that encodes a transporter
protein provided herein and one or more components. Pharmaceutical
compositions can include a virus provided herein and a
pharmaceutical carrier. Combinations can include two or more
viruses, a virus and a detectable substrate that is transported
into cells that express the transporter encoded by the virus, a
virus and a detectable compound, a virus and a viral expression
modulating compound, a virus and a therapeutic compound, or any
combination thereof. Kits can include the pharmaceutical
compositions and/or combinations provided herein, and one or more
components, such as instructions for use, a device for detecting a
virus in a subject, a device for administering a compound to a
subject and a device for administering a compound to a subject.
[0330] 1. Pharmaceutical Compositions
[0331] Provided herein are pharmaceutical compositions containing a
virus provided herein and a suitable pharmaceutical carrier.
Pharmaceutical compositions provided herein can be in various
forms, e.g., in solid, liquid, powder, aqueous, or lyophilized
form. Examples of suitable pharmaceutical carriers are known in the
art and include but are not limited to water, buffers, saline
solutions, phosphate buffered saline solutions, various types of
wetting agents, sterile solutions, alcohols, gum arabic, vegetable
oils, benzyl alcohols, gelatin, glycerin, carbohydrates such as
lactose, sucrose, amylose or starch, magnesium stearate, talc,
silicic acid, viscous paraffin, perfume oil, fatty acid
monoglycerides and diglycerides, pentaerythritol fatty acid esters,
hydroxy methylcellulose, powders, among others. Pharmaceutical
compositions provided herein can contain other additives including,
for example, antioxidants and preservatives, analgesic agents,
binders, disintegrants, coloring, diluents, excipients, extenders,
glidants, solubilizers, stabilizers, tonicity agents, vehicles,
viscosity agents, flavoring agents, emulsions, such as oil/water
emulsions, emulsifying and suspending agents, such as acacia, agar,
alginic acid, sodium alginate, bentonite, carbomer, carrageenan,
carboxymethylcellulose, cellulose, cholesterol, gelatin,
hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl
methylcellulose, methylcellulose, octoxynol 9, oleyl alcohol,
povidone, propylene glycol monostearate, sodium lauryl sulfate,
sorbitan esters, stearyl alcohol, tragacanth, xanthan gum, and
derivatives thereof, solvents, and miscellaneous ingredients such
as crystalline cellulose, microcrystalline cellulose, citric acid,
dextrin, dextrose, liquid glucose, lactic acid, lactose, magnesium
chloride, potassium metaphosphate, starch, among others. Such
carriers and/or additives can be formulated by conventional methods
and can be administered to the subject at a suitable dose.
Stabilizing agents such as lipids, nuclease inhibitors, polymers,
and chelating agents can preserve the compositions from degradation
within the body.
[0332] Colloidal dispersion systems that can be used for delivery
of viruses include macromolecule complexes, nanocapsules,
microspheres, beads and lipid-based systems including oil-in-water
emulsions (mixed), micelles, liposomes and lipoplexes. An exemplary
colloidal system is a liposome. Organ-specific or cell-specific
liposomes can be used in order to achieve delivery only to the
desired tissue. The targeting of liposomes can be carried out by
the person skilled in the art by applying commonly known methods.
This targeting includes passive targeting (utilizing the natural
tendency of the liposomes to distribute to cells of the RES in
organs which contain sinusoidal capillaries) or active targeting
(for example, by coupling the liposome to a specific ligand, for
example, an antibody, a receptor, sugar, glycolipid and protein by
methods know to those of skill in the art). In the present methods,
monoclonal antibodies can be used to target liposomes to specific
tissues, for example, tumor tissue, via specific cell-surface
ligands.
[0333] 2. Host Cells
[0334] Also provided herein are host cells that contain a virus
provided herein, such as a modified vaccinia virus. Such cells can
be group of a single type of cells or a mixture of different types
of cells. Host cells can include cultured cell lines, primary cells
and proliferative cells. These host cells can include any of a
variety of animal cells, such as mammalian, avian and insect cells
and tissues that are susceptible to the virus, such as vaccinia
virus, infection, including chicken embryo, rabbit, hamster and
monkey kidney cells. Suitable host cells include but are not
limited to hematopoietic cells (totipotent, stem cells, leukocytes,
lymphocytes, monocytes, macrophages, APC, dendritic cells,
non-human cells and the like), pulmonary cells, tracheal cells,
hepatic cells, epithelial cells, endothelial cells, muscle cells
(e.g., skeletal muscle, cardiac muscle or smooth muscle),
fibroblasts, and cell lines including, for example, CV-1, BSC40,
Vero, BSC40 and BSC-1, and human HeLa cells. Methods for
transforming these host cells, phenotypically selecting
transformants, and other such methods are known in the art.
[0335] 3. Combinations
[0336] Provided are combinations of the viruses provided herein and
a second agent, such as a second virus or other therapeutic or
diagnostic agent, such as a tranporter substrate protein. A
combination can include any virus or reagent for effecting
attenuation thereof in accord with the methods provided herein.
Combinations can include a virus provided herein with one or more
additional viruses. Combinations of the viruses provided can also
contain pharmaceutical compositions containing the viruses or host
cells containing the viruses as described herein.
[0337] In one embodiment, the virus in a combination is an
attenuated virus, such as for example, an attenuated vaccinia virus
that encodes a transporter protein. Exemplary attenuated viruses
include vaccinia viruses provided herein, such as, but not limited
to, for example, vaccinia viruses described in the Examples (e.g.,
GLV-1h99, GLV-1h100, GLV-1h101, GLV-1h139, GLV-1h146 and GLV-1h150,
GLV-1h151, GLV-1h152 and GLV-1h153).
[0338] Combinations provided herein can contain a virus and a
therapeutic compound. Therapeutic compounds for the compositions
provided herein can be, for example, an anti-cancer or
chemotherapeutic compound. Exemplary therapeutic compounds include,
for example, cytokines, growth factors, photosensitizing agents,
radionuclides, toxins, siRNA molecules, enzyme/pro E drug pairs,
anti-metabolites, signaling modulators, anti-cancer antibiotics,
anti-cancer antibodies, angiogenesis inhibitors, chemotherapeutic
compounds or a combination thereof. Viruses provided herein can be
combined with an anti-cancer compound, such as a platinum
coordination complex. Exemplary platinum coordination complexes
include, for example, cisplatin, carboplatin, oxaliplatin,
DWA2114R, NK121, IS 3 295, and 254-S. Additional exemplary
therapeutic compounds for the use in pharmaceutical composition
combinations can be found elsewhere herein (see e.g., Section I for
exemplary cytokines, growth factors, photosensitizing agents,
radionuclides, toxins, siRNA molecules, enzyme/pro-drug pairs,
anti-metabolites, signaling modulators, anti-cancer antibiotics,
anti-cancer antibodies, angiogenesis inhibitors, and
chemotherapeutic compounds). Exemplary chemotherapeutic agents
include methotrexate, vincristine, adriamycin, non-sugar containing
chloroethylnitrosoureas, 5-fluorouracil, mitomycin C, bleomycin,
doxorubicin, dacarbazine, taxol, fragyline, Meglamine GLA,
valrubicin, carmustaine and poliferposan, MM1270, BAY 12-9566, RAS
farnesyl transferase inhibitor, farnesyl transferase inhibitor,
MMP, MTA/LY231514, LY264618/Lometexol, Glamolec, CI-994, TNP-470,
Hycamtin/Topotecan, PKC412, Valspodar/PSC833,
Novantrone/Mitroxantrone, Metaret/Suramin, Batimastat, E7070,
BCH-4556, CS-682, 9-AC, AG3340, AG3433, Incel/VX-710, VX-853,
ZD0101, IS1641, ODN 698, TA 2516/Marmistat, BB2516/Marmistat, CDP
845, D2163, PD183805, DX8951f, Lemonal DP 2202, FK 317,
Picibanil/OK-432, AD 32/Valrubicin, Metastron/strontium derivative,
Temodal/Temozolomide, Evacet/liposomal doxorubicin,
Yewtaxan/Placlitaxel, Taxol/Paclitaxel, Xeload/Capecitabine,
Furtulon/Doxifluridine, Cyclopax/oral paclitaxel, Oral Taxoid,
SPU-077/Cisplatin, HMR 1275/Flavopiridol, CP-358 (774)/EGFR, CP-609
(754)/RAS oncogene inhibitor, BMS-182751/oral platinum,
UFT(Tegafur/Uracil), Ergamisol/Levamisole, Eniluracil/776C85/5FU
enhancer, Campto/Levamisole, Camptosar/Irinotecan,
Tumodex/Ralitrexed, Leustatin/Cladribine, Paxex/Paclitaxel,
Doxil/liposomal doxorubicin, Caelyx/liposomal doxorubicin,
Fludara/Fludarabine, Pharmarubicin/Epirubicin, DepoCyt, ZD1839, LU
79553/Bis-Naphtalimide, LU 103793/Dolastain, Caetyx/liposomal
doxorubicin, Gemzar/Gemcitabine, ZD 0473/Anormed, YM 116, Iodine
seeds, CDK4 and CDK2 inhibitors, PARP inhibitors,
D4809/Dexifosamide, Ifes/Mesnex/Ifosamide, Vumon/Teniposide,
Paraplatin/Carboplatin, Plantinol/cisplatin, Vepeside/Etoposide, ZD
9331, Taxotere/Docetaxel, prodrug of guanine arabinoside, Taxane
Analog, nitrosoureas, alkylating agents such as melphelan and
cyclophosphamide, Aminoglutethimide, Asparaginase, Busulfan,
Carboplatin, Chlorombucil, Cytarabine HCl, Dactinomycin,
Daunorubicin HCl, Estramustine phosphate sodium, Etoposide (VP
16-213), Floxuridine, Fluorouracil (5-FU), Flutamide, Hydroxyurea
(hydroxycarbamide), Ifosfamide, Interferon Alfa-2a, Alfa-2b,
Leuprolide acetate (LHRH-releasing factor analogue), Lomustine
(CCNU), Mechlorethamine HCl (nitrogen mustard), Mercaptopurine,
Mesna, Mitotane (o.p'-DDD), Mitoxantrone HCl, Octreotide,
Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifen citrate,
Thioguanine, Thiotepa, Vinblastine sulfate, Amsacrine (m-AMSA),
Azacitidine, Erythropoietin, Hexamethylmelamine (HMM), Interleukin
2, Mitoguazone (methyl-GAG; methyl glyoxal bis-guanylhydrazone;
MGBG), Pentostatin (2'deoxycoformycin), Semustine (methyl-CCNU),
Teniposide (VM-26) and Vindesine sulfate.
[0339] In a further embodiment, the combination can include
additional therapeutic compounds such as, for example, compounds
that are substrates for enzymes encoded and expressed by the virus,
or other therapeutic compounds provided herein or known in the art
to act in concert with a virus. For example, the virus can express
an enzyme that converts a prodrug into an active chemotherapy drug
for killing the cancer cell. Hence, combinations provided herein
can contain therapeutic compounds, such as prodrugs. An exemplary
virus/therapeutic compound combination can include a virus encoding
Herpes simplex virus thymidine kinase with the prodrug gancyclovir.
Additional exemplary enzyme/pro-drug pairs, for the use in
combinations provided include, but are not limited to, varicella
zoster thymidine kinase/gancyclovir, cytosine
deaminase/5-fluorouracil, purine nucleoside
phosphorylase/6-methylpurine deoxyriboside, beta
lactamase/cephalosporin-doxorubicin, carboxypeptidase
G2/4-[(2-chloroethyl)(2-mesuloxyethyl)amino]benzoyl-L-glutamic
acid, cytochrome P450/acetominophen, horseradish
peroxidase/indole-3-acetic acid, nitroreductase/CB 1954, rabbit
carboxylesterase/7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycam-
potothecin, mushroom
tyrosinase/bis-(2-chloroethyl)amino-4-hydroxyphenylaminomethanone
28, beta
galactosidase/1-chloromethyl-5-hydroxy-1,2-dihydro-3H-benz[e]indole,
beta glucuronidase/epirubicin-glucoronide, thymidine
phosphorylase/5'-deoxy-5-fluorouridine, deoxycytidine
kinase/cytosine arabinoside, beta-lactamase and
linamerase/linamarin. Additional exemplary prodrugs, for the use in
combinations can also be found elsewhere herein (see e.g., Section
I). Any of a variety of known combinations provided herein or
otherwise known in the art can be included in the combinations
provided herein.
[0340] In a further embodiment, combinations can include compounds
that can kill or inhibit viral growth or toxicity. Combinations
provided herein can contain antibiotic, antifungal, anti-parasitic
or antiviral compounds for treatment of infections. Exemplary
antibiotics which can be included in a combination with a virus
provided herein include, but are not limited to, ceftazidime,
cefepime, imipenem, aminoglycoside, vancomycin and antipseudomonal
.beta.-lactam. Exemplary antifungal agents which can be included in
a combination with a virus provided herein include, but are not
limited to, amphotericin B, dapsone, fluconazole, flucytosine,
griseofluvin, intraconazole, ketoconazole, miconazole,
clotrimazole, nystatin, and combinations thereof. Exemplary
antiviral agents can be included in a combination with a virus
provided herein include, but are not limited to, cidofovir,
alkoxyalkyl esters of cidofovir (CDV), cyclic CDV, and
(S)-9-(3-hydroxy-2 phosphonylmethoxypropyl)adenine,
5-(Dimethoxymethyl)-2'-deoxyuridine, isatin-beta-thiosemicarbazone,
N-methanocarbathymidine, brivudin, 7-deazaneplanocin A, ST-246,
Gleevec, 2'-beta-fluoro-2',3'-dideoxyadenosine, indinavir,
nelfinavir, ritonavir, nevirapine, AZT, ddI, ddC, and combinations
thereof. Typically, combinations with an antiviral agent contain an
antiviral agent known to be effective against the virus of the
combination. For example, combinations can contain a vaccinia virus
with an antiviral compound, such as cidofovir, alkoxyalkyl esters
of cidofovir, gancyclovir, acyclovir, ST-246, and Gleevec.
[0341] In another embodiment, the combination can further include a
detectable compound. A detectable compound can include a ligand or
substrate or other compound that can interact with and/or bind
specifically to a virally expressed protein or RNA molecule, and
can provide a detectable signal, such as a signal detectable by
tomographic, spectroscopic, magnetic resonance, or other known
techniques. Exemplary detectable compounds can be, or can contain,
an imaging agent such as a magnetic resonance, ultrasound or
tomographic imaging agent, including a radionuclide. The detectable
compound can include any of a variety of compounds as provided
elsewhere herein or are otherwise known in the art. Typically, the
detectable compound included with a virus in the combinations
provided herein will be a compound that is a substrate, a ligand,
or can otherwise specifically interact with, a protein or RNA
encoded by the virus; in some examples, the protein or RNA is an
exogenous protein or RNA. Exemplary viruses/detectable compounds
include a virus encoding luciferase/luciferin,
.alpha.-galactosidase/(4,7,10-tri(acetic
acid)-1-(2-.beta.-galactopyranosylethoxy)-1,4,7,10-tetraazacyclododecane)
gadolinium (Egad), and other combinations known in the art.
[0342] In another embodiment, the combination can further include a
virus gene expression modulating compound. Compounds that modulate
gene expression are known in the art, and include, but are not
limited to, transcriptional activators, inducers, transcriptional
suppressors, RNA polymerase inhibitors and RNA binding compounds
such as siRNA or ribozymes. Any of a variety of gene expression
modulating compounds known in the art can be included in the
combinations provided herein. Typically, the gene expression
modulating compound included with a virus in the combinations
provided herein will be a compound that can bind, inhibit or react
with one or more compounds, active in gene expression such as a
transcription factor or RNA of the virus of the combination. An
exemplary virus/expression modulator can be a virus encoding a
chimeric transcription factor complex having a mutant human
progesterone receptor fused to a yeast GAL4 DNA-binding domain an
activation domain of the herpes simplex virus protein VP16 and also
containing a synthetic promoter containing a series of GAL4
recognition sequences upstream of the adenovirus major late E1B
TATA box, where the compound can be RU486 (see, e.g., Yu et al.,
(2002) Mol Genet Genomics 268:169-178). A variety of other
virus/expression modulator combinations known in the art also can
be included in the combinations provided herein.
[0343] In a further embodiment, combination can further contain
nanoparticles. Nanoparticles can be designed such that they carry
one or more therapeutic agents provided herein. Additionally,
nanoparticles can be designed to carry a molecule that targets the
nanoparticle to the tumor cells. In one non-limiting example,
nanoparticles can be coated with a radionuclide and, optionally, an
antibody immunoreactive with a tumor-associated antigen.
[0344] 4. Kits
[0345] The viruses, cells, pharmaceutical compositions or
combinations provided herein can be packaged as kits. Kits can
optionally include one or more components such as instructions for
use, devices and additional reagents, and components, such as
tubes, containers and syringes for practice of the methods.
Exemplary kits can include the viruses provided herein, and can
optionally include instructions for use, a device for detecting a
virus in a subject, a device for administering the virus to a
subject, and a device for administering a compound to a
subject.
[0346] In one example, a kit can contain instructions. Instructions
typically include a tangible expression describing the virus and,
optionally, other components included in the kit, and methods for
administration, including methods for determining the proper state
of the subject, the proper dosage amount, and the proper
administration method, for administering the virus. Instructions
can also include guidance for monitoring the subject over the
duration of the treatment time.
[0347] In another example, a kit can contain a device for detecting
a virus in a subject. Devices for detecting a virus in a subject
can include a low light imaging device for detecting light, for
example, emitted from luciferase, or fluoresced from fluorescent
protein, such as a green or red fluorescent protein, a magnetic
resonance measuring device such as an MRI or NMR device, a
tomographic scanner, such as a PET, CT, CAT, SPECT or other related
scanner, an ultrasound device, or other device that can be used to
detect a protein expressed by the virus within the subject.
Typically, the device of the kit will be able to detect one or more
proteins expressed by the virus of the kit. Any of a variety of
kits containing viruses and detection devices can be included in
the kits provided herein, for example, a virus expressing
luciferase and a low light imager or a virus expressing fluorescent
protein, such as a green or red fluorescent protein, and a low
light imager.
[0348] Kits provided herein also can include a device for
administering a virus to a subject. Any of a variety of devices
known in the art for administering medications or vaccines can be
included in the kits provided herein. Exemplary devices include,
but are not limited to, a hypodermic needle, an intravenous needle,
a catheter, a needle-less injection device, an inhaler and a liquid
dispenser, such as an eyedropper. Typically, the device for
administering a virus of the kit will be compatible with the virus
of the kit; for example, a needle-less injection device such as a
high pressure injection device can be included in kits with viruses
not damaged by high pressure injection, but is typically not
included in kits with viruses damaged by high pressure
injection.
[0349] Kits provided herein also can include a device for
administering a compound to a subject. Any of a variety of devices
known in the art for administering medications to a subject can be
included in the kits provided herein. Exemplary devices include a
hypodermic needle, an intravenous needle, a catheter, a needle-less
injection, but are not limited to, a hypodermic needle, an
intravenous needle, a catheter, a needle-less injection device, an
inhaler and a liquid dispenser such as an eyedropper. Typically the
device for administering the compound of the kit will be compatible
with the desired method of administration of the compound. For
example, a compound to be delivered subcutaneously can be included
in a kit with a hypodermic needle and syringe.
I. DIAGNOSTIC AND THERAPEUTIC METHODS
[0350] Provided are diagnostic and therapeutic methods, including
methods of detecting, imaging, treating and/or preventing
immunoprivileged cells or tissue, including cancerous cells, tumors
and metastases. Such sites, diseases and disorders include sites of
cell proliferation, proliferative conditions, neoplasms, tumors,
neoplastic disease, wounds and inflammation. The diagnostic and
therapeutic methods provided herein include, but are not limited
to, administering a virus provided herein that encodes a
transporter protein to a subject containing a tumor and/or
metastases. Viruses provided herein include viruses that have been
modified using the methods provided herein to generate viruses that
encode transporter proteins. Target cells that are infected with
such viruses express the transporter proteins, which permits uptake
of corresponding transporter substrates, which can be diagnostic
and or therapeutic agents or conjugated to a diagnostic or
therapeutic agents. Selection and modification of such transporter
substrates are described elsewhere herein. The uptake of labeled
substrates by virally infected tumor cells allows visualization of
the tumor tissue and can be used to monitor tumor therapy.
[0351] The administered viruses also can posses one or more
characteristics including attenuated pathogenicity, low toxicity,
preferential accumulation in tumor, ability to activate an immune
response against tumor cells, immunogenicity, replication
competence, ability to express additional exogenous diagnostic
and/or therapeutic genes, and an ability to elicit antibody
production against an expressed gene product. The viruses can be
administered for diagnosis and/or therapy of subjects, such as, but
not limited to humans and other mammals, including rodents, dogs,
cats, primates, or livestock.
[0352] The viruses provided herein can accumulate in tumors or
metastases. In some examples, the administration of a virus
provided herein results in a slowing of tumor growth. In other
examples, the administration of a virus provided herein results in
a decrease in tumor volume. The therapeutic methods provided
herein, however, do not require the administered virus to kill
tumor cells or decrease the tumor size. Instead, the methods
provided herein include administering to a subject a virus provided
herein that can cause or enhance an anti-tumor immune response in
the subject. In some examples, the viruses provided herein can be
administered to a subject without causing viral-induced disease in
the subject. In some examples, the viruses can elicit an anti-tumor
immune response in the subject, where typically the viral-mediated
anti-tumor immune response can develop, for example, over several
days, a week or more, 10 days or more, two weeks or more, or a
month or more. In some exemplary methods, the virus can be present
in the tumor, and can cause an anti-tumor immune response without
the virus itself causing enough tumor cell death to prevent tumor
growth. In some examples, the tumor is a monotherapeutic tumor or
monotherapeutic cancer, where the tumor or cancer does not decrease
in volume when treated with the virus or a therapeutic agent
alone.
[0353] In some examples, provided herein are methods for eliciting
or enhancing antibody production against a selected antigen or a
selected antigen type in a subject, where the methods include
administering to a subject a virus that can accumulate in a tumor
and/or metastasis, and can cause release of a selected antigen or
selected antigen type from the tumor, resulting in antibody
production against the selected antigen or selected antigen type.
Any of a variety of antigens can be targeted in the methods
provided herein, including a selected antigen such as an exogenous
gene product expressed by the virus, or a selected antigen type
such as one or more tumor antigens release from the tumor as a
result of viral infection of the tumor (e.g., by lysis, apoptosis,
secretion or other mechanism of causing antigen release from the
tumor).
[0354] In some embodiments, it can be desirable to maintain release
of the selected antigen or selected antigen type over a series of
days, for example, at least a week, at least ten days, at least two
weeks or at least a month. Provided herein are methods for
providing a sustained antigen release within a subject, where the
methods include administering to a subject a virus that can
accumulate in a tumor and/or metastasis, and can cause sustained
release of an antigen, resulting in antibody production against the
antigen. The sustained release of antigen can result in an immune
response by the viral-infected host, in which the host can develop
antibodies against the antigen, and/or the host can mount an immune
response against cells expressing the antigen, including an immune
response against tumor cells. Thus, the sustained release of
antigen can result in immunization against tumor cells. In some
embodiments, the viral-mediated sustained antigen release-induced
immune response against tumor cells can result in complete removal
or killing of all tumor cells.
[0355] In some embodiments, the therapeutic methods provided herein
inhibit tumor growth in a subject, where the methods include
administering to a subject a virus that can accumulate in a tumor
and/or metastasis, and can cause or enhance an anti-tumor immune
response. The anti-tumor immune response induced as a result of
tumor or metastases-accumulated viruses can result in inhibition of
tumor growth.
[0356] In some embodiments, the therapeutic methods provided herein
inhibit growth or formation of a metastasis in a subject, where the
methods include administering to a subject a virus provided herein
that can accumulate in a tumor and/or metastasis, and can cause or
enhance an anti-tumor immune response. The anti-tumor immune
response induced as a result of tumor or metastasis-accumulated
viruses can result in inhibition of metastasis growth or
formation.
[0357] In other embodiments, the therapeutic methods provided
herein decrease the size of a tumor and/or metastasis in a subject,
where the methods include administering to a subject a virus
provided herein that can accumulate in a tumor and/or metastasis,
and can cause or enhance an anti-tumor immune response. The
anti-tumor immune response induced as a result of tumor or
metastasis-accumulated viruses can result in a decrease in the size
of the tumor and/or metastasis.
[0358] In some embodiments, the therapeutic methods provided herein
eliminate a tumor and/or metastasis from a subject, where the
methods include administering to a subject a virus provided herein
that can accumulate in a tumor and/or metastasis, and can cause or
enhance an anti-tumor immune response. The anti-tumor immune
response induced as a result of tumor or metastasis-accumulated
viruses can result in elimination of the tumor and/or metastasis
from the subject.
[0359] Methods of reducing or inhibiting tumor growth, inhibiting
metastasis growth and/or formation, decreasing the size of a tumor
or metastasis, eliminating a tumor or metastasis, or other tumor
therapeutic methods provided herein include causing or enhancing an
anti-tumor immune response in the host. The immune response of the
host, being anti-tumor in nature, can be mounted against tumors
and/or metastases in which viruses have accumulated, and can also
be mounted against tumors and/or metastases in which viruses have
not accumulated, including tumors and/or metastases that form after
administration of the virus to the subject. Accordingly, a tumor
and/or metastasis whose growth or formation is inhibited, or whose
size is decreased, or that is eliminated, can be a tumor and/or
metastasis in which the viruses have accumulated, or also can be a
tumor and/or metastasis in which the viruses have not accumulated.
Accordingly, provided herein are methods of reducing or inhibiting
tumor growth, inhibiting metastasis growth and/or formation,
decreasing the size of a tumor or metastasis, eliminating a tumor
or metastasis, or other tumor therapeutic methods, where the method
includes administering to a subject a virus provided herein, where
the virus accumulates in at least one tumor or metastasis and
causes or enhances an anti-tumor immune response in the subject,
and the immune response also is mounted against a tumor and/or
metastasis in which the virus cell did not accumulate. In another
embodiment, methods are provided for inhibiting or preventing
recurrence of a neoplastic disease or inhibiting or preventing new
tumor growth, where the methods include administering to a subject
a virus provided herein that can accumulate in a tumor and/or
metastasis, and can cause or enhance an anti-tumor immune response,
and the anti-tumor immune response can inhibit or prevent
recurrence of a neoplastic disease or inhibit or prevent new tumor
growth.
[0360] The tumor or neoplastic disease therapeutic methods provided
herein, such as methods of reducing or inhibiting tumor growth,
inhibiting metastasis growth and/or formation, decreasing the size
of a tumor or metastasis, eliminating a tumor or metastasis, or
other tumor therapeutic methods, also can include administering to
a subject a virus provided herein that can cause tumor cell lysis
or tumor cell death. Such a virus can be the same virus as the
virus that can cause or enhance an anti-tumor immune response in
the subject. Viruses, such as the viruses provided herein, can
cause cell lysis or tumor cell death as a result of expression of
an endogenous gene or as a result of an exogenous gene. Endogenous
or exogenous genes can cause tumor cell lysis or inhibit cell
growth as a result of direct or indirect actions, as is known in
the art, including lytic channel formation or activation of an
apoptotic pathway. Gene products, such as exogenous gene products
can function to activate a prodrug to an active, cytotoxic form,
resulting in cell death where such genes are expressed.
[0361] Such methods of antigen production or tumor and/or
metastasis treatment can include administration of a virus provided
herein for therapy, such as for gene therapy, for cancer gene
therapy, or for vaccine therapy. Such a virus can be used to
stimulate humoral and/or cellular immune response, induce strong
cytotoxic T lymphocytes responses in subjects who can benefit from
such responses. For example, the virus can provide prophylactic and
therapeutic effects against a tumor infected by the virus or other
infectious diseases, by rejection of cells from tumors or lesions
using viruses that express immunoreactive antigens (Earl et al.,
Science 234: 728-831 (1986); Lathe et al., Nature (London) 32:
878-880 (1987)), cellular tumor-associated antigens (Bernards et
al., Proc. Natl. Acad. Sci. USA 84: 6854-6858 (1987); Estin et al.,
Proc. Natl. Acad. Sci. USA 85: 1052-1056 (1988); Kantor et al., J.
Natl. Cancer Inst. 84: 1084-1091 (1992); Roth et al., Proc. Natl.
Acad. Sci. USA 93: 4781-4786 (1996)) and/or cytokines (e.g., IL-2,
IL-12), costimulatory molecules (B7-1, B7-2) (Rao et al., J.
Immunol. 156: 3357-3365 (1996); Chamberlain et al., Cancer Res. 56:
2832-2836 (1996); Oertli et al., J. Gen. Virol. 77: 3121-3125
(1996); Qin and Chatterjee, Human Gene Ther. 7: 1853-1860 (1996);
McAneny et al., Ann. Surg. Oncol. 3: 495-500 (1996)), or other
therapeutic proteins.
[0362] As shown previously, solid tumors can be treated with
viruses, such as vaccinia viruses, resulting in an enormous
tumor-specific virus replication, which can lead to tumor protein
antigen and viral protein production in the tumors (U.S. Patent
Publication No. 2005/0031643). Vaccinia virus administration to
mice resulted in lysis of the infected tumor cells and a resultant
release of tumor-cell-specific antigens. Continuous leakage of
these antigens into the body led to a very high level of antibody
titer (in approximately 7-14 days) against tumor proteins, viral
proteins, and the virus encoded engineered proteins in the mice.
The newly synthesized anti-tumor antibodies and the enhanced
macrophage, neutrophils count were continuously delivered via the
vasculature to the tumor and thereby provided for the recruitment
of an activated immune system against the tumor. The activated
immune system then eliminated the foreign compounds of the tumor
including the viral particles. This interconnected release of
foreign antigens boosted antibody production and continuous
response of the antibodies against the tumor proteins to function
like an autoimmunizing vaccination system initiated by vaccinia
viral infection and replication, followed by cell lysis, protein
leakage and enhanced antibody production. Thus, the viruses
provided herein and the viruses generated using the methods
provided herein can be administered in a complete process that can
be applied to all tumor systems with immunoprivileged tumor sites
as site of privileged viral growth, which can lead to tumor
elimination by the host's own immune system.
[0363] In other embodiments, methods are provided for immunizing a
subject, where the methods include administering to the subject a
virus that expresses one or more antigens against which antigens
the subject will develop an immune response. The immunizing
antigens can be endogenous to the virus, such as vaccinia antigens
on a vaccinia virus used to immunize against smallpox, measles,
mumps, or the immunizing antigens can be exogenous antigens
expressed by the virus, such as influenza or HIV antigens expressed
on a viral capsid surface. In the case of smallpox, for example, a
tumor specific protein antigen can be carried by an attenuated
vaccinia virus (encoded by the viral genome) for a smallpox
vaccine. Thus, the viruses provided herein, including the modified
vaccinia viruses can be used as vaccines.
[0364] In one embodiment, the tumor treated is a cancer such as
pancreatic cancer, non-small cell lung cancer, multiple myeloma or
leukemia, although the cancer is not limited in this respect, and
other metastatic diseases can be treated by the combinations
provided herein. For example, the tumor treated can be a solid
tumor, such as of the lung and bronchus, breast, colon and rectum,
kidney, stomach, esophagus, liver and intrahepatic bile duct,
urinary bladder, brain and other nervous system, head and neck,
oral cavity and pharynx, cervix, uterine corpus, thyroid, ovary,
testes, prostate, malignant melanoma, cholangiocarcinoma, thymoma,
non-melanoma skin cancers, as well as hematologic tumors and/or
malignancies, such as childhood leukemia and lymphomas, multiple
myeloma, Hodgkin's disease, lymphomas of lymphocytic and cutaneous
origin, acute and chronic leukemia such as acute lymphoblastic,
acute myelocytic or chronic myelocytic leukemia, plasma cell
neoplasm, lymphoid neoplasm and cancers associated with AIDS.
Exemplary tumors include, for example, pancreatic tumors, ovarian
tumors, lung tumors, colon tumors, prostate tumors, cervical tumors
and breast tumors. In one embodiment, the tumor is a carcinoma such
as, for example, an ovarian tumor or a pancreatic tumor.
[0365] 1. Administration
[0366] In performing the therapeutic methods provided herein, a
virus provided herein that encodes a transporter protein can be
administered to a subject, including a subject having a tumor or
having neoplastic cells, or a subject to be immunized. An
administered virus can be a virus provided herein or any other
virus generated using the methods provided herein. In some
embodiments, the virus administered is a virus containing a
characteristic such as attenuated pathogenicity, low toxicity,
preferential accumulation in tumor, ability to activate an immune
response against tumor cells, high immunogenicity, replication
competence and ability to express exogenous proteins, and
combinations thereof. A substrate that is transported by the
transporter protein can be administered simultaneously or
sequentially to administration of the virus for detection, imaging
or treatment.
[0367] a. Steps Prior to Administering the Virus
[0368] In some embodiments, one or more steps can be performed
prior to administration of the virus to the subject. Any of a
variety of preceding steps can be performed, including, but not
limited to diagnosing the subject with a condition appropriate for
virus administration, determining the immunocompetence of the
subject, immunizing the subject, treating the subject with a
chemotherapeutic agent, treating the subject with radiation, or
surgically treating the subject.
[0369] For embodiments that include administering a virus to a
tumor-bearing subject for therapeutic purposes, the subject has
typically been previously diagnosed with a neoplastic condition.
Diagnostic methods also can include determining the type of
neoplastic condition, determining the stage of the neoplastic
conditions, determining the size of one or more tumors in the
subject, determining the presence or absence of metastatic or
neoplastic cells in the lymph nodes of the subject, or determining
the presence of metastases of the subject. Some embodiments of
therapeutic methods for administering a virus to a subject can
include a step of determination of the size of the primary tumor or
the stage of the neoplastic disease, and if the size of the primary
tumor is equal to or above a threshold volume, or if the stage of
the neoplastic disease is at or above a threshold stage, a virus is
administered to the subject. In a similar embodiment, if the size
of the primary tumor is below a threshold volume, or if the stage
of the neoplastic disease is at or below a threshold stage, the
virus is not yet administered to the subject; such methods can
include monitoring the subject until the tumor size or neoplastic
disease stage reaches a threshold amount, and then administering
the virus to the subject. Threshold sizes can vary according to
several factors, including rate of growth of the tumor, ability of
the virus to infect a tumor, and immunocompetence of the subject.
Generally the threshold size will be a size sufficient for a virus
to accumulate and replicate in or near the tumor without being
completely removed by the host's immune system, and will typically
also be a size sufficient to sustain a virus infection for a time
long enough for the host to mount an immune response against the
tumor cells, typically about one week or more, about ten days or
more, or about two weeks or more. Exemplary threshold tumor sizes
for viruses, such as vaccinia viruses, are at least about 100
mm.sup.3, at least about 200 mm.sup.3, at least about 300 mm.sup.3,
at least about 400 mm.sup.3, at least about 500 mm.sup.3, at least
about 750 mm.sup.3, at least about 1000 mm.sup.3, or at least about
1500 mm.sup.3. Threshold neoplastic disease stages also can vary
according to several factors, including specific requirement for
staging a particular neoplastic disease, aggressiveness of growth
of the neoplastic disease, ability of the virus to infect a tumor
or metastasis, and immunocompetence of the subject. Generally the
threshold stage will be a stage sufficient for a virus to
accumulate and replicate in a tumor or metastasis without being
completely removed by the host's immune system, and will typically
also be a size sufficient to sustain a virus infection for a time
long enough for the host to mount an immune response against the
neoplastic cells, typically about one week or more, about ten days
or more, or about two weeks or more. Exemplary threshold stages are
any stage beyond the lowest stage (e.g., Stage I or equivalent), or
any stage where the primary tumor is larger than a threshold size,
or any stage where metastatic cells are detected.
[0370] In other embodiments, prior to administering to the subject
a virus, the immunocompetence of the subject can be determined. The
methods of administering a virus to a subject provided herein can
include causing or enhancing an immune response in a subject.
Accordingly, prior to administering a virus to a subject, the
ability of a subject to mount an immune response can be determined.
Any of a variety of tests of immunocompetence known in the art can
be performed in the methods provided herein. Exemplary
immunocompetence tests can examine ABO hemagglutination titers
(IgM), leukocyte adhesion deficiency (LAD), granulocyte function
(NBT), T and B cell quantitation, tetanus antibody titers, salivary
IgA, skin test, tonsil test, complement C3 levels, and factor B
levels, and lymphocyte count. One skilled in the art can determine
the desirability to administer a virus to a subject according to
the level of immunocompetence of the subject, according to the
immunogenicity of the virus, and, optionally, according to the
immunogenicity of the neoplastic disease to be treated. Typically,
a subject can be considered immunocompetent if the skilled artisan
can determine that the subject is sufficiently competent to mount
an immune response against the virus.
[0371] In some embodiments, the subject can be immunized prior to
administering to the subject a virus according to the methods
provided herein. Immunization can serve to increase the ability of
a subject to mount an immune response against the virus, or
increase the speed at which the subject can mount an immune
response against a virus. Immunization also can serve to decrease
the risk to the subject of pathogenicity of the virus. In some
embodiments, the immunization can be performed with an immunization
virus that is similar to the therapeutic virus to be administered.
For example, the immunization virus can be a
replication-incompetent variant of the therapeutic virus. In other
embodiments, the immunization material can be digests of the
therapeutic virus to be administered. Any of a variety of methods
for immunizing a subject against a known virus are known in the art
and can be used herein. In one example, vaccinia viruses treated
with, for example, 1 microgram of psoralen and ultraviolet light at
365 nm for 4 minutes, can be rendered replication incompetent. In
another embodiment, the virus can be selected as the same or
similar to a virus against which the subject has been previously
immunized, e.g., in a childhood vaccination.
[0372] In another embodiment, the subject can have administered
thereto a virus without any previous steps of cancer treatment such
as chemotherapy, radiation therapy or surgical removal of a tumor
and/or metastases. The methods provided herein take advantage of
the ability of the viruses to enter or localize near a tumor, where
the tumor cells can be protected from the subject's immune system;
the viruses can then proliferate in such an immunoprotected region
and can also cause the release, typically a sustained release, of
tumor antigens from the tumor to a location in which the subject's
immune system can recognize the tumor antigens and mount an immune
response. In such methods, existence of a tumor of sufficient size
or sufficiently developed immunoprotected state can be advantageous
for successful administration of the virus to the tumor, and for
sufficient tumor antigen production. If a tumor is surgically
removed, the viruses may not be able to localize to other
neoplastic cells (e.g., small metastases) because such cells have
not yet have matured sufficiently to create an immunoprotective
environment in which the viruses can survive and proliferate, or
even if the viruses can localize to neoplastic cells, the number of
cells or size of the mass can be too small for the viruses to cause
a sustained release of tumor antigens in order for the host to
mount an anti-tumor immune response. Thus, for example, provided
herein are methods of treating a tumor or neoplastic disease in
which viruses are administered to a subject with a tumor or
neoplastic disease without removing the primary tumor, or to a
subject with a tumor or neoplastic disease in which at least some
tumors or neoplastic cells are intentionally permitted to remain in
the subject. In other typical cancer treatment methods such as
chemotherapy or radiation therapy, such methods typically have a
side effect of weakening the subject's immune system. This
treatment of a subject by chemotherapy or radiation therapy can
reduce the subject's ability to mount an anti-tumor immune
response. Thus, for example, provided herein are methods of
treating a tumor or neoplastic disease in which viruses are
administered to a subject with a tumor or neoplastic disease
without treating the subject with an immune system-weakening
therapy, such as chemotherapy or radiation therapy.
[0373] In an alternative embodiment, prior to administration of a
virus to the subject, the subject can be treated in one or more
cancer treatment steps that do not remove the primary tumor or that
do not weaken the immune system of the subject. A variety of more
sophisticated cancer treatment methods are being developed in which
the tumor can be treated without surgical removal or immune-system
weakening therapy. Exemplary methods include administering a
compound that decreases the rate of proliferation of the tumor or
neoplastic cells without weakening the immune system (e.g., by
administering tumor suppressor compounds or by administering tumor
cell-specific compounds) or administering an
angiogenesis-inhibiting compound. Thus, combined methods that
include administering a virus to a subject can further improve
cancer therapy. Thus, provided herein are methods of administering
a virus to a subject, along with prior to or subsequent to, for
example, administering a compound that slows tumor growth without
weakening the subject's immune system or a compound that inhibits
vascularization of the tumor.
[0374] b. Mode of Administration
[0375] Any mode of administration of a virus to a subject can be
used, provided the mode of administration permits the virus to
enter a tumor or metastasis. Modes of administration can include,
but are not limited to, systemic, intravenous, intraperitoneal,
subcutaneous, intramuscular, transdermal, intradermal,
intra-arterial (e.g., hepatic artery infusion), intravesicular
perfusion, intrapleural, intraarticular, topical, intratumoral,
intralesional, multipuncture (e.g., as used with smallpox
vaccines), inhalation, percutaneous, subcutaneous, intranasal,
intratracheal, oral, intracavity (e.g., administering to the
bladder via a catheter, administering to the gut by suppository or
enema), vaginal, rectal, intracranial, intraprostatic,
intravitreal, aural, or ocular administration. Transporter
substrate also can be similarly administered. In some examples,
both the virus and transporter substrates are administered
systemically for detection, imaging and/or treatment of a
tumor.
[0376] One skilled in the art can select any mode of administration
compatible with the subject and the virus, and that also is likely
to result in the virus reaching tumors and/or metastases. The route
of administration can be selected by one skilled in the art
according to any of a variety of factors, including the nature of
the disease, the kind of tumor, and the particular virus contained
in the pharmaceutical composition. Administration to the target
site can be performed, for example, by ballistic delivery, as a
colloidal dispersion system, or systemic administration can be
performed by injection into an artery.
[0377] c. Dosages
[0378] The dosage regimen can be any of a variety of methods and
amounts, and can be determined by one skilled in the art according
to known clinical factors. As is known in the medical arts, dosages
for any one patient can depend on many factors, including the
subject's species, size, body surface area, age, sex,
immunocompetence, and general health, the particular virus to be
administered, duration and route of administration, the kind and
stage of the disease, for example, tumor size, and other treatments
or compounds, such as chemotherapeutic drugs, being administered
concurrently. In addition to the above factors, such levels can be
affected by the infectivity of the virus, and the nature of the
virus, as can be determined by one skilled in the art. In the
present methods, appropriate minimum dosage levels of viruses can
be levels sufficient for the virus to survive, grow and replicate
in a tumor or metastasis. Exemplary minimum levels for
administering a virus to a 65 kg human can include at least about
1.times.10.sup.5 plaque forming units (PFU), at least about
5.times.10.sup.5 PFU, at least about 1.times.10.sup.6 PFU, at least
about 5.times.10.sup.6 PFU, at least about 1.times.10.sup.7 PFU, at
least about 1.times.10.sup.8 PFU, at least about 1.times.10.sup.9
PFU, or at least about 1.times.10.sup.10 PFU. In the present
methods, appropriate maximum dosage levels of viruses can be levels
that are not toxic to the host, levels that do not cause
splenomegaly of 3 times or more, levels that do not result in
colonies or plaques in normal tissues or organs after about 1 day
or after about 3 days or after about 7 days. Exemplary maximum
levels for administering a virus to a 65 kg human can include no
more than about 1.times.10.sup.11 PFU, no more than about
5.times.1010 PFU, no more than about 1.times.10.sup.10 PFU, no more
than about 5.times.10.sup.9 PFU, no more than about
1.times.10.sup.9 PFU, or no more than about 1.times.10.sup.8
PFU.
[0379] For combination therapies with chemotherapeutic compounds,
dosages for the administration of such compounds are known in the
art or can be determined by one skilled in the art according to
known clinical factors (e.g., subject's species, size, body surface
area, age, sex, immunocompetence, and general health, duration and
route of administration, the kind and stage of the disease, for
example, tumor size, and other viruses, treatments, or compounds,
such as other chemotherapeutic drugs, being administered
concurrently). In addition to the above factors, such levels can be
affected by the infectivity of the virus, and the nature of the
virus, as can be determined by one skilled in the art. For example,
Cisplatin (also called cis-platinum, platinol;
cis-diamminedichloroplatinum; and cDDP) is representative of a
broad class of water-soluble, platinum coordination compounds
frequently employed in the therapy of testicular cancer, ovarian
tumors and a variety of other cancers. (See, e.g., Blumenreich et
al. Cancer 55(5): 1118-1122 (1985); Forastiere et al. J. Clin.
Oncol. 19(4): 1088-1095 (2001)). Methods of employing cisplatin
clinically are well known in the art. For example, cisplatin has
been administered in a single day over a six hour period, once per
month, by slow intravenous infusion. For localized lesions,
cisplatin can be administered by local injection. Intraperitoneal
infusion can also be employed. Cisplatin can be administered in
doses as low as 10 mg/m.sup.2 per treatment if part of a multi-drug
regimen, or if the patient has an adverse reaction to higher
dosing. In general, a clinical dose is from about 30 to about 120
or 150 mg/m.sup.2 per treatment.
[0380] Typically, platinum-containing chemotherapeutic agents are
administered parenterally, for example by slow intravenous
infusion, or by local injection, as discussed above. The effects of
intralesional (intra-tumoral) and IP administration of cisplatin is
described in (Nagase et al. Cancer Treat. Rep. 71(9): 825-829
(1987); and Theon et al. J. Am. Vet. Med. Assoc. 202(2): 261-7.
(1993)).
[0381] In one exemplary embodiment, the mutant vaccinia virus is
administered once or 2-4 times with 0-60 days apart, followed by
1-30 days where no anti-cancer treatment, then cisplatin is
administered daily for 1-5 days, followed by 1-30 days where no
anti-cancer treatment is administered. Each component of the
therapy, virus or cisplatin treatment, or the virus and cisplatin
combination therapy can be repeated.
[0382] In another exemplary embodiment, cisplatin is administered
daily for 1 to 5 days, followed by 1-10 days where no anti-cancer
treatment is administered, then the mutant vaccinia virus is
administered once or 2-4 times with 0-60 days apart. Such treatment
scheme can be repeated. In another exemplary embodiment, cisplatin
is administered daily for 1 to 5 days, followed by 1-10 days where
no anti-cancer treatment is administered, then the mutant vaccinia
virus is administered once or 2-4 times with 0-60 days apart. This
is followed by 5-60 days where no anti-cancer treatment is
administered, then cisplatin is administered again for 1-5 days.
Such treatment scheme can be repeated.
[0383] Gemcitabine (GEMZAR.RTM.) is another compound employed in
the therapy of breast cancer, non-small cell lung cancer, and
pancreatic cancer. Gemcitabine is a nucleoside analogue that
exhibits antitumor activity. Methods of employing gemcitabine
clinically are well known in the art. For example, gemcitabine has
been administered by intravenous infusion at a dose of 1000
mg/m.sup.2 over 30 minutes once weekly for up to 7 weeks (or until
toxicity necessitates reducing or holding a dose), followed by a
week of rest from treatment of pancreatic cancer. Subsequent cycles
can consist of infusions once weekly for 3 consecutive weeks out of
every 4 weeks. Gemcitabine has also been employed in combination
with cisplatin in cancer therapy.
[0384] In one exemplary embodiment, the mutant vaccinia virus is
administered once or 2-4 times with 0-60 days apart, followed by
1-30 days where no anti-cancer treatment is administered, then
gemcitabine is administered 1-7 times with 0-30 days apart,
followed by 1-30 days where no anti-cancer treatment is
administered. Such treatment scheme can be repeated. In another
exemplary embodiment, gemcitabine is administered 1-7 times with
0-30 days apart, followed by 1-10 days where no anti-cancer
treatment is administered, then the mutant vaccinia virus is
administered once or 2-4 times with 0-60 days apart. This is
followed by 5-60 days where no anti-cancer treatment is
administered. Such treatment scheme can be repeated. In another
exemplary embodiment, gemcitabine is administered 1-7 times with
0-30 days apart, followed by 1-10 days where no anti-cancer
treatment is administered, then the mutant vaccinia virus is
administered once or 2-4 times with 0-60 days apart. This is
followed by 5-60 days where no anti-cancer treatment is
administered, then gemcitabine is administered again for 1-7 times
with 0-30 days apart. Such treatment scheme can be repeated.
[0385] As will be understood by one of skill in the art, the
optimal treatment regimen will vary and it is within the scope of
the treatment methods to evaluate the status of the disease under
treatment and the general health of the patient prior to, and
following one or more cycles of combination therapy in order to
determine the optimal therapeutic combination.
[0386] d. Number of Administrations
[0387] The methods provided herein can include a single
administration of a virus to a subject or multiple administrations
of a virus to a subject. In some embodiments, a single
administration is sufficient to establish a virus in a tumor, where
the virus can proliferate and can cause or enhance an anti-tumor
response in the subject; such methods do not require additional
administrations of a virus in order to cause or enhance an
anti-tumor response in a subject, which can result, for example in
inhibition of tumor growth, inhibition of metastasis growth or
formation, reduction in tumor or size, elimination of a tumor or
metastasis, inhibition or prevention of recurrence of a neoplastic
disease or new tumor formation, or other cancer therapeutic
effects. In other embodiments, a virus can be administered on
different occasions, separated in time typically by at least one
day. Separate administrations can increase the likelihood of
delivering a virus to a tumor or metastasis, where a previous
administration has been ineffective in delivering a virus to a
tumor or metastasis. Separate administrations can increase the
locations on a tumor or metastasis where virus proliferation can
occur or can otherwise increase the titer of virus accumulated in
the tumor, which can increase the scale of release of antigens or
other compounds from the tumor in eliciting or enhancing a host's
anti-tumor immune response, and also can, optionally, increase the
level of virus-based tumor lysis or tumor cell death. Separate
administrations of a virus can further extend a subject's immune
response against viral antigens, which can extend the host's immune
response to tumors or metastases in which viruses have accumulated,
and can increase the likelihood of a host mounting an anti-tumor
immune response.
[0388] When separate administrations are performed, each
administration can be a dosage amount that is the same or different
relative to other administration dosage amounts. In one embodiment,
all administration dosage amounts are the same. In other
embodiments, a first dosage amount can be a larger dosage amount
than one or more subsequent dosage amounts, for example, at least
10.times. larger, at least 100.times. larger, or at least
1000.times. larger than subsequent dosage amounts. In one example
of a method of separate administrations in which the first dosage
amount is greater than one or more subsequent dosage amounts, all
subsequent dosage amounts can be the same, smaller amount relative
to the first administration.
[0389] Separate administrations can include any number of two or
more administrations, including two, three, four, five or six
administrations. One skilled in the art can readily determine the
number of administrations to perform or the desirability of
performing one or more additional administrations according to
methods known in the art for monitoring therapeutic methods and
other monitoring methods provided herein. Accordingly, the methods
provided herein include methods of providing to the subject one or
more administrations of a virus, where the number of
administrations can be determined by monitoring the subject, and,
based on the results of the monitoring, determining whether or not
to provide one or more additional administrations. Deciding on
whether or not to provide one or more additional administrations
can be based on a variety of monitoring results, including, but not
limited to, indication of tumor growth or inhibition of tumor
growth, appearance of new metastases or inhibition of metastasis,
the subject's anti-virus antibody titer, the subject's anti-tumor
antibody titer, the overall health of the subject, the weight of
the subject, the presence of virus solely in tumor and/or
metastases, the presence of virus in normal tissues or organs.
[0390] The time period between administrations can be any of a
variety of time periods. The time period between administrations
can be a function of any of a variety of factors, including
monitoring steps, as described in relation to the number of
administrations, the time period for a subject to mount an immune
response, the time period for a subject to clear the virus from
normal tissue, or the time period for virus proliferation in the
tumor or metastasis. In one example, the time period can be a
function of the time period for a subject to mount an immune
response; for example, the time period can be more than the time
period for a subject to mount an immune response, such as more than
about one week, more than about ten days, more than about two
weeks, or more than about a month; in another example, the time
period can be less than the time period for a subject to mount an
immune response, such as less than about one week, less than about
ten days, less than about two weeks, or less than about a month. In
another example, the time period can be a function of the time
period for a subject to clear the virus from normal tissue; for
example, the time period can be more than the time period for a
subject to clear the virus from normal tissue, such as more than
about a day, more than about two days, more than about three days,
more than about five days, or more than about a week. In another
example, the time period can be a function of the time period for
virus proliferation in the tumor or metastasis; for example, the
time period can be more than the amount of time for a detectable
signal to arise in a tumor or metastasis after administration of a
virus expressing a detectable marker, such as about 3 days, about 5
days, about a week, about ten days, about two weeks, or about a
month.
[0391] e. Co-Administrations
[0392] Also provided are methods in which an additional therapeutic
substance, such as a different therapeutic virus or a therapeutic
compound is administered. These can be administered simultaneously,
sequentially or intermittently with the first virus. The additional
therapeutic substance can interact with the virus or a gene product
thereof, or the additional therapeutic substance can act
independently of the virus.
[0393] Combination therapy treatment has advantages in that: 1) it
avoids single agent resistance; 2) in a heterogeneous tumor
population, it can kill cells by different mechanisms; and 3) by
selecting drugs with non-overlapping toxicities, each agent can be
used at full dose to elicit maximal efficacy and synergistic
effect. Combination therapy can be done by combining a
diagnostic/therapeutic virus with one or more of the following
anti-cancer agents: chemotherapeutic agents, therapeutic
antibodies, siRNAs, toxins, enzyme-prodrug pairs or radiation.
[0394] i. Administering a Plurality of Viruses
[0395] Methods are provided for administering to a subject two or
more viruses. Administration can be effected simultaneously,
sequentially or intermittently. The plurality of viruses can be
administered as a single composition or as two or more
compositions. The two or more viruses can include at least two
viruses. In a particular embodiment, where there are two viruses,
both viruses are vaccinia viruses.
[0396] In another embodiment, one viruses is a vaccinia virus and
the second viruses is any one of an adenovirus, an adeno-associated
virus, a retrovirus, a herpes simplex virus, a reovirus, a mumps
virus, a foamy virus, an influenza virus, a myxoma virus, a
vesicular stomatitis virus, or any other virus described herein or
known in the art. Viruses can be chosen based on the pathway on
which they act. For example, a virus that targets an activated Ras
pathway can be combined with a virus that targets tumor cells
defective in p53 expression.
[0397] The plurality of viruses can be provided as combinations of
compositions containing and/or as kits that include the viruses
packaged for administration and optionally including instructions
therefore. The compositions can contain the viruses formulated for
single dosage administration (i.e., for direct administration) and
can require dilution or other additions.
[0398] In one embodiment, at least one of the viruses is a modified
virus such as those provided herein, having a characteristic such
as low pathogenicity, low toxicity, preferential accumulation in
tumor, ability to activate an immune response against tumor cells,
immunogenic, replication competent, ability to express exogenous
proteins, and combinations thereof. The viruses can be administered
at approximately the same time, or can be administered at different
times. The viruses can be administered in the same composition or
in the same administration method, or can be administered in
separate composition or by different administration methods.
[0399] The time period between administrations can be any time
period that achieves the desired effects, as can be determined by
one skilled in the art. Selection of a time period between
administrations of different viruses can be determined according to
parameters similar to those for selecting the time period between
administrations of the same virus, including results from
monitoring steps, the time period for a subject to mount an immune
response, the time period for a subject to clear virus from normal
tissue, or the time period for virus proliferation in the tumor or
metastasis. In one example, the time period can be a function of
the time period for a subject to mount an immune response; for
example, the time period can be more than the time period for a
subject to mount an immune response, such as more than about one
week, more than about ten days, more than about two weeks, or more
than about a month; in another example, the time period can be less
than the time period for a subject to mount an immune response,
such as less than about one week, less than about ten days, less
than about two weeks, or less than about a month. In another
example, the time period can be a function of the time period for a
subject to clear the virus from normal tissue; for example, the
time period can be more than the time period for a subject to clear
the virus from normal tissue, such as more than about a day, more
than about two days, more than about three days, more than about
five days, or more than about a week. In another example, the time
period can be a function of the time period for virus proliferation
in the tumor or metastasis; for example, the time period can be
more than the amount of time for a detectable signal to arise in a
tumor or metastasis after administration of a virus expressing a
detectable marker, such as about 3 days, about 5 days, about a
week, about ten days, about two weeks, or about a month.
[0400] ii. Therapeutic Compounds
[0401] Any therapeutic or anti-cancer agent can be used as the
second, therapeutic or anti-cancer agent in the combined cancer
treatment methods provided herein. The methods can include
administering one or more therapeutic compounds to the subject in
addition to administering a virus or plurality thereof to a
subject. Therapeutic compounds can act independently, or in
conjunction with the virus, for tumor therapeutic effects.
[0402] Therapeutic compounds that can act independently include any
of a variety of known chemotherapeutic compounds that can inhibit
tumor growth, inhibit metastasis growth and/or formation, decrease
the size of a tumor or metastasis, eliminate a tumor or metastasis,
without reducing the ability of a virus to accumulate in a tumor,
replicate in the tumor, and cause or enhance an anti-tumor immune
response in the subject.
[0403] Therapeutic compounds that act in conjunction with the
viruses include, for example, compounds that alter the expression
of the viruses or compounds that can interact with a
virally-expressed gene, or compounds that can inhibit virus
proliferation, including compounds toxic to the virus. Therapeutic
compounds that can act in conjunction with the virus include, for
example, therapeutic compounds that increase the proliferation,
toxicity, tumor cell killing or immune response eliciting
properties of a virus, and also can include, for example,
therapeutic compounds that decrease the proliferation, toxicity or
cell killing properties of a virus. Optionally, the therapeutic
agent can exhibit or manifest additional properties, such as,
properties that permit its use as an imaging agent, as described
elsewhere herein.
[0404] Therapeutic compounds also include, but are not limited to,
chemotherapeutic agents, nanoparticles, radiation therapy, siRNA
molecules, enzyme/pro-drug pairs, photosensitizing agents, toxins,
microwaves, a radionuclide, an angiogenesis inhibitor, a mitosis
inhibitor protein (e.g., cdc6), an antitumor oligopeptide (e.g.,
antimitotic oligopeptides, high affinity tumor-selective binding
peptides), a signaling modulator, anti-cancer antibiotics, or a
combination thereof.
[0405] Exemplary photosensitizing agents include, but are not
limited to, for example, indocyanine green, toluidine blue,
aminolevulinic acid, texaphyrins, benzoporphyrins, phenothiazines,
phthalocyanines, porphyrins such as sodium porfimer, chlorins such
as tetra(m-hydroxyphenyl)chlorin or tin(IV) chlorin e6, purpurins
such as tin ethyl etiopurpurin, purpurinimides, bacteriochlorins,
pheophorbides, pyropheophorbides or cationic dyes. In one
embodiment, a vaccinia virus, such as a vaccinia virus provided
herein, is administered to a subject having a tumor, cancer or
metastasis in combination with a photosensitizing agent.
[0406] Radionuclides, which depending up the radionuclide, amount
and application can be used for diagnosis and/or for treatment.
They include, but are not limited to, for example, a compound or
molecule containing .sup.32Phosphate, .sup.60Cobalt,
.sup.90Yttirum, .sup.99Technicium, .sup.103 Palladium,
.sup.106Ruthenium, .sup.111Indium, .sup.117Lutetium,
.sup.125Iodine, .sup.131Iodine, .sup.137Cesium, .sup.153Samarium,
.sup.186Rhenium, .sup.188Rhenium, .sup.192Iridium, 198Gold,
.sup.211Astatine, .sup.212Bismuth or .sup.213Bismuth. In one
embodiment, a vaccinia virus, such as a vaccinia virus provided
herein, is administered to a subject having a tumor, cancer or
metastasis in combination with a radionuclide.
[0407] Toxins include, but are not limited to, chemotherapeutic
compounds such as, but not limited to, 5-fluorouridine,
calicheamicin and maytansine. Signaling modulators include, but are
not limited to, for example, inhibitors of macrophage inhibitory
factor, toll-like receptor agonists and stat 3 inhibitors. In one
embodiment, a vaccinia virus, such as a vaccinia virus provided
herein, is administered to a subject having a tumor, cancer or
metastasis in combination with a toxin or a signaling
modulator.
[0408] Combination therapy between chemotherapeutic agents and
therapeutic viruses can be effective/curative in situations when
single agent treatment is not effective. Chemotherapeutic compounds
include, but are not limited to, alkylating agents such as thiotepa
and cyclosphosphamide; alkyl sulfonates such as busulfan,
improsulfan and piposulfan; aziridines such as benzodopa,
carboquone, meturedopa and uredopa; ethylenimines and
methylamelamines including altretamine, triethylenemelamine,
trietylenephosphoramide, triethylenethiophosphaoramide and
trimethylolomelamime nitrogen mustards such as chiorambucil,
chlomaphazine, cholophosphamide, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,
novembichin, phenesterine, prednimustine, trofosfamide, uracil
mustard; nitrosureas such as carmustine, chlorozotocin,
fotemustine, lomustine, nimustine, ranimustine; antibiotics such as
aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,
cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin,
chromomycins, dactinomycin, daunorubicin, detorubicin,
6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,
idarubicin, marcellomycin, mitomycins, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate and 5-fluorouracil (5-FU); folic acid analogues such
as denopterin, methotrexate, pteropterin, trimetrexate; purine
analogs such as fludarabine, 6-mercaptopurine, thiamiprine,
thioguanine; pyrimidine analogs such as ancitabine, azacitidine,
6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,
enocitabine, floxuridine; androgens such as calusterone,
dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;
demecolcine; diaziquone; elfornithine; elliptinium acetate;
etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine;
mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin;
phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide;
procarbazine; polysaccharide-K; razoxane; sizofuran;
spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; urethan; vindesine; dacarbazine;
mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;
cytosine arabinoside; cyclophosphamide; thiotepa; taxoids, e.g.,
paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine;
mercaptopurine; methotrexate; platinum analogs such as cisplatin
and carboplatin; vinblastine; platinum; etoposide (VP-16);
ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine;
navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda;
ibandronate; CPT11; topoisomerase inhibitor RFS 2000;
difluoromethylomithine (DMFO); retinoic acid; esperamicins;
capecitabine; and pharmaceutically acceptable salts, acids or
derivatives of any of the above. Also included are anti-hormonal
agents that act to regulate or inhibit hormone action on tumors
such as anti-estrogens including for example tamoxifen, raloxifene,
aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen,
trioxifene, keoxifene, LY117018, onapristone and toremifene
(Fareston); and antiandrogens such as flutamide, nilutamide,
bicalutamide, leuprolide and goserelin; and pharmaceutically
acceptable salts, acids or derivatives of any of the above. Such
chemotherapeutic compounds that can be used herein include
compounds whose toxicities preclude use of the compound in general
systemic chemotherapeutic methods. Chemotherapeutic agents also
include new classes of targeted chemotherapeutic agents such as,
for example, imatinib (sold by Novartis under the trade name
Gleevec in the United States), gefitinib (developed by Astra Zeneca
under the trade name Iressa) and erlotinib. Particular
chemotherapeutic agents include, but are not limited to, cisplatin,
carboplatin, oxaliplatin, DWA2114R, NK121, IS 3 295, and 254-S
vincristine, prednisone, doxorubicin and L-asparaginase;
mechoroethamine, vincristine, procarbazine and prednisone (MOPP),
cyclophosphamide, vincristine, procarbazine and prednisone
(C-MOPP), bleomycin, vinblastine, gemcitabine and 5-fluorouracil.
Exemplary chemotherapeutic agents are, for example, cisplatin,
carboplatin, oxaliplatin, DWA2114R, NK121, IS 3 295, and 254-S. In
a non-limiting embodiment, a vaccinia virus, such as a vaccinia
virus provided herein, is administered to a subject having a tumor,
cancer or metastasis in combination with a platinum coordination
complex, such as cisplatin, carboplatin, oxaliplatin, DWA2114R,
NK121, IS 3 295, and 254-S. Tumors, cancers and metastasis can be
any of those provided herein, and in particular, can be a
pancreatic tumor, an ovarian tumor, a lung tumor, a colon tumor, a
prostate tumor, a cervical tumor or a breast tumor; exemplary
tumors are pancreatic and ovarian tumors. Tumors, cancers and
metastasis can be a monotherapy-resistant tumor such as, for
example, one that does not respond to therapy with virus alone or
anti-cancer agent alone, but that does respond to therapy with a
combination of virus and anti-cancer agent. Typically, a
therapeutically effective amount of virus is systemically
administered to the subject and the virus localizes and accumulates
in the tumor. Subsequent to administering the virus, the subject is
administered a therapeutically effective amount of an anti-cancer
agent, such as cisplatin. In one example, cisplatin is administered
once-daily for five consecutive days. One of skill in the art could
determine when to administer the anti-cancer agent subsequent to
the virus using, for example, in vivo animal models. Using the
methods provided herein, administration of a virus and anti-cancer
agent, such as cisplatin can cause a reduction in tumor volume, can
cause tumor growth to stop or be delayed or can cause the tumor to
be eliminated from the subject. The status of tumors, cancers and
metastasis following treatment can be monitored using any of the
methods provided herein and known in the art.
[0409] Exemplary anti-cancer antibiotics include, but are not
limited to, anthracyclines such as doxorubicin hydrochloride
(adriamycin), idarubicin hydrochloride, daunorubicin hydrochloride,
aclarubicin hydrochloride, epirubicin hydrochloride and purarubicin
hydrochloride, pleomycins such as pleomycin and peplomycin sulfate,
mitomycins such as mitomycin C, actinomycins such as actinomycin D,
zinostatinstimalamer and polypeptides such as neocarzinostatin. In
one embodiment, a vaccinia virus, such as a vaccinia virus provided
herein, is administered to a subject having a tumor, cancer or
metastasis in combination with an anti-cancer antibiotic.
[0410] In one embodiment, nanoparticles can be designed such that
they carry one or more therapeutic agents provided herein.
Additionally, nanoparticles can be designed to carry a molecule
that targets the nanoparticle to the tumor cells. In one
non-limiting example, nanoparticles can be coated with a
radionuclide and, optionally, an antibody immunoreactive with a
tumor-associated antigen. In one embodiment, a vaccinia virus, such
as a vaccinia virus provided herein, is administered to a subject
having a tumor, cancer or metastasis in combination with a
nanoparticle carrying any of the therapeutic agents provided
herein.
[0411] Radiation therapy has become a foremost choice of treatment
for a majority of cancer patients. The wide use of radiation
treatment stems from the ability of gamma-irradiation to induce
irreversible damage in targeted cells with the preservation of
normal tissue function. Ionizing radiation triggers apoptosis, the
intrinsic cellular death machinery in cancer cells, and the
activation of apoptosis seems to be the principal mode by which
cancer cells die following exposure to ionizing radiation. In one
embodiment, a vaccinia virus, such as a vaccinia virus provided
herein, is administered to a subject having a tumor, cancer or
metastasis in combination with radiation therapy.
[0412] Thus, provided herein are methods of administering to a
subject one or more therapeutic compounds that can act in
conjunction with the virus to increase the proliferation, toxicity,
tumor cell killing, or immune response eliciting properties of a
virus. Also provided herein are methods of administering to a
subject one or more therapeutic compounds that can act in
conjunction with the virus to decrease the proliferation, toxicity,
or cell killing properties of a virus. Therapeutic compounds to be
administered can be any of those provided herein or in the art.
[0413] Therapeutic compounds that can act in conjunction with the
virus to increase the proliferation, toxicity, tumor cell killing
or immune response eliciting properties of a virus are compounds
that can alter gene expression, where the altered gene expression
can result in an increased killing of tumor cells or an increased
anti-tumor immune response in the subject. A gene
expression-altering compound can, for example, cause an increase or
decrease in expression of one or more viral genes, including
endogenous viral genes and/or exogenous viral genes. For example, a
gene expression-altering compound can induce or increase
transcription of a gene in a virus such as an exogenous gene that
can cause cell lysis or cell death, that can provoke an immune
response, that can catalyze conversion of a prodrug-like compound,
or that can inhibit expression of a tumor cell gene. Any of a wide
variety of compounds that can alter gene expression are known in
the art, including IPTG and RU486. Exemplary genes whose expression
can be up-regulated include proteins and RNA molecules, including
toxins, enzymes that can convert a prodrug to an anti-tumor drug,
cytokines, transcription regulating proteins, siRNA and ribozymes.
In another example, a gene expression-altering compound can inhibit
or decrease transcription of a gene in a virus such as a
heterologous gene that can reduce viral toxicity or reduces viral
proliferation. Any of a variety of compounds that can reduce or
inhibit gene expression can be used in the methods provided herein,
including siRNA compounds, transcriptional inhibitors or inhibitors
of transcriptional activators. Exemplary genes whose expression can
be down-regulated include proteins and RNA molecules, including
viral proteins or RNA that suppress lysis, nucleotide synthesis or
proliferation, and cellular proteins or RNA molecules that suppress
cell death, immunoreactivity, lysis, or viral replication.
[0414] In another embodiment, therapeutic compounds that can act in
conjunction with the virus to increase the proliferation, toxicity,
tumor cell killing, or immune response eliciting properties of a
virus are compounds that can interact with a virally expressed gene
product, and such interaction can result in an increased killing of
tumor cells or an increased anti-tumor immune response in the
subject. A therapeutic compound that can interact with a
virally-expressed gene product can include, for example a prodrug
or other compound that has little or no toxicity or other
biological activity in its subject-administered form, but after
interaction with a virally expressed gene product, the compound can
develop a property that results in tumor cell death, including but
not limited to, cytotoxicity, ability to induce apoptosis, or
ability to trigger an immune response. In one non-limiting example,
the virus carries an enzyme into the cancer cells. Once the enzyme
is introduced into the cancer cells, an inactive form of a
chemotherapy drug (i.e., a prodrug) is administered. When the
inactive prodrug reaches the cancer cells, the enzyme converts the
prodrug into the active chemotherapy drug, so that it can kill the
cancer cell. Thus, the treatment is targeted only to cancer cells
and does not affect normal cells. The prodrug can be administered
concurrently with, or sequentially to, the virus. A variety of
prodrug-like substances are known in the art and an exemplary set
of such compounds are disclosed elsewhere herein, where such
compounds can include gancyclovir, 5-fluorouracil, 6-methylpurine
deoxyriboside, cephalosporin-doxorubicin,
4-[(2-chloroethyl)(2-mesuloxyethyl)amino]benzoyl-L-glutamic acid,
acetaminophen, indole-3-acetic acid, CB1954,
7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin,
bis-(2-chloroethyl)amino-4-hydroxyphenyl-aminomethanone 28,
1-chloromethyl-5-hydroxy-1,2-dihydro-3H-benz[e]indole,
epirubicin-glucuronide, 5'-deoxy-5-fluorouridine, cytosine
arabinoside, linamarin, and a nucleoside analogue (e.g.,
fluorouridine, fluorodeoxyuridine, fluorouridine arabinoside,
cytosine arabinoside, adenine arabinoside, guanine arabinoside,
hypoxanthine arabinoside, 6-mercaptopurineriboside, theoguanosine
riboside, nebularine, 5-iodouridine, 5-iododeoxyuridine,
5-bromodeoxyuridine, 5-vinyldeoxyuridine,
9-[(2-hydroxy)ethoxy]methylguanine (acyclovir),
9-[(2-hydroxy-1-hydroxymethyl)-ethoxy]methylguanine (DHPG),
azauridien, azacytidine, azidothymidine, dideoxyadenosine,
dideoxycytidine, dideoxyinosine, dideoxyguanosine,
dideoxythymidine, 3'-deoxyadenosine, 3'-deoxycytidine,
3'-deoxyinosine, 3'-deoxyguanosine, 3'-deoxythymidine).
[0415] In another embodiment, therapeutic compounds that can act in
conjunction with the virus to decrease the proliferation, toxicity
or cell killing properties of a virus are compounds that can
inhibit viral replication, inhibit viral toxins or cause viral
death. A therapeutic compound that can inhibit viral replication,
inhibit viral toxins, or cause viral death can generally include a
compound that can block one or more steps in the viral life cycle,
including, but not limited to, compounds that can inhibit viral DNA
replication, viral RNA transcription, viral coat protein assembly,
outer membrane or polysaccharide assembly. Any of a variety of
compounds that can block one or more steps in a viral life cycle
are known in the art, including any known antiviral compound (e.g.,
cidofovir), viral DNA polymerase inhibitors, viral RNA polymerase
inhibitors, inhibitors of proteins that regulate viral DNA
replication or RNA transcription. In another example, a virus can
contain a gene encoding a viral life cycle protein, such as DNA
polymerase or RNA polymerase that can be inhibited by a compound
that is, optionally, non-toxic to the host organism.
[0416] In addition to combination therapy between chemotherapeutic
agents and a virus provided herein, other more complex combination
therapy strategies could be applied as well. For example, a
combination therapy can include chemotherapeutic agents,
therapeutic antibodies, and a virus provided herein. Alternatively,
another combination therapy can be the combination of radiation,
therapeutic antibodies, and a virus provided herein. Therefore, the
concept of combination therapy also can be based on the application
of a virus provided herein virus along with one or more of the
following therapeutic modalities, namely, chemotherapeutic agents,
radiation therapy, therapeutic antibodies, hyper- or hypothermia
therapy, siRNA, diagnostic/therapeutic bacteria,
diagnostic/therapeutic mammalian cells, immunotherapy, and/or
targeted toxins (delivered by antibodies, liposomes and
nanoparticles).
[0417] Effective delivery of each components of the combination
therapy is an important aspect of the methods provided herein. In
accordance with one aspect, the modes of administration discussed
below exploit one of more of the key features: (i) delivery of a
virus provided herein to the tumors by a mode of administration
effect to achieve highest titer of virus and highest therapeutic
effect; (ii) delivery of any other mentioned therapeutic modalities
to the tumor by a mode of administration to achieve the optimal
therapeutic effect. The dose scheme of the combination therapy
administered is such that the combination of the two or more
therapeutic modalities is therapeutically effective. Dosages will
vary in accordance with such factors as the age, health, sex, size
and weight of the patient, the route of administration, the
toxicity of the drugs, frequency of treatment and the relative
susceptibilities of the cancer to each of the therapeutic
modalities.
[0418] iii. Immunotherapies and Biological Therapies
[0419] Therapeutic compounds also include, but are not limited to,
compounds that exert an immunotherapeutic effect, stimulate the
immune system, carry a therapeutic compound, or a combination
thereof. Optionally, the therapeutic agent can exhibit or manifest
additional properties, such as, properties that permit its use as
an imaging agent, as described elsewhere herein. Such therapeutic
compounds include, but are not limited to, anti-cancer antibodies,
radiation therapy, siRNA molecules and compounds that suppress the
immune system. Immunotherapy includes for example,
immune-stimulating molecules (protein-based or non-protein-based),
cells and antibodies. Immunotherapy treatments can include
stimulating immune cells to act more effectively or to make the
tumor cells or tumor associated antigens recognizable to the immune
system (i.e., break tolerance).
[0420] Cytokines and growth factors include, but are not limited
to, interleukins, such as, for example, interleukin-1,
interleukin-2, interleukin-6 and interleukin-12, tumor necrosis
factors, such as tumor necrosis factor alpha (TNF-.alpha.),
interferons such as interferon gamma (IFN-.gamma.), granulocyte
macrophage colony stimulating factors (GM-CSF), angiogenins, and
tissue factors.
[0421] Anti-cancer antibodies include, but are not limited to,
Rituximab, ADEPT, Trastuzumab (Herceptin), Tositumomab (Bexxar),
Cetuximab (Erbitux), Ibritumomab (Zevalin), Alemtuzumab
(Campath-1H), Epratuzumab (Lymphocide), Gemtuzumab ozogamicin
(Mylotarg), Bevacimab (Avastin), Tarceva (Erlotinib), SUTENT
(sunitinib malate), Panorex (Edrecolomab), RITUXAN (Rituximab),
Zevalin (.sup.90Y-ibritumomab tiuexetan), Mylotarg (Gemtuzumab
Ozogamicin) and Campath (Alemtuzumab).
[0422] Thus, provided herein are methods of administering to a
subject one or more therapeutic compounds that can act in
conjunction with the virus to stimulate or enhance the immune
system, thereby enhancing the effect of the virus. Such
immunotherapy can be either delivered as a separate therapeutic
modality or could be encoded (if the immunotherapy is
protein-based) by the administered virus.
[0423] Biological therapies are treatments that use natural body
substances or drugs made from natural body substances. They can
help to treat a cancer and control side effects caused by other
cancer treatments such as chemotherapy. Biological therapies are
also sometimes called Biological Response Modifiers (BRM's),
biologic agents or simply "biologics" because they stimulate the
body to respond biologically (or naturally) to cancer.
Immunotherapy is treatment using natural substances that the body
uses to fight infection and disease. Because it uses natural
substances, immunotherapy is also a biological therapy. There are
several types of drugs that come under the term biological therapy:
these include, for example, monoclonal antibodies (mAbs), cancer
vaccines, growth factors for blood cells, cancer growth inhibitors,
anti-angiogenic factors, interferon alpha, interleukin-2 (IL-2),
gene therapy and BCG vaccine for bladder cancer
[0424] Monoclonal antibodies (mAbs) are of particular interest for
treating cancer because of the specificity of binding to a unique
antigen and the ability to produce large quantities in the
laboratory for mass distribution. Monoclonal antibodies can be
engineered to act in the same way as immune system proteins: that
is, to seek out and kill foreign matter in your body, such as
viruses. Monoclonal antibodies can be designed to recognize
epitopes on the surface of cancer cells. The antibodies target
specifically bind to the epitopes and either kill the cancer cells
or deliver a therapeutic agent to the cancer cell. Methods of
conjugating therapeutic agents to antibodies is well-known in the
art. Different antibodies have to be made for different types of
cancer; for example, Rituximab recognizes CD20 protein on the
outside of non Hodgkin's lymphoma cells; ADEPT is a treatment using
antibodies that recognize bowel (colon) cancer; and Trastuzumab
(Herceptin) recognizes breast cancer cells that produce too much of
the protein HER 2 ("HER 2 positive"). Other antibodies include, for
example, Tositumomab (Bexxar), Cetuximab (Erbitux), Ibritumomab
(Zevalin), Alemtuzumab (Campath-1H), Epratuzumab (Lymphocide),
Gemtuzumab ozogamicin (Mylotarg) and Bevacimab (Avastin). Thus, the
viruses provided herein can be administered concurrently with, or
sequentially to, one or more monoclonal antibodies in the treatment
of cancer. In one embodiment, additional therapy is administered in
the form of one or more of any of the other treatment modalities
provided herein.
[0425] Rather than attempting to prevent infection, such as is the
case with the influenza virus, cancer vaccines help treat the
cancer once it has developed. The aim of cancer vaccines is to
stimulate the immune response. Cancer vaccines include, for
example, antigen vaccines, whole cell vaccines, dendritic cell
vaccines, DNA vaccines and anti-idiotype vaccines. Antigen vaccines
are vaccines made from tumor-associated antigens in, or produced
by, cancer cells. Antigen vaccines stimulate a subject's immune
system to attack the cancer. Whole cell vaccines are vaccines that
use the whole cancer cell, not just a specific antigen from it, to
make the vaccine. The vaccine is made from a subject's own cancer
cells, another subject's cancer cells or cancer cells grown in a
laboratory. The cells are treated in the laboratory, usually with
radiation, so that they can't grow, and are administered to the
subject via injection or through an intravenous drip into the
bloodstream so they can stimulate the immune system to attack the
cancer. One type of whole cell vaccine is a dendritic cell vaccine,
which help the immune system to recognize and attack abnormal
cells, such as cancer cells. Dendritic cell vaccines are made by
growing dendritic cells alongside the cancer cells in the lab. The
vaccine is administered to stimulate the immune system to attack
the cancer. Anti-idiotype vaccines are vaccines that stimulate the
body to make antibodies against cancer cells. Cancer cells make
some tumor-associated antigens that the immune system recognizes as
foreign. But because cancer cells are similar to non-cancer cells,
the immune system can respond weakly. DNA vaccines boost the immune
response. DNA vaccines are made from DNA from cancer cells that
carry the genes for the tumor-associated antigens. When a DNA
vaccine is injected, it enables the cells of the immune system to
recognize the tumor-associated antigens, and activates the cells in
the immune system (i.e., breaking tolerance). The most promising
results from using DNA vaccines are in treating melanoma. Thus, the
viruses provided herein can be administered concurrently with, or
sequentially to, a whole cell vaccine in the treatment of cancer.
In one embodiment, additional therapy is administered in the form
of one or more of any of the other treatment modalities provided
herein.
[0426] Growth factors are natural substances that stimulate the
bone marrow to make blood cells. Recombinant technology can be used
to generate growth factors which can be administered to a subject
to increase the number of white blood cells, red blood cells and
stem cells in the blood. Growth factors used in cancer treatment to
boost white blood cells include Granulocyte Colony Stimulating
Factor (G-CSF) also called filgrastim (Neupogen) or lenograstim
(Granocyte) and Granulocyte and Macrophage Colony Stimulating
Factor (GM-CSF), also called molgramostim. A growth factor to help
treat anemia is erythropoietin (EPO). EPO encourages the body to
make more red blood cells, which in turn, increases hemoglobin
levels and the levels of oxygen in body tissues. Other growth
factors are being developed which can boost platelets. Thus, the
viruses provided herein can be administered concurrently with, or
sequentially to, a growth factor such as GM-CSF, in the treatment
of cancer. In one embodiment, additional therapy is administered in
the form of one or more of any of the other treatment modalities
provided herein.
[0427] Cancer growth inhibitors use cell-signaling molecules which
control the growth and multiplication of cells, such as cancer
cells. Drugs that block these signaling molecules can stop cancers
from growing and dividing. Cancer growth factors include, but are
not limited to, tyrosine kinases. Thus, drugs that block tyrosine
kinases are tyrosine kinase inhibitors (TKIs). Examples of TKIs
include, but are not limited to, Erlotinib (Tarceva, OSI-774),
Iressa (Gefitinib, ZD 1839) and Imatinib (Glivec, STI 571). Another
type of growth inhibitor is Bortezomib (Velcade) for multiple
myeloma and for some other cancers. Velcade is a proteasome
inhibitor. Proteasomes are found in all cells and help break down
proteins in cells. Interfering with the action of proteosomes
causes a build up of proteins in the cell to toxic levels; thereby
killing the cancer cells. Cancer cells are more sensitive to
Velcade than normal cells. Thus, the viruses provided herein can be
administered concurrently with, or sequentially to, a cancer growth
inhibitor, such as Velcade, in the treatment of cancer. In one
embodiment, additional therapy is administered in the form of one
or more of any of the other treatment modalities provided
herein.
[0428] Cancers need a blood supply to expand and grow their own
blood vessels as they get bigger. Without its own blood supply, a
cancer cannot grow due to lack of nutrients and oxygen.
Anti-angiogenic drugs stop tumors from developing their own blood
vessels. Examples of these types of drugs include, but are not
limited to, Thalidomide, mainly for treating myeloma but also in
trials for other types of cancer, and Bevacizumab (Avastin), a type
of monoclonal antibody that has been investigated for bowel cancer.
Thus, the viruses provided herein can be administered concurrently
with, or sequentially to, an anti-angiogenic drug in the treatment
of cancer. In one embodiment, additional therapy is administered in
the form of one or more of any of the other treatment modalities
provided herein.
[0429] Interferon-alpha (IFN-.alpha.) is a natural substance
produced in the body, in very small amounts, as part of the immune
response. IFN-.alpha. is administered as a treatment to boost the
immune system and help fight cancers such as renal cell (kidney)
cancer, malignant melanoma, multiple myeloma and some types of
leukemias. IFN-.alpha. works in several ways: it can help to stop
cancer cells growing, it can also boost the immune system to help
it attack the cancer, and it can affect the blood supply to the
cancer cells. Thus, the viruses provided herein can be administered
concurrently with, or sequentially to, IFN-.alpha. in the treatment
of cancer. In one embodiment, additional therapy is administered in
the form of one or more of any of the other treatment modalities
provided herein.
[0430] Administration of IL-2 is a biological therapy drug because
it is naturally produced by the immune system. Thus, it is also an
immunotherapy. Interleukin 2 is used in treating renal cell
(kidney) cancer, and is being tested in clinical trials for several
other types of cancers. IL-2 works directly on cancer cells by
interfering with cell grow and proliferation; it stimulates the
immune system by promoting the growth of killer T cells and other
cells that attack cancer cells; and it also stimulates cancer cells
to secrete chemoattractants that attract immune system cells. IL-2
is generally administered as a subcutaneous injection just under
the skin once daily for 5 days, followed by 2 days rest. The cycle
of injections is repeated for 4 weeks followed by a week without
treatment. The treatment regiment and the number of cycles
administered depends on the type of cancer and how it responds to
the treatment. IL-2 can be self-administered or administered by a
health professional. Alternatively, IL-2 can be administered
intravenously via injection or drip. Thus, the viruses provided
herein can be administered concurrently with, or sequentially to,
IL-2 in the treatment of cancer. In one embodiment, additional
therapy is administered in the form of one or more of any of the
other treatment modalities provided herein.
[0431] Gene therapy involves treating cancer by blocking abnormal
genes in cancer cells, repairing or replacing abnormal genes in
cancer cells, encouraging even more genes to become abnormal in
cancer cells so that they die or become sensitive to treatment,
using viruses to carry treatment-activating enzymes into the cancer
cells, or a combination thereof. As a result, cancer cells die due
to damage in the cell. Cancer cells develop as a result of several
types of mutations in several of their genes. Targeted genes
include, but are not limited to, those that encourage the cell to
multiply (i.e., oncogenes), genes that stop the cell multiplying
(i.e., tumor suppressor genes) and genes that repair other damaged
genes. Gene therapy can involve repair of damaged oncogenes or
blocking the proteins that the oncogenes produce. The tumor
suppressor gene, p53, is damaged in many human cancers. Viruses
have been used in to deliver an undamaged p53 gene into cancer
cells, and early clinical trials are now in progress looking at
treating cancers with modified p53-producing viruses. Gene therapy
could be used to replace the damaged DNA repairing genes. In an
alternative embodiment, methods of increasing DNA damage within a
tumor cell can promote death of the tumor cell or cause increased
susceptibility of the tumor cell to other cancer treatments, such
as radiotherapy or chemotherapy. Thus, the viruses provided herein
can be administered concurrently with, or sequentially to, any of
the gene therapy methods provided herein or known in the art in the
treatment of cancer. In one embodiment, additional therapy is
administered in the form of one or more of any of the other
treatment modalities provided herein.
[0432] Treatment of early stage bladder cancer is called
intravesical treatment, which is mainly used to treat stage T1
bladder cancers that are high grade (grade 3 or G3) or carcinoma in
situ of the bladder (also known as T is or CIS). BCG is a vaccine
for tuberculosis (TB), which also has been found to be effective in
treating CIS and preventing bladder cancers from recurring. In some
cases, BCG vaccines have been used for treating grade 2 early
bladder cancer. Because bladder cancer can occur anywhere in the
bladder lining, it cannot be removed in the same way as the
papillary early bladder cancers. Rather a BCG vaccine is
administered using intravesical therapy; that is, first, a catheter
(tube) put is inserted into the bladder, followed by intra-catheter
administration of a BCG vaccine and/or a chemotherapy. BCG
treatment occurs weekly for 6 weeks or more depending on the effect
on the bladder cancer. BCG treatment of bladder cancer can be
combined with other types of treatments, such as administration of
chemotherapy (intravesical), IL-2, treatment with drugs that make
cells sensitive to light, vitamins, and photodynamic therapy. Thus,
the viruses provided herein can be administered concurrently with,
or sequentially to, BCG vaccines in the treatment of cancer. In one
embodiment, additional therapy is administered in the form of one
or more of any of the other treatment modalities provided
herein.
[0433] f. State of Subject
[0434] In another embodiment, the methods provided herein for
administering a virus to a subject can be performed on a subject in
any of a variety of states, including an anesthetized subject, an
alert subject, a subject with elevated body temperature, a subject
with reduced body temperature, or other state of the subject that
is known to affect the accumulation of a virus in the tumor. As
provided herein, it has been determined that a subject that is
anesthetized can have a decreased rate of accumulation of a virus
in a tumor relative to a subject that is not anesthetized. Further
provided herein, it has been determined that a subject with
decreased body temperature can have a decreased rate of
accumulation of a virus in a tumor relative to a subject with a
normal body temperature. Accordingly, provided herein are methods
of administering a virus to a subject, where the methods can
include administering a virus to a subject where the subject is not
under anesthesia, such as general anesthesia; for example, the
subject can be under local anesthesia, or can be unanesthetized.
Also provided herein are methods of administering a virus to a
subject, where the methods can include administering a virus to a
subject with altered body temperature, where the alteration of the
body temperature can influence the ability of the virus to
accumulate in a tumor; typically, a decrease in body temperature
can decrease the ability of a virus to accumulate in a tumor. Thus,
in one exemplary embodiment, a method is provided for administering
a virus to a subject, where the method includes elevating the body
temperature of the subject to a temperature above normal, and
administering a virus to the subject, where the virus can
accumulate in the tumor more readily in the subject with higher
body temperature relative to the ability of the virus to accumulate
in a tumor of a subject with a normal body temperature. In another
embodiment, localized elevations in temperature in the area
surrounding the tumor can be used to increase the accumulation of
the virus in the tumor.
[0435] 2. Monitoring
[0436] The methods provided herein can further include one or more
steps of monitoring the subject, monitoring the tumor, and/or
monitoring the virus administered to the subject. Any of a variety
of monitoring steps can be included in the methods provided herein,
including, but not limited to, monitoring tumor size, monitoring
anti-(tumor antigen) antibody titer, monitoring the presence and/or
size of metastases, monitoring the subject's lymph nodes,
monitoring the subject's weight or other health indicators
including blood or urine markers, monitoring anti-(viral antigen)
antibody titer, monitoring viral expression of a detectable gene
product, and directly monitoring viral titer in a tumor, tissue or
organ of a subject.
[0437] The purpose of the monitoring can be simply for assessing
the health state of the subject or the progress of therapeutic
treatment of the subject, or can be for determining whether or not
further administration of the same or a different virus is
warranted, or for determining when or whether or not to administer
a compound to the subject where the compound can act to increase
the efficacy of the therapeutic method, or the compound can act to
decrease the pathogenicity of the virus administered to the
subject.
[0438] a. Monitoring Viral Gene Expression
[0439] In some embodiments, the methods provided herein can include
monitoring one or more virally expressed genes. Viruses, such as
those provided herein that encode a tranporter protein or otherwise
known in the art, can express one or more detectable gene products,
including but not limited to, detectable proteins. The infected
cells/tissue can thus be imaged by one more imaging methods. For
example the infected cells can be imaged by administering a labeled
transporter substrate that is transported into the infected cell by
the expressed transporter protein. The localization of the
accumulated transporter protein can detected, thereby imaging the
infected tissue. The viruses also can encode one more additional
detectable proteins that can be imaged by optical or non-optical
imaging methods.
[0440] As provided herein, measurement of a detectable gene product
expressed by a virus can provide an accurate determination of the
level of virus present in the subject. As further provided herein,
measurement of the location of the detectable gene product, for
example, by imaging methods including, but not limited to, magnetic
resonance, fluorescence, and tomographic methods, can determine the
localization of the virus in the subject. Accordingly, the methods
provided herein that include monitoring a detectable viral gene
product can be used to determine the presence or absence of the
virus in one or more organs or tissues of a subject, and/or the
presence or absence of the virus in a tumor or metastases of a
subject. Further, the methods provided herein that include
monitoring a detectable viral gene product can be used to determine
the titer of virus present in one or more organs, tissues, tumors
or metastases. Methods that include monitoring the localization
and/or titer of viruses in a subject can be used for determining
the pathogenicity of a virus; since viral infection, and
particularly the level of infection, of normal tissues and organs
can indicate the pathogenicity of the probe, methods of monitoring
the localization and/or amount of viruses in a subject can be used
to determine the pathogenicity of a virus. Since methods provided
herein can be used to monitor the amount of viruses at any
particular location in a subject, the methods that include
monitoring the localization and/or titer of viruses in a subject
can be performed at multiple time points, and, accordingly can
determine the rate of viral replication in a subject, including the
rate of viral replication in one or more organs or tissues of a
subject; accordingly, the methods of monitoring a viral gene
product can be used for determining the replication competence of a
virus. The methods provided herein also can be used to quantitate
the amount of virus present in a variety of organs or tissues, and
tumors or metastases, and can thereby indicate the degree of
preferential accumulation of the virus in a subject; accordingly,
the viral gene product monitoring methods provided herein can be
used in methods of determining the ability of a virus to accumulate
in tumor or metastases in preference to normal tissues or organs.
Since the viruses used in the methods provided herein can
accumulate in an entire tumor or can accumulate at multiple sites
in a tumor, and can also accumulate in metastases, the methods
provided herein for monitoring a viral gene product can be used to
determine the size of a tumor or the number of metastases that are
present in a subject. Monitoring such presence of viral gene
product in tumor or metastasis over a range of time can be used to
assess changes in the tumor or metastasis, including growth or
shrinking of a tumor, or development of new metastases or
disappearance of metastases, and also can be used to determine the
rate of growth or shrinking of a tumor, or development of new
metastases or disappearance of metastases, or the change in the
rate of growth or shrinking of a tumor, or development of new
metastases or disappearance of metastases. Accordingly, the methods
of monitoring a viral gene product can be used for monitoring a
neoplastic disease in a subject, or for determining the efficacy of
treatment of a neoplastic disease, by determining rate of growth or
shrinking of a tumor, or development of new metastases or
disappearance of metastases, or the change in the rate of growth or
shrinking of a tumor, or development of new metastases or
disappearance of metastases.
[0441] Any of a variety of detectable proteins can be detected in
the monitoring methods provided herein; an exemplary, non-limiting
list of such detectable proteins includes any of a variety of
fluorescent proteins (e.g., green or red fluorescent proteins), any
of a variety of luciferases, transferrin or other iron binding
proteins; or receptors, binding proteins, and antibodies, where a
compound that specifically binds the receptor, binding protein or
antibody can be a detectable agent or can be labeled with a
detectable substance (e.g., a radionuclide or imaging agent).
Viruses expressing a detectable protein can be detected by a
combination of the method provided herein and know in the art.
Viruses expressing more than one detectable protein or two or more
viruses expressing various detectable protein can be detected and
distinguished by dual imaging methods. For example, a virus
expressing a fluorescent protein and an iron binding protein can be
detected in vitro or in vivo by low light fluorescence imaging and
magnetic resonance, respectively. In another example, a virus
expressing two or more fluorescent proteins can be detected by
fluorescence imaging at different wavelength. In vivo dual imaging
can be performed on a subject that has been administered a virus
expressing two or more detectable gene products or two or more
viruses each expressing one or more detectable gene products. In a
particular example, the viruses such as the hNET encoding viruses
provided herein can be imaged by detection of the accumulation of a
radiolabeled MIBG substrate that is transported by the hNET
transporter into a tumor cell. Such viruses can also be imaged by
optical methods such as detection of GFP expression by fluorescence
imaging.
[0442] b. Monitoring Tumor Size
[0443] Also provided herein are methods of monitoring tumor and/or
metastasis size and location. Tumor and or metastasis size can be
monitored by any of a variety of methods known in the art,
including external assessment methods or tomographic or magnetic
imaging methods. In addition to the methods known in the art,
methods provided herein, for example, monitoring viral gene
expression, can be used for monitoring tumor and/or metastasis
size.
[0444] Monitoring size over several time points can provide
information regarding the increase or decrease in size of a tumor
or metastasis, and can also provide information regarding the
presence of additional tumors and/or metastases in the subject.
Monitoring tumor size over several time points can provide
information regarding the development of a neoplastic disease in a
subject, including the efficacy of treatment of a neoplastic
disease in a subject.
[0445] c. Monitoring Antibody Titer
[0446] The methods provided herein also can include monitoring the
antibody titer in a subject, including antibodies produced in
response to administration of a virus to a subject. The viruses
administered in the methods provided herein can elicit an immune
response to endogenous viral antigens. The viruses administered in
the methods provided herein also can elicit an immune response to
exogenous genes expressed by a virus. The viruses administered in
the methods provided herein also can elicit an immune response to
tumor antigens. Monitoring antibody titer against viral antigens,
viral expressed exogenous gene products, or tumor antigens can be
used in methods of monitoring the toxicity of a virus, monitoring
the efficacy of treatment methods, or monitoring the level of gene
product or antibodies for production and/or harvesting.
[0447] In one embodiment, monitoring antibody titer can be used to
monitor the toxicity of a virus. Antibody titer against a virus can
vary over the time period after administration of the virus to the
subject, where at some particular time points, a low anti-(viral
antigen) antibody titer can indicate a higher toxicity, while at
other time points a high anti-(viral antigen) antibody titer can
indicate a higher toxicity. The viruses used in the methods
provided herein can be immunogenic, and can, therefore, elicit an
immune response soon after administering the virus to the subject.
Generally, a virus against which a subject's immune system can
quickly mount a strong immune response can be a virus that has low
toxicity when the subject's immune system can remove the virus from
all normal organs or tissues. Thus, in some embodiments, a high
antibody titer against viral antigens soon after administering the
virus to a subject can indicate low toxicity of a virus. In
contrast, a virus that is not highly immunogenic can infect a host
organism without eliciting a strong immune response, which can
result in a higher toxicity of the virus to the host. Accordingly,
in some embodiments, a high antibody titer against viral antigens
soon after administering the virus to a subject can indicate low
toxicity of a virus.
[0448] In other embodiments, monitoring antibody titer can be used
to monitor the efficacy of treatment methods. In the methods
provided herein, antibody titer, such as anti-(tumor antigen)
antibody titer, can indicate the efficacy of a therapeutic method
such as a therapeutic method to treat neoplastic disease.
Therapeutic methods provided herein can include causing or
enhancing an immune response against a tumor and/or metastasis.
Thus, by monitoring the anti-(tumor antigen) antibody titer, it is
possible to monitor the efficacy of a therapeutic method in causing
or enhancing an immune response against a tumor and/or metastasis.
The therapeutic methods provided herein also can include
administering to a subject a virus that can accumulate in a tumor
and can cause or enhance an anti-tumor immune response.
Accordingly, it is possible to monitor the ability of a host to
mount an immune response against viruses accumulated in a tumor or
metastasis, which can indicate that a subject has also mounted an
anti-tumor immune response, or can indicate that a subject is
likely to mount an anti-tumor immune response, or can indicate that
a subject is capable of mounting an anti-tumor immune response.
[0449] In other embodiments, monitoring antibody titer can be used
for monitoring the level of gene product or antibodies for
production and/or harvesting. As provided herein, methods can be
used for producing proteins, RNA molecules or other compounds by
expressing an exogenous gene in a virus that has accumulated in a
tumor. Further provided herein are methods for producing antibodies
against a protein, RNA molecule or other compound produced by
exogenous gene expression of a virus that has accumulated in a
tumor. Monitoring antibody titer against the protein, RNA molecule
or other compound can indicate the level of production of the
protein, RNA molecule or other compound by the tumor-accumulated
virus, and also can directly indicate the level of antibodies
specific for such a protein, RNA molecule or other compound.
[0450] d. Monitoring General Health Diagnostics
[0451] The methods provided herein also can include methods of
monitoring the health of a subject. Some of the methods provided
herein are therapeutic methods, including neoplastic disease
therapeutic methods. Monitoring the health of a subject can be used
to determine the efficacy of the therapeutic method, as is known in
the art. The methods provided herein also can include a step of
administering to a subject a virus. Monitoring the health of a
subject can be used to determine the pathogenicity of a virus
administered to a subject. Any of a variety of health diagnostic
methods for monitoring disease such as neoplastic disease,
infectious disease, or immune-related disease can be monitored, as
is known in the art. For example, the weight, blood pressure,
pulse, breathing, color, temperature or other observable state of a
subject can indicate the health of a subject. In addition, the
presence or absence or level of one or more components in a sample
from a subject can indicate the health of a subject. Typical
samples can include blood and urine samples, where the presence or
absence or level of one or more components can be determined by
performing, for example, a blood panel or a urine panel diagnostic
test. Exemplary components indicative of a subject's health
include, but are not limited to, white blood cell count,
hematocrit, or reactive protein concentration.
[0452] e. Monitoring Coordinated with Treatment
[0453] Also provided herein are methods of monitoring a therapy,
where therapeutic decisions can be based on the results of the
monitoring. Therapeutic methods provided herein can include
administering to a subject a virus, where the virus can
preferentially accumulate in a tumor and/or metastasis, and where
the virus can cause or enhance an anti-tumor immune response. Such
therapeutic methods can include a variety of steps including
multiple administrations of a particular virus, administration of a
second virus, or administration of a therapeutic compound.
Determination of the amount, timing or type of virus or compound to
administer to the subject can be based on one or more results from
monitoring the subject. For example, the antibody titer in a
subject can be used to determine whether or not it is desirable to
administer a virus or compound, the quantity of virus or compound
to administer, and the type of virus or compound to administer,
where, for example, a low antibody titer can indicate the
desirability of administering additional virus, a different virus,
or a therapeutic compound such as a compound that induces viral
gene expression. In another example, the overall health state of a
subject can be used to determine whether or not it is desirable to
administer a virus or compound, the quantity of virus or compound
to administer, and the type of virus or compound to administer,
where, for example, determining that the subject is healthy can
indicate the desirability of administering additional virus, a
different virus, or a therapeutic compound such as a compound that
induces viral gene expression. In another example, monitoring a
detectable virally expressed gene product can be used to determine
whether or not it is desirable to administer a virus or compound,
the quantity of virus or compound to administer, and the type of
virus or compound to administer. Such monitoring methods can be
used to determine whether or not the therapeutic method is
effective, whether or not the therapeutic method is pathogenic to
the subject, whether or not the virus has accumulated in a tumor or
metastasis, and whether or not the virus has accumulated in normal
tissues or organs. Based on such determinations, the desirability
and form of further therapeutic methods can be derived.
[0454] In one embodiment, determination of whether or not a
therapeutic method is effective can be used to derive further
therapeutic methods. Any of a variety of methods of monitoring can
be used to determine whether or not a therapeutic method is
effective, as provided herein or otherwise known in the art. If
monitoring methods indicate that the therapeutic method is
effective, a decision can be made to maintain the current course of
therapy, which can include further administrations of a virus or
compound, or a decision can be made that no further administrations
are required. If monitoring methods indicate that the therapeutic
method is ineffective, the monitoring results can indicate whether
or not a course of treatment should be discontinued (e.g., when a
virus is pathogenic to the subject), or changed (e.g., when a virus
accumulates in a tumor without harming the host organism, but
without eliciting an anti-tumor immune response), or increased in
frequency or amount (e.g., when little or no virus accumulates in
tumor).
[0455] In one example, monitoring can indicate that a virus is
pathogenic to a subject. In such instances, a decision can be made
to terminate administration of the virus to the subject, to
administer lower levels of the virus to the subject, to administer
a different virus to a subject, or to administer to a subject a
compound that reduces the pathogenicity of the virus. In one
example, administration of a virus that is determined to be
pathogenic can be terminated. In another example, the dosage amount
of a virus that is determined to be pathogenic can be decreased for
subsequent administration; in one version of such an example, the
subject can be pre-treated with another virus that can increase the
ability of the pathogenic virus to accumulate in tumor, prior to
re-administering the pathogenic virus to the subject. In another
example, a subject can have administered thereto a virus that is
pathogenic to the subject; administration of such a pathogenic
virus can be accompanied by administration of, for example, an
antiviral compound (e.g., cidofovir), pathogenicity attenuating
compound (e.g., a compound that down-regulates the expression of a
lytic or apoptotic gene product), or other compound that can
decrease the proliferation, toxicity, or cell killing properties of
a virus, as described herein elsewhere. In one variation of such an
example, the localization of the virus can be monitored, and, upon
determination that the virus is accumulated in tumor and/or
metastases but not in normal tissues or organs, administration of
the antiviral compound or pathogenicity attenuating compound can be
terminated, and the pathogenic activity of the virus can be
activated or increased, but limited to the tumor and/or metastasis.
In another variation of such an example, after terminating
administration of the antiviral compound or pathogenicity
attenuating compound, the presence of the virus and/or
pathogenicity of the virus can be further monitored, and
administration of such a compound can be reinitiated if the virus
is determined to pose a threat to the host by, for example,
spreading to normal organs or tissues, releasing a toxin into the
vasculature, or otherwise having pathogenic effects reaching beyond
the tumor or metastasis.
[0456] In another example, monitoring can determine whether or not
a virus has accumulated in a tumor or metastasis of a subject. Upon
such a determination, a decision can be made to further administer
additional virus, a different virus or a compound to the subject.
In another example, monitoring the presence of a virus in a tumor
can be used in deciding to administer to the subject a compound,
where the compound can increase the pathogenicity, proliferation,
or immunogenicity of a virus or the compound can otherwise act in
conjunction with the virus to increase the proliferation, toxicity,
tumor cell killing, or immune response eliciting properties of a
virus; in one variation of such an example, the virus can, for
example, have little or no lytic or cell killing capability in the
absence of such a compound; in a further variation of such an
example, monitoring of the presence of the virus in a tumor or
metastasis can be coupled with monitoring the absence of the virus
in normal tissues or organs, where the compound is administered if
the virus is present in tumor or metastasis and not at all present
or substantially not present in normal organs or tissues; in a
further variation of such an example, the amount of virus in a
tumor or metastasis can be monitored, where the compound is
administered if the virus is present in tumor or metastasis at
sufficient levels.
H. OTHER MICROORGANISMS AND CELLS
[0457] In some examples, an isolated cell, such as a mammalian
cell, can contain any of the viruses provided herein that encode a
transporter protein. For example, an isolated cell can be infected
with a virus provided herein. Exemplary of such cells are tumor
cells, stem cells, immune cells or other cells that can localize in
tumor tissues. The virally infected cells also can be administered
to patient or subject with a tumor for diagnosis and/or treatment
of the tumor.
I. EXAMPLES
[0458] The following examples are included for illustrative
purposes only and are not intended to limit the scope of the
invention.
Example 1
Recombinant Viruses Generation
A. Construction of Modified Vaccinia Viruses
[0459] Modified vaccinia viruses containing DNA encoding several
types of transporter proteins were generated by removing and
inserting nucleic acid at several gene loci in a vaccinia virus
genome, including F14.5L (also referred to as F3; see U.S. Patent
Publication No. 2005/0031643), thymidine kinase (TK; J2R) and/or
hemagglutinin (HA; A56R) gene loci. The heterologous DNA inserted
into the virus genome included expression cassettes containing
protein-encoding DNA operably linked to a vaccinia virus promoter.
Modified vaccinia strains encoding the following transporter
proteins were generated: Human norepinephrine transporter (hNET)
and the human sodium iodide symporter (hNIS). Modified vaccinia
viruses containing a transporter gene, hNET, and a anti-cancer
therapeutic gene, IL-24, also were generated.
[0460] The starting strain for the modified vaccinia viruses
described herein was vaccinia virus (VV) strain GLV-1h68 (also
named RVGL21, SEQ ID NO: 1). This genetically engineered strain,
which has been described in U.S. Patent Publication No.
2005/0031643, contains DNA insertions in the F14.5L, thymidine
kinase (TK) and hemagglutinin (HA) genes. GLV-1h68 was prepared
from the vaccinia virus strain designated LIVP (a vaccinia virus
strain, originally derived by adapting the vaccinia Lister strain
(ATCC Catalog No. VR-1549) to calf skin (Research Institute of
Viral Preparations, Moscow, Russia, Al'tshtein et al. (1983) Dokl.
Akad. Nauk USSR 285:696-699). The LIVP strain, whose genome
sequence is set forth in SEQ ID NO: 2 and from which GLV-1h68 was
generated, contains a mutation in the coding sequence of the TK
gene, in which a substitution of a guanine nucleotide with a
thymidine nucleotide (nucleotide position 80207 of SEQ ID NO: 2)
introduces a premature STOP codon within the coding sequence.
[0461] As described in U.S. Patent Publication No. 2005/0031643
(see particularly, Example 1 of the application), GLV-1h68 was
generated by inserting expression cassettes encoding detectable
marker proteins into the F14.5L (also designated in LIVP as F3)
gene, thymidine kinase (TK) gene, and hemagglutinin (HA) gene loci
of the vaccinia virus LIVP strain. Specifically, an expression
cassette containing a Ruc-GFP cDNA (a fusion of DNA encoding
Renilla luciferase and DNA encoding GFP) under the control of a
vaccinia synthetic early/late promoter P.sub.SEL was inserted into
the F14.5L gene; an expression cassette containing DNA encoding
beta-galactosidase under the control of the vaccinia early/late
promoter P.sub.7.5k (denoted (P.sub.7.5k)LacZ) and DNA encoding a
rat transferrin receptor positioned in the reverse orientation for
transcription relative to the vaccinia synthetic early/late
promoter P.sub.SEL (denoted (P.sub.SEL)rTrfR) was inserted into the
TK gene (the resulting virus does not express transferrin receptor
protein since the DNA encoding the protein is positioned in the
reverse orientation for transcription relative to the promoter in
the cassette); and an expression cassette containing DNA encoding
.beta.-glucuronidase under the control of the vaccinia late
promoter P.sub.11k (denoted (P.sub.11k)gusA) was inserted into the
HA gene.
[0462] Insertion of the expression cassettes into the LIVP genome
to generate the GLV-1h68 strain resulted in disruption of the
coding sequences for each of the F14.5L, TK and HA genes;
accordingly, all three genes in the resulting strains are
nonfunctional in that they do not encode the corresponding
full-length proteins. As described in U.S. Patent Publication No.
2005/0031643, disruption of these genes not only attenuates the
virus, but also enhances its tumor-specific accumulation. Previous
data have shown that systemic delivery of the GLV-1h68 virus in a
mouse model of breast cancer resulted in the complete eradication
of large subcutaneous GI-101A human breast carcinoma xenograft
tumors in nude mice (see U.S. Patent Publication No.
2005/0031643).
[0463] 1. Modified Viral Strains
[0464] Modified recombinant vaccinia viruses containing
heterologous DNA inserted into one or more loci of the vaccinia
virus genome were generated via homologous recombination between
DNA sequences in the genome and a transfer vector using methods
described herein and known to those of skill in the art (see, e.g.,
Falkner and Moss (1990) J. Virol. 64:3108-2111; Chakrabarti et al.
(1985) Mol. Cell. Biol. 5:3403-3409; and U.S. Pat. No. 4,722,848).
In these methods, the existing target gene in the starting vaccinia
virus genome is replaced by an interrupted copy of the gene
contained in the transfer vector through two crossover events: a
first crossover event of homologous recombination between the
vaccinia virus genome and the transfer vector and a second
crossover event of homologous recombination between direct repeats
within the target locus. The interrupted version of the target gene
that is in the transfer vector contains the insertion DNA flanked
on each side by DNA corresponding to the left portion of the target
gene and right portion of the target gene, respectively. The
transfer vector also contains a dominant selection marker, e.g.,
the E. coli guanine phosphoribosyltransferase (gpt) gene, under the
control of a vaccinia virus early promoter (e.g., P.sub.7.5kE).
Including such a marker in the vector enables a transient dominant
selection process to identify recombinant virus grown under
selective pressure that has incorporated the transfer vector within
its genome. Because the marker gene is not stably integrated into
the genome, it is deleted from the genome in a second crossover
event that occurs when selection is removed. Thus, the final
recombinant virus contains the interrupted version of the target
gene as a disruption of the target loci, but does not retain the
selectable marker from the transfer vector.
[0465] Homologous recombination between a transfer vector and a
starting vaccinia virus genome occurred upon introduction of the
transfer vector into cells that have been infected with the
starting vaccinia virus. A series of transfer vectors was
constructed as described below and the following modified vaccinia
strains were constructed: GLV-1h140, GLV-1h141, GLV-1h142,
GLV-1h143, GLV-1h144, GLV-1h145, GLV-1h111, GLV-1h152, GLV-1h153.
The construction of these strains is summarized in the following
Table, which lists the modified vaccinia virus strains, including
the previously described GLV-1h68, their respective genotypes, and
the transfer vectors used to engineer the viruses:
TABLE-US-00005 TABLE 6 Generation of engineered vaccinia viruses
Name of Parental VV Transfer Virus Virus Vector Genotype GLV-1h68
-- -- F14.5L: (P.sub.SEL)Ruc-GFP TK: (P.sub.SEL)rTrfR-
(P.sub.7.5k)LacZ HA: (P.sub.11k)gusA GLV-1h99 GLV-1h68 FSE-hNET
F14.5L: (P.sub.SE)hNET TK: (P.sub.SEL)rTrfR- (P.sub.7.5k)LacZ HA:
(P.sub.11k)gusA GLV-1h100 GLV-1h68 TK-SE-hNET3 F14.5L:
(P.sub.SEL)Ruc-GFP TK: (P.sub.SE)hNET HA: (P.sub.11k)gusA GLV-1h101
GLV-1h68 TK-SL-hNET3 F14.5L: (P.sub.SEL)Ruc-GFP TK: (P.sub.SL)hNET
HA: (P.sub.11k)gusA GLV-1h139 GLV-1h68 HA-SE-hNET-1 F14.5L:
(P.sub.SEL)Ruc-GFP TK: (P.sub.SEL)rTrfR- (P.sub.7.5k)LacZ HA:
(P.sub.SE)hNET GLV-1h146 GLV-1h100 HA-SE-IL-24-1 F14.5L:
(P.sub.SEL)Ruc-GFP TK: (P.sub.SE)hNET HA: (P.sub.SE)IL-24 GLV-1h150
GLV-1h101 HA-SE-IL-24-1 F14.5L: (P.sub.SEL)Ruc-GFP TK:
(P.sub.SL)hNET HA: (P.sub.SE)IL-24 GLV-1h151 GLV-1h68 HA-SE-hNIS-1
F14.5L: (P.sub.SEL)Ruc-GFP TK: (P.sub.SEL)rTrfR- (P.sub.7.5k)LacZ
HA: (P.sub.SE)hNIS GLV-1h152 GLV-1h68 HA-SEL-hNIS-2 F14.5L:
(P.sub.SEL)Ruc-GFP TK: (P.sub.SEL)rTrfR- (P.sub.7.5k)LacZ HA:
(P.sub.SEL)hNIS GLV-1h153 GLV-1h68 HA-SL-hNIS-1 F14.5L:
(P.sub.SEL)Ruc-GFP TK: (P.sub.SEL)rTrfR- (P.sub.7.5k)LacZ HA:
(P.sub.SL)hNIS
Briefly, the strains listed in Table 2 were generated as follows
(further details are provided below):
[0466] GLV-1h99 was generated by insertion of an expression
cassette encoding hNET under the control of the vaccinia P.sub.SE
promoter into the F14.5L locus of starting strain GLV-1h68, thereby
deleting the Ruc-GFP fusion gene expression cassette at the F14.5L
locus of starting GLV-1h68. Thus, in strain GLV-1h99, the vaccinia
F14.5L gene is interrupted within the coding sequence by a DNA
fragment containing DNA encoding hNET operably linked to the
vaccinia synthetic early promoter.
[0467] GLV-1h100 was generated by insertion of an expression
cassette encoding hNET under the control of the vaccinia P.sub.SE
promoter into the TK locus of starting strain GLV-1h68 thereby
deleting the LacZ/rTFr expression cassette at the TK locus of
starting GLV-1h68. Thus, in strain GLV-1h100, the vaccinia TK gene
is interrupted within the coding sequence by a DNA fragment
containing DNA encoding hNET operably linked to the vaccinia
synthetic early promoter.
[0468] GLV-1h101 was generated by insertion of an expression
cassette encoding hNET under the control of the vaccinia P.sub.SL
promoter into the TK locus of starting strain GLV-1h68 thereby
deleting the LacZ/rTFr expression cassette at the TK locus of
starting GLV-1h68. Thus, in strain GLV-1h101, the vaccinia TK gene
is interrupted within the coding sequence by a DNA fragment
containing DNA encoding hNET operably linked to the vaccinia
synthetic late promoter.
[0469] GLV-1h139 was generated by insertion of an expression
cassette encoding hNET under the control of the vaccinia P.sub.SE
promoter into the HA locus of starting strain GLV-1h68 thereby
deleting the gusA expression cassette at the HA locus of starting
GLV-1h68. Thus, in strain GLV-1h139, the vaccinia HA gene is
interrupted within the coding sequence by a DNA fragment containing
DNA encoding hNET operably linked to the vaccinia synthetic early
promoter.
[0470] GLV-1h146 was generated by insertion of an expression
cassette encoding IL-24 under the control of the vaccinia P.sub.SE
promoter into the HA locus of starting strain GLV-1h100, thereby
deleting the gusA expression cassette at the HA locus of starting
GLV-1h100. Thus, in strain GLV-1h146, the vaccinia HA gene is
interrupted within the coding sequence by a DNA fragment containing
DNA encoding IL-24 operably linked to the vaccinia synthetic early
promoter and the vaccinia TK gene is interrupted within the coding
sequence by a DNA fragment containing DNA encoding hNET operably
linked to the vaccinia synthetic early promoter.
[0471] GLV-1h150 was generated by insertion of an expression
cassette encoding IL-24 under the control of the vaccinia P.sub.SE
promoter into the HA locus of starting strain GLV-1h101, thereby
deleting the gusA expression cassette at the HA locus of starting
GLV-1h101. Thus, in strain GLV-1h150, the vaccinia HA gene is
interrupted within the coding sequence by a DNA fragment containing
DNA encoding IL-24 operably linked to the vaccinia synthetic early
promoter and the vaccinia TK gene is interrupted within the coding
sequence by a DNA fragment containing DNA encoding hNET operably
linked to the vaccinia synthetic late promoter.
[0472] GLV-1h151 was generated by insertion of an expression
cassette encoding hNIS under the control of the vaccinia P.sub.SE
promoter into the HA locus of starting strain GLV-1h68 thereby
deleting the gusA expression cassette at the HA locus of starting
GLV-1h68. Thus, in strain GLV-1h151, the vaccinia HA gene is
interrupted within the coding sequence by a DNA fragment containing
DNA encoding hNIS operably linked to the vaccinia synthetic early
promoter.
[0473] GLV-1h152 was generated by insertion of an expression
cassette encoding hNIS under the control of the vaccinia P.sub.SEL
promoter into the HA locus of starting strain GLV-1h68 thereby
deleting the gusA expression cassette at the HA locus of starting
GLV-1h68. Thus, in strain GLV-1h152, the vaccinia HA gene is
interrupted within the coding sequence by a DNA fragment containing
DNA encoding hNIS operably linked to the vaccinia synthetic
early/late promoter.
[0474] GLV-1h153 was generated by insertion of an expression
cassette encoding hNIS under the control of the vaccinia P.sub.SL
promoter into the HA locus of starting strain GLV-1h68 thereby
deleting the gusA expression cassette at the HA locus of starting
GLV-1h68. Thus, in strain GLV-1h153, the vaccinia HA gene is
interrupted within the coding sequence by a DNA fragment containing
DNA encoding hNIS operably linked to the vaccinia synthetic late
promoter.
[0475] 2. VV Transfer Vectors Employed for the Production of
Modified Vaccinia Viruses
[0476] The following vectors were constructed and employed as
described below to generate the recombinant vaccinia viral
strains.
[0477] a. FSE-hNET: For Insertion of an Expression Cassette
Encoding hNET Under the Control of the Vaccinia P.sub.SE Promoter
into the Vaccinia F14.5L Locus
[0478] The FSE-hNET vector (SEQ ID NO.: 3) was employed to create
vaccinia virus strain GLV-1h99, having the following genotype:
F14.5L: (P.sub.SE)hNET, TK: (P.sub.SEL)rTrfR-(P.sub.7.5k)LacZ, HA:
(P.sub.11k)gusA. FSE-hNET contains the human norepinephrine
transporter (hNET) under the control of the vaccinia P.sub.SE
promoter, flanked by sequences of the F14.5L gene.
[0479] To generate the FSE-hNET vector, DNA encoding hNET was PCR
amplified from the plasmid pBluescript II KS+-hNET as the template
with the following primers:
hNET5 (5'-GTCGACGCCACCATGCTTCTGGCGCGGATGAA-3', SEQ ID NO: 4) (Sal I
restriction site underlined) and hNET3
(5'-GATATCTCAGATGGCCAGCCAGTGTT-3', SEQ ID NO: 5) (EcoR V site
underlined). The PCR product was gel-purified, and cloned into the
pCR-Blunt II-TOPO vector (SEQ ID NO: 6) using the Zero Blunt TOPO
PCR Cloning Kit (Invitrogen). The resulting construct pCRII-hNET1
confirmed by sequencing. The hNET cDNA was released from
pCRII-hNET1 with Sal I and EcoR V enzyme digest, and subcloned into
the intermediate vector pCR-SE1 (SEQ ID NO: 7), precut with SalI
and SmaI. This step puts hNET cDNA downstream of the sequence for
vaccinia virus synthetic early promoter (P.sub.SE). The viral hNET
expression cassette (SE-hNET) was released from this intermediate
construct by BamH I and Hind III enzyme digest, and inserted into
the same cut viral transfer vector pNCVVf14.5T (SEQ ID NO: 8). The
final construct FSE-hNET1 was confirmed by sequencing and used for
insertion of SE-hNET into the F14.5L locus in GLV-1h68.
[0480] b. TK-SE-hNET3: For Insertion of an Expression Cassette
Encoding hNET Under the Control of the Vaccinia P.sub.SE Promoter
into the Vaccinia TK Locus
[0481] The TK-SE-hNET3 vector (SEQ ID NO.: 9) was employed to
create vaccinia virus strain GLV-1h100, having the following
genotype: F14.5L: (P.sub.SEL)Ruc-GFP, TK: (P.sub.SE)hNET, HA:
(P.sub.11k)gusA. TK-SE-hNET3 contains the human norepinephrine
transporter (hNET) under the control of the vaccinia P.sub.SE
promoter, flanked by sequences of the TK gene. To generate vector
TK-SE-hNET3, hNET cDNA was released from FSE-hNET1 with Sal I and
Pac I enzyme digestion, and inserted into same cut vector TK-SE-mIP
10 (SEQ ID NO: 10). The resulting construct TK-SE-hNET3 was
confirmed by sequencing.
[0482] c. TK-SL-hNET3: For Insertion of an Expression Cassette
Encoding hNET Under the Control of the Vaccinia P.sub.SL Promoter
into the Vaccinia TK Locus
[0483] The TK-SL-hNET3 vector (SEQ ID NO.: 11) was employed to
create vaccinia virus strain GLV-1h101, having the following
genotype: F14.5L:(P.sub.SEL)Ruc-GFP, TK: (P.sub.SL)hNET, HA:
(P.sub.11k)gusA. TK-SE-hNET3 contains the human norepinephrine
transporter (hNET) under the control of the vaccinia P.sub.SL
promoter, flanked by sequences of the TK gene. To generate vector
TK-SL-hNET3, hNET cDNA was released from FSE-hNET1 with Sal I and
Pac I enzyme digestion, and inserted into same cut vector
TK-SL-mIP10 (SEQ ID NO: 12). The resulting construct TK-SL-hNET3
was confirmed by sequencing.
[0484] d. HA-SE-hNET-1: For Insertion of an Expression Cassette
Encoding hNET Under the Control of the Vaccinia P.sub.SE Promoter
into the Vaccinia HA Locus
[0485] The HA-SE-hNET-1 vector (SEQ ID NO.: 13) was employed to
create vaccinia virus strain GLV-1h139, having the following
genotype: F14.5L: (P.sub.SEL)Ruc-GFP, TK:
(P.sub.SEL)rTrfR-(P.sub.7.5k)LaCZ, HA: (P.sub.SE)hNET. HA-SE-hNET-1
contains the human norepinephrine transporter (hNET) under the
control of the vaccinia P.sub.SE promoter, flanked by sequences of
the HA gene. To generate vector HA-SE-hNET-1, hNET cDNA was
released from TK-SL-hNET-3 (SEQ ID NO.: 11) by Sal I and Pac I
enzyme digest, and subcloned into same cut vector HA-SE-RLN-7 (SEQ
ID NO.: 14), thereby replacing RLN cDNA with the hNET cDNA. The
resulting construct HA-SE-hNET-1 was confirmed by sequencing.
[0486] e. HA-SE-IL24-1: For Insertion of an Expression Cassette
Encoding IL-24 Under the Control of the Vaccinia P.sub.SEPromoter
into the vaccinia HA Locus
[0487] The HA-SE-IL24-1 vector (SEQ ID NO.: 15) was employed to
create vaccinia virus strains GLV-1h146 and GLV-1h150, having the
following genotypes: F14.5L: (P.sub.SEL)Ruc-GFP, TK:
(P.sub.SE)hNET, HA: (P.sub.SE)IL-24 and F14.5L: (P.sub.SEL)Ruc-GFP,
TK: (P.sub.SL)hNET, HA: (P.sub.SE)IL-24, respectively. HA-SE-IL24-1
contains the human IL-24 gene under the control of the vaccinia
P.sub.SE promoter, flanked by sequences of the HA gene. To generate
vector HA-SE-IL24-1, human IL24 cDNA was PCR amplified using Homo
sapiens interleukin 24, transcript variant 1 (cDNA clone MGC:8926)
from Origene as the template with the following primers:
mda-5 (5'-GTCGACCACCATGAATTTTCAACAGAGGCTGC-3', SEQ ID NO.: 16) (Sal
I site underlined) and mda-3
(5'-CCCGGGTTATCAGAGCTTGTAGAATTTCTGCATC-3', SEQ ID NO.: 17) (Sma I
site underlined)). The resulting PCR product was gel purified, and
cloned into the pCR-Blunt 1'-TOPO vector (SEQ ID NO.: 6), using
Zero Blunt TOPO PCR Cloning Kit (Invitrogen). The resulting
construct pCRII-IL24-3 was sequence confirmed. The IL24 cDNA was
released from pCRII-IL24-3 by Sal I and Sma I digest, and subcloned
into same cut vector pCR-SE1 (SEQ ID NO.: 7), placing IL24 under
the control of vaccinia synthetic promoter (P.sub.SE). The
resulting construct pCR-SE-IL24-2 was sequence confirmed. The IL24
was then released by Sal I and Pac I enzyme digest, and subcloned
into same cut vector HA-SE-RLN-7 (SEQ ID NO.: 14) thereby replacing
RLN cDNA with IL-24 cDNA. The resulting constructs HA-SE-IL24-1 was
sequence confirmed.
[0488] f. HA-SE-hNIS-1: For Insertion of an Expression Cassette
Encoding hNIS Under the Control of the Vaccinia P.sub.SE Promoter
into the Vaccinia HA Locus
[0489] The HA-SE-hNIS-1 vector (SEQ ID NO.: 18) was employed to
create vaccinia virus strain GLV-1h151, having the following
genotype: F14.5L: (P.sub.SEL)Ruc-GFP, TK:
(P.sub.SEL)rTrfR-(P.sub.7.5k)LacZ, HA: (P.sub.SE)hNIS. HA-SE-hNIS-1
contains the human sodium iodide symporter (hNIS) under the control
of the vaccinia P.sub.SE promoter, flanked by sequences of the HA
gene. To generate vector HA-SE-hNIS-1, hNIS cDNA was PCR amplified
using human cDNA clone TC124097 (SLC5A5) from OriGene as the
template with following primers:
TABLE-US-00006 hNIS-5 (5'-GTCGACCACCATGGAGGCCGTGGAGACCGG-3', SEQ ID
NO.: 19) (Sal I site underlined) and hNIS-3
(5'-TTAATTAATCAGAGGTTTGTCTCCTGCTGGTCTCGA-3', SEQ ID NO.: 20) (Pac I
site underlined).
The PCR product was gel-purified, and cloned into the pCR-Blunt
1'-TOPO vector (SEQ ID NO.: 6) using Zero Blunt TOPO PCR Cloning
Kit (Invitrogen). The resulting construct pCRII-hNIS-2 confirmed by
sequencing. The hNIS cDNA was released from pCRII-hNIS-2 with Sal I
and Pac I enzyme digestion, and subcloned into same cut vector
HA-SE-RLN-7 (SEQ ID NO.: 14), thereby replacing RLN cDNA. The
resulting construct HA-SE-hNIS-1 was confirmed by sequencing.
[0490] g. HA-SEL-hNIS-2: For Insertion of an Expression Cassette
Encoding hNIS Under the Control of the Vaccinia P.sub.SEL Promoter
into the Vaccinia HA Locus
[0491] The HA-SEL-hNIS-2 vector (SEQ ID NO.: 21) was employed to
create vaccinia virus strain GLV-1h1 52, having the following
genotype: F14.5L: (P.sub.SEL)Ruc-GFP, TK:
(P.sub.SEL)rTrfR-(P.sub.7.5k)LacZ, HA: (P.sub.SEL)hNIS.
HA-SEL-hNIS-2 contains the human sodium iodide symporter (hNIS)
under the control of the vaccinia P.sub.SELpromoter, flanked by
sequences of the HA gene. To generate vector HA-SEL-hNIS-2, the
hNIS cDNA was released from pCRII-hNIS-2 with Sal I and Pac I
enzyme digestion, and subcloned into same cut vector HA-SEL-RLN-2
(SEQ ID NO.: 22), thereby replacing RLN cDNA. The resulting
construct HA-SEL-hNIS-2 was confirmed by sequencing.
[0492] h. HA-SL-hNIS-1: For Insertion of an Expression Cassette
Encoding hNIS Under the Control of the Vaccinia P.sub.SL Promoter
into the Vaccinia HA Locus
[0493] The HA-SL-hNIS-1 vector (SEQ ID NO.: 23) was employed to
create vaccinia virus strain GLV-1h153, having the following
genotype: F14.5L: (P.sub.SEL)Ruc-GFP, TK:
(P.sub.SEL)rTrfR-(P.sub.7.5k)LacZ, HA: (P.sub.SL)hNIS. HA-SL-hNIS-1
contains the human sodium iodide symporter (hNIS) under the control
of the vaccinia P.sub.SL promoter, flanked by sequences of the HA
gene. To generate vector HA-SL-hNIS-1, the hNIS cDNA was released
from pCRII-hNIS-2 with Sal I and Pac I enzyme digestion, and
subcloned into same cut vector HA-SL-RLN-3 (SEQ ID NO.: 24),
thereby replacing RLN cDNA. The resulting construct HA-SL-hNIS-1
was confirmed by sequencing.
[0494] 3. Preparation of Recombinant Vaccinia Viruses
[0495] African green monkey kidney fibroblast CV-1 cells (American
Type Culture Collection (Manassas, Va.); CCL-70) were employed for
viral generation and production. The cells were grown in Dulbecco's
modified Eagle's medium (DMEM) supplemented with 1%
antibiotic-antimycotic solution (Mediatech, Inc., Herndon, Va.) and
10% fetal bovine serum (FBS; Mediatech, Inc., Herndon, Va.) at
37.degree. C. under 5% CO.sub.2. For virus generation of
recombinant viruses, the CV-1 cells were infected with GLV-1h68 (or
designated parental virus, see Table 2) at m.o.i. of 0.1 for 1 hr.
The infected cells were then transfected using Fugene (Roche,
Indianapolis, Ind.) with the designated transfer vector (see Table
2 and description of viral transfer vectors above). At two days
post infection, infected/transfected cells were harvested and the
recombinant viruses were selected and plaque purified using
standard methods as described previously (Falkner and Moss (1990)
J. Virol. 64:3108-3111).
[0496] 4. Verification of Vaccinia Virus Strain Genotypes
[0497] The genotype of the vaccinia viruses was verified by PCR and
restriction enzyme digestion. The lack of expression of gusA gene
in viruses GLV-1h139, GLV-1h146, GLV-1h150, GLV-1h151, GLV-1h152
and GLV-1h153 was confirmed X-GlcA
(5-bromo-4-chloro-3-indolyl-.beta.-D-glucuronic acid) staining of
the infected cells. Viruses lacking gusA expression are unable to
convert the X-GlcA substrate as indicated by lack of development of
blue color in the assay as compared to a control strain (e.g.
GLV-1h68). Lack of expression of the GFP gene in GLV-1h99 was
confirmed by fluorescence microscopy as compared to a control
strain (e.g. GLV-1h68). The lack of expression of the LacZ gene for
viruses GLV-1h100, GLV-1h101, GLV-1h146 and GLV-1h150 was confirmed
by X-gal (5-bromo-4-chloro-3-indolyl-.beta.-D-galactopyranoside)
staining of the infected cells. Viruses lacking lacZ expression are
unable to convert the X-gal substrate as indicated by lack of
development of blue color in the assay as compared to a control
strain (e.g. GLV-1h68). Standard techniques for X-GlcA and X-gal
viral staining and fluorescence microscopy were employed and are
well-known in the art.
B. Vaccinia Virus Purification
[0498] Ten T225 flasks of confluent CV-1 cells (seeded at
2.times.10.sup.7 cells per flask the day before infection) were
infected with each virus at m.o.i. of 0.1. The infected cells were
harvested two days post infection and lysed using a glass Dounce
homogenizer. The cell lysate was clarified by centrifugation at
1,800g for 5 min, and then layered on a cushion of 36% sucrose, and
centrifuged at 13,000 rpm in a HB-6 rotor, Sorvall RC-5B
Refrigerated Superspeed Centrifuge for 2 hours. The virus pellet
was resuspended in 1 ml of 1 mM Tris, pH 9.0, loaded on a sterile
24% to 40% continuous sucrose gradient, and centrifuged at 26,000g
for 50 min. The virus band was collected and diluted using 2
volumes of 1 mM Tris, pH 9.0, and then centrifuged at 13,000 rpm in
a HB-6 rotor for 60 min. The final virus pellet was resuspended in
1 ml of 1 mM Tris, pH 9.0 and the titer was determined in CV-1
cells (ATCC No. CCL-70).
Example 2
In Vitro Virus Infection Studies with GLV-1h99
A. Analysis of Vaccinia Virus Replication in Tissue Culture
[0499] To evaluate any effects of hNET expression on vaccinia virus
replication, PANC-1 cells were infected with either GLV-1h99 or its
parent virus, GLV-1h68, at moi of 0.01 for 1 h at 37.degree. C. The
inoculum was then aspirated, and the cell monolayers were washed
twice with 2 ml of DPBS (Mediatech, Inc., Herndon, Va.). Two
milliliters of cell culture medium containing 2% FBS were added
into each well of a multiwell plate. Three wells each were
harvested at 24, 48, and 72 hours post infection. The harvested
cells were subject to three cycles of freeze-thaw and were
sonicated three times for 1 minute at full power before titration.
The viral titers at each time point were determined in CV-1 cells
using a standard plaque assay.
[0500] GLV-1h99 gave significantly higher viral yields
(approximately 10 fold) at all three time points in comparison with
its parental virus, GLV-1h68. The enhanced viral replication for
GLV-1h99 may be due to the difference in the strength of the
vaccinia viral promoter at the F14.5L locus in the viruses.
GLV-1h68 contains a strong vaccinia P.sub.SEL promoter, which is
100 times stronger than a P.sub.SE promoter (Chakrabarti et al.
(1997) Biotechniques 23(6):1094-7). Previous studies have shown
that virus replication in cell culture is inversely proportional
with strength of promoters inserted into the viral genome. Thus,
the use of a P.sub.SE promoter in the GLV-1h99 can result in the
higher viral titers observed.
[0501] (The PANC-1 cells used in the study were obtained from the
American Type Culture Collection (Manassas, Va.) and maintained in
Dulbecco's modified Eagle's medium (DMEM) supplemented with 1%
antibiotic-antimycotic solution (Mediatech, Inc., Herndon, Va.) and
10% fetal bovine serum (FBS; Mediatech, Inc., Herndon, Va.) at
37.degree. C. under 5% CO.sub.2).
B. Cytotoxicity Assay
[0502] Mesothelioma (MSTO-211H, H2373, JMN and H2052) and
pancreatic cancer (PANC-1, MiaPaCa2, BxPC3 and HS766T) cell lines
were plated at 2.times.10.sup.4 per well in 12-well plates in 1 ml
of media per well. After incubation for 6 hours, cells were
infected with GLV-1h99 or GLV-1h68 at MOI's (multiplicity of
infection) of 1.0, 0.10, and 0.01 and 0 (control wells). Viral
cytotoxicity was measured daily for 7 days. Cells were washed with
PBS and lysed in 200 .mu.l per well of 1.5% Triton X (Sigma, St.
Louis, Mo.) to release intracellular lactate dehydrogenase, which
was quantified with a Cytotox 96 kit (Promega, Madison, Wis.) on a
spectrophotometer (EL321e, Bio-Tek Instruments) at 490 nm. Results
were measured as the percentage of surviving cells. This percentage
was determined by comparing the measured lactate dehydrogenase of
each infected sample to that in uninfected, control cells. All
samples were analyzed in triplicate. Four mesothelioma and four
pancreatic cancer cell lines demonstrated lytic cytotoxicity
following exposure to GLV-1h99 (hNET-expressing virus) and to
GLV-1h68 (non-hNET containing virus). Similar cytotoxicity was
observed with GLV-1h99 and GLV-1h68 at a MOI of 1.0 and a
dose-dependent lytic effect was also demonstrated for m.o.i.
ranging from 0.01 to 1.00. At a MOI of 0.1, all MSTO-211H and H2052
mesothelioma cells as well as 80% of the PANC1 pancreatic cancer
cells were dead at day 7. Oncolysis appeared to be more gradual
over time in PANC1 cells, compared to the more sigmoidal lytic-time
profile in MSTO-211H cells. The mesothelioma cell line JMN and the
pancreatic cancer cell line HS766T were more resistant to cell
death (80% cell death by day 7 at a MOI of 1.0). MiaPaCa2 and BxPC3
(pancreatic cancer cell lines) and H2373 (mesothelioma cell line)
were sensitive to the virus at a higher MOI of 10.
C. Immunoblot Analysis of hNET Expression
[0503] To evaluate the level of hNET protein expression in cells
CV-1 cells in 60 mm dishes were infected with GLV-1h68 or GLV-1h99
at m.o.i of approximately 10. Approximately two days post
infection, cells were harvested and solubilized in RIPA buffer (10
mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100,
1% sodium deoxycholate and proteinase inhibitor cocktail). The cell
lysates were separated on a 4-12% Bis-Tris Gel (Invitrogen), and
proteins were transferred onto a PVDF membrane (Amersham
Biosciences). The membrane was incubated with an anti-hNET
monoclonal antibody (NET17-1, Mab Technologies, Stone Mountain,
Calif.) at a dilution of approximately 1:500, and detected using
WesternBreeze Chromogenic Western Blot Immunodetection Kit
(Invitrgen). GLV-1h99-infected cells expressed hNET protein,
whereas cells either mock-infected or infected with GLV-1h68 did
not. Five major bands (35, 47, 50, 65, 73 kDa) were detected in
GLV-1h99-infected CV-1 cells.
[0504] In a separate experiment, the two most sensitive
mesothelioma cell lines (MSTO-211H and H2052) and the most
sensitive pancreatic cancer cell line (PANC 1), based on the
cytotoxicity assays, were chosen for immunoblot analysis and
compared to the endogenous hNET-expressing neuroblastoma cell line,
SK-N-SH. hNET protein expression was evaluated in cells infected
with either GLV-1h99 or GLV-1h68 viruses at a MOI of 1.0 at 12, 24,
48 and 72h after infection. A purified mouse antibody against hNET
(NET17-1, Mab Tech Inc. GA, USA) was used at a final dilution of
1:500 and incubated for 12 hours at +4.degree. C. The secondary
antibody (peroxidase conjugated anti-mouse IgG (Vector labs Inc.,
CA, USA)) exposure was for 1 hour at a 1:2000 dilution.
Peroxidase-bound protein bands were visualized using the ECL method
(Amersham Pharmacia Biotech, Little Chalfont, UK).
[0505] In addition to the .about.80-kD hNET band, two low-molecular
weight immunoreactive bands (.about.50-55 kD and .about.37-40 kD,
respectively) are seen in the blots of the GLV-1h99-infected cells;
these bands are barely visible in the blots of the SK-N-SH
neuroblastoma cells. Similar to the cytotoxicity assay, there was a
viral dose-dependent expression of hNET at different MOI's (0.1,
1.0, 5 and 10). Strong hNET expression was found in all the three
cell lines by 12 hours after GLV-1h99 viral infection, peaking at
24 hours followed by a gradual decline over 72 hours. A similar
pattern of hNET expression was observed in the other cell lines,
although the hNET immunoblot bands were less intense. The
neuroblastoma cell line (SK-N-SH), expressing endogenous hNET,
served as a positive control for the immunoblot analysis and
radiotracer uptake studies; the GLV-1h68 virus-infected (non-hNET
containing virus) and the uninfected mesothelioma and pancreatic
cancer cell lines served as negative controls, showing no hNET
expression.
D. In Vitro Radiotracer Uptake Assay
[0506] [.sup.123I]MIBG radiotracer uptake studies were performed in
MSTO-211h and PANC1 cells after infection with virus (GLV-1h99 or
GLV-1h68) at a MOI of 1.0 as well as in the neuroblastoma cell
line, SK-N-SH, using previously described methods (Che J et al.
(2005) Mol Imaging 4:128-36. For these studies, clinical grade
[.sup.123I]MIBG was obtained from MDS Nordion (Canada). The average
radiochemical purity was in excess of 97% (determined by MDS
Nordion using the Sep Pak cartridge method), and the specific
activity .about.320 MBq/.mu.mol (8.7 mCi/.mu.mol) according to the
vendor. [.sup.124I]MIBG was prepared using minor modifications to
the reported nucleophilic isotopic exchange method (Eersels J et
al. (2005) J Labelled Compds Radiopharm 48:241-57), following a
procedure previously reported (Moroz M A, et al. (2007) J Nucl Med
48:827-36). The radiochemical purity of the final product was
>95% with an overall yield of >80% and the specific activity
18.5.+-.5.2 MBq/.mu.mol (0.5.+-.0.14 mCi/.mu.mol). The maximum
specific activities (no carrier-added synthesis) for the
[.sup.123I]- and [.sup.124I]-labeled compounds were 8.9 and 1.2
TBq/.mu.mol (241 and 33 Ci/.mu.mol), respectively, due to the
7.4-fold difference in the decay rate of the two isotopes.
[0507] For the uptake assay, cells were plated at 1.times.10.sup.6
per well in 6-well plates in 2 ml of media per well. After
incubation for 6 hours, cells were infected with GLV-1h68 or
GLV1h99 at MOIs of 1.0 and 0 (control wells). Following 12-, 24-,
48- and 72-h incubation periods with virus at 37.degree. C. and 5%
CO.sub.2, the medium was aspirated and the cells were washed with
PBS (pH 7.4). [.sup.123 I]MIBG uptake was initiated by adding 2 ml
of DME containing 0.0185 MBq/ml (0.5 .mu.Ci/ml) carrier-free
[.sup.123I]MIBG. Cells were harvested after a 60-minute incubation
period, and the cell pellet-to-medium activity ratio (cpm/gm of
pellet/cpm/ml of medium) was calculated from the radioactivity
measurements assayed in a gamma counter (Packard, United
Technologies). All studies were performed in triplicate. A
two-tailed unpaired t-test was applied to determine the
significance of differences between values using the MS Office 2003
Excel 11.0 statistical package (Microsoft, Redmond, Wash.,
USA).
[0508] [.sup.123I]MIBG accumulation in non-infected MSTO-211H and
PANC1 cells was low. There was no significant increase in
radiotracer uptake 24 h after infection of the cells with GLV-1h68
(non-hNET containing virus, negative control). In contrast, there
was a significant (p<0.01) increase in [.sup.123 I]MIBG
accumulation in both cancer cell lines at all time points (12 h, 24
h, 48 h and 72 h) after infection with GLV-1h99. Peak radiotracer
uptake was observed at 48 h after virus infection in both cell
lines. The natural hNET-expressing neuroblastoma cell line
(SK-N-SH) served as a positive control. Total cell protein in the
[.sup.123I]MIBG uptake assays were unchanged over the first 24
hours following GLV-1h99 infection, compared to uninfected cells.
At 48 and 72 h after viral infection, there was a decrease in
measured cell protein.
Example 3
In Vivo Animal Model Studies of [.sup.123I]MIBG Uptake by GLV-1h99
Infected Tumor Cells
A. Malignant Pleural Mesothelioma Xenograft Model
[0509] Athymic nu/nu female mice were purchased from the National
Cancer Institute (NCI, MD) and were housed five per cage and
allowed food and water ad libitum in the MSKCC Vivarium for 1 week
before tumor cell implantation. All animal studies were performed
in compliance with all applicable policies, procedures and
regulatory requirements of the Institutional Animal Care and Use
Committee (IACUC), the Research Animal Resource Center (RARC) of
MSKCC and the National Institutes of Health (NIH) "Guide for the
Care and Use of Laboratory Animals". All animal procedures were
performed under anesthesia induced by inhalation of 2% isoflurane.
After the studies all animals were sacrificed by CO.sub.2
asphyxiation.
[0510] An incision 3 to 5 mm in length was made over the fourth to
fifth intercostal space of the right chest. The underlying
inflating and deflating lung was thereby easily visualized through
the thin fascia. Slowly, 100 .mu.l of MSTO-211H malignant
mesothelioma cellular suspension (5.times.10.sup.6 cells) were
injected. After the injection the skin was closed with surgical
staples and mice were returned to their cages.
[0511] Intrapleural treatment with virus was performed in a similar
fashion as described above 10 days after tumor cell instillation
into the pleural cavity. GLV-1h99 or GLV-1h68 (1.times.10.sup.7
pfu) was administered in 100 .mu.L PBS and animals were gently
rotated from side to side to help distribute the virus throughout
the pleural cavity. Control animals (no virus) received only 100
.mu.L PBS.
B. [.sup.123I]MIBG Gamma-Camera In Vivo Imaging
[0512] Each animal was injected intravenously with .about.18.5 MBq
(500 .mu.Ci) of [.sup.123 I]MIBG 48 or 72 hours after intrapleural
GLV-1h99 injection. Sequential PET imaging was performed 1 to 48
hours after radiotracer administration using a X-SPECT.TM.
dedicated small-animal gamma camera SPECT-CT scanner (Gamma Medica,
Northridge, Calif.). A photopeak energy window of 143-175 keV and a
low-energy high-resolution (LEHR) parallel-hole collimator was used
to acquire ten-minute .sup.123I images at 2 hours post-[.sup.123
I]MIBG administration.
[0513] The X-SPECT.TM. gamma camera system was calibrated by
imaging a mouse-size (30-ml) cylinder filled with an independently
measured concentration (MBq/ml) of technetium-99m using a photopeak
energy window of 126-154 keV and LEHR collimation. The resulting
.sup.99mTc images were exported to Intefile and then imported into
the ASIPro.TM. (Siemens Pre-clinical Solutions, Knoxville, Tenn.)
image-processing software environment. By region of interest (ROI)
analysis, a system calibration factor (in cpm/pixel per MBq/ml) was
derived. Animal images were likewise exported to Interfile and then
imported into ASIPro.TM. and parameterized in terms of the
decay-corrected percent injected dose per gram (% ID/gm) based on
the foregoing calibration factor, the administered activity, the
time post-administration of imaging, and the image duration.
Implicit in the foregoing analysis is the reasonable assumption
that the sensitivities of the X-SPECT.TM. gamma camera system for
.sup.123I and .sup.99mTc are comparable.
C. [.sup.124I]MIBG microPET In Vivo Imaging
[0514] In a group of 5 animals (10 days after MSTO-211H tumor cell
instillation into the pleural cavity), each animal was injected via
the tail vein with 9.25 MBq (250 .mu.Ci) of [.sup.18F]FDG.
(Clinical grade [.sup.18F]FDG was obtained from IBA Molecular
(Somerset, N.J.) with a specific activity>41 MBq/.mu.mol (>11
mCi/.mu.mol) and a radiochemical purity of >98%). [.sup.18F]FDG
PET scanning was performed 1 h after tracer administration using a
10-minute list-mode acquisition. Animals were fasted 12 h before
tracer administration and kept under anesthesia between FDG
injection and imaging.
[0515] In a group of 16 animals, 4 sub-groups of 3-5 animals each
were studied (5 animals in sub-group 1 and 2; 3 animals in
sub-group 3 and 4). Each animal was injected via the tail vein with
9.25 MBq (250 .mu.Ci) of [.sup.124I]MIBG. Animals in sub-groups 1
and 2 were injected with GLV-1h99 48 and 72 h prior to
[.sup.124I]MIBG administration. Sub-group 3 animals received
GLV-1h68 48 h prior to radiotracer administration; sub-group 4
animals was not injected with virus, receiving only 100 .mu.l PBS).
Potassium iodide was used to block the uptake of radioactive iodine
by the thyroid. [.sup.124I]MIBG PET was performed for 10 minute 1,
2, and 4 h after tracer administration, for 15 minute at 12h, for
30 minute at 24h, and for 60 minute at 48h. After tracer
administration and between imaging time points, the animals were
allowed to wake up and maintain normal husbandry.
[0516] Imaging was performed using a Focus 120 microPET.TM.
dedicated small-animal PET scanner (Concorde Microsystems Inc,
Knoxville, Tenn.). Three-dimensional (3D) list-mode data were
acquired using an energy window of 350-700 keV for .sup.18F and
410-580 keV for .sup.124I, respectively, and a coincidence timing
window of 6 ns. These data were then sorted into two-dimensional
(2D) histograms by Fourier re-binning. The image data were
corrected for (a) non-uniformity of scanner response using a
uniform cylinder source-based normalization, (b) dead time count
losses using a singles count rate-based global correction, (c)
physical decay to the time of injection, and (d) the .sup.124I
branching ratio. The count rates in the reconstructed images were
converted to activity concentration (% of injected dose per gram of
tissue, % ID/g) using a system calibration factor (MBq/ml per
cps/voxel) derived from imaging of a mouse-size phantom filled with
a uniform aqueous solution of .sup.18F.
[0517] Image analysis was performed using ASIPro.TM. (Siemens
Pre-clinical Solutions, Knoxville, Tenn.). ROI's were manually
drawn over tumor, lung, liver and skeletal muscle. For each tissue
and time point post-injection, the measured radioactivity was
expressed as % ID/g. The maximum value was recorded for each tissue
and tumor-to-organ ratios for lung, liver and skeletal muscle were
then calculated. A two-tailed unpaired t-test was applied to
determine the significance of differences between values using the
MS Office 2003 Excel 11.0 statistical package (Microsoft, Redmond,
Wash., USA).
D. In Vivo Imaging Results
[0518] Tumor radioactivity values (% ID/g) were measured and
tumor-to-organ ratios were calculated. The highest levels of
radioactivity in the pleural tumors were found 48 h after injection
of GLV-1h99 (hNET-expressing virus), followed by tumors that were
injected with GLV-1h99 72h prior to [.sup.1241]MIBG administration.
Low levels of radioactivity were observed in tumors that were
injected with GLV-1h68 (non-hNET containing virus) and in tumors
that were not injected with virus. Maximum activity in both the
pleural tumors and remote organs (background) were observed at the
time of the initial measurement, 1 hour after radiotracer
administration. Tumor and remote organ activity decreased over time
(1 to 72 hours) in all four groups of animals. The decrease in
tumor activity was more rapid over the first 12 hours after
[.sup.124I]MIBG administration in the two control groups; tumors
injected with GLV-1h68 (non-hNET containing virus) or no virus.
[0519] Tumor-to-organ (lung, liver, muscle) ratios were calculated
from the PET image data and the highest values were obtained for
the group of animals that were infected with GLV-1h99
(hNET-expressing virus) 48h before radiotracer administration.
Comparing the animals that were treated with GLV-1h99 48 h before
[.sup.124I]MIBG administration to the animals that received no
virus, the ratio differences were highly significant (p<0.01) at
the 2h imaging time point and significant (p<0.05) at the 1 h
imaging time point. Nearly the same low tumor-to-organ ratios were
found for the two control groups of animals and the tumor-to-organ
ratios decreased over time.
[0520] For localization of the tumors and for comparison to a
clinically used imaging technique, [.sup.18F]FDG PET imaging was
also performed. [.sup.124I]MIBG PET and [.sup.18F]FDG PET imaging
were compared. The pleural tumors were visualized by [.sup.18F]FDG
PET imaging, but image contrast at 48 and 72 hours after GLV-1h99
virus (hNET-expressing virus) injection was greater with
[.sup.124I]MIBG PET compared to [.sup.18F]FDG PET. The
[.sup.124I]MIBG and [.sup.18F]FDG tumor-to-lung, tumor-to-liver and
tumor-to-muscle ratios in control animals were similar.
[0521] In vivo hNET expression in the pleural tumors after GLV-1h99
(hNET-expressing) virus administration was also imaged by
[.sup.123I]MIBG planar scintigraphy as described above. All
GLV-1h99 injected animals showed localized accumulation of
[.sup.123I]MIBG radioactivity in the virus-injected pleural tumors
compared to the control animals that received no virus. The
tumor-to-background ratios for the GLV-1h99 infected animals was
with 2.4.+-.0.2, significantly (p<0.01) higher compared to the
group that received no virus, 1.5.+-.0.1.
E. Immunohistochemistry
[0522] After each final image was taken, the animals were
sacrificed and the tumors harvested and frozen in Tissue-Tek
Optimal Cutting Temperature (O.C.T.) Compound (Sakura Finetek USA,
Inc., Torrance, Calif.). Tissues were cut into 5-.mu.m thick
sections and mounted on glass slides. Cryosections were fixed and
stained with hematoxylin and eosin (H & E) and
5-bromo-4-chloro-3-indolyl-B-D-galactopyranoside (X-gal; 1 mg/ml)
in an iron solution of 5 mmol/l K.sub.4Fe(CN).sub.6, 5 mmol/l
K.sub.3Fe(CN).sub.6, and 2 mmol/l MgCl.sub.2, as previously
described (Kelly K, et al. (2008) FASEB J 22:1839-48), to identify
virally mediated lacZ expression.
[0523] All pleural lesions were shown to be malignant pleural
mesothelioma on H & E staining. In addition, all tumors
infected with vaccinia virus stained positive for lacZ, confirming
the presence of the virus in tumors and indicating that all tumors
visualized by [.sup.124I]MIBG PET or [.sup.123I]MIBG scintigraphy
reflected GLV-1h99 expression of a functional hNET transporter
protein.
Example 4
Effect of an hNET-Expressing Virus on Tumor Growth In Vivo
[0524] The hNET expressing virus, GLV-1h99, is derived from the
parental virus strain GLV-1h68, which can eradicate solid human
breast tumors in nude mice with a single intravenous (i.v.)
injection (see U.S. Patent Publication 2005/0031643). The effect of
replacing the Ruc-GFP expression cassette at the F14.5L gene locus
in GLV-1h68 with hNET expression cassette on the anti-tumor
properties of the virus were examined. A mouse xenograft model of
human pancreatic cancer was employed for the study.
[0525] PANC-1 xenograft tumors were developed in 6-8 weeks old male
nude mice (NCl:Hsd:Athymic Nude-Foxn1.sup.nu, Harlan) by implanting
5.times.106 PANC-1 cells subcutaneously on the right hind leg.
Tumor growth was recorded once a week in three dimensions using a
digital caliper. Tumor volume was calculated as
[(length.times.width.times.height)/2] and reported in mm.sup.3.
Twenty-seven days after tumor cell implantation, groups of 8 mice
were injected with a single i.v. dose of 5.times.10.sup.6 pfu of
GLV-1h68 or GLV-1h99 in 100 .mu.l of PBS. As described previously,
the growth of tumors treated with GLV-1h68 can be divided into
three phases: growth, inhibition, and regression (Zhang et al
(2007) Cancer Res. 67(20):10038-46). The tumors treated with
GLV-1h99 showed similar growth pattern to the GLV-1h68 tumors,
however, the tumors started to shrink one week earlier for. the
GLV-1h99 injected mice as compared with GLV-1h68-treated tumors.
The tumor started to shrink after day 13 following virus
administration for the GLV-1h99 mice, whereas the GLV-1h68 mice did
not exhibit tumor shrinkage until afterday 21 following virus
administration. The degree of initial tumor shrinkage also slightly
faste in the GLV-199 injected mice. Near complete eradication of
the tumor was observed around 53-57 days following viral
administration. Expression of hNET did not have any negative
effects on vaccinia virotherapy. The accelerated tumor shrinkage by
GLV-1h99 is consistent with the enhanced viral replication in
tissue culture in comparison with GLV-1h68.
[0526] Similar results also were observed in a human breast tumor
(GLV-101A) xenograft nude mouse model treated with GLV-1h68 and
GLV-1h99. To develop subcutaneous (s.c) breast tumors in mice,
human breast cancer G1-101A cells (Rumbaugh-Goodwin Institute for
Cancer Research Inc. Plantation, Fla.; U.S. Pat. No. 5,693,533) at
a dose of 5.times.10.sup.6 cells/0.1 ml/mouse were injected s.c.
into the right hind leg of 6-8 week old female athymic mice. On day
31 after GI-101A cell implantation, when median tumor size was
about 500 mm.sup.3, GLV-1h68 and GLV-1h99 viruses at the dose of
10.sup.7 PFU/mouse were injected i.v. into the femoral vein. Tumor
shrinkage for the GLV-1h99 virus was observed at around day 24,
whereas the GLV-1h68 virus did not show tumor shrinkage in this
experiment until after day 30. Previously published results for the
GLV-1h68 virus which showed that GLV-1h68 viruses show tumor
shrinkage of G101A tumors earlier (around day 25) by either tail
vein or femoral vein administration of the virus (see Zhang et al.
(2007) Cancer Res. 67(20):10038-46); though no comparison was made
to GLV-1h99 viruses in the particular experiment. When compared to
GLV-1h68, overall GLV-1h99 appeared to shrink tumors faster in both
xenograft models.
Example 5
Toxicity Study on GLV-1h99 Viruses
[0527] The percentage of body weight change following intravenous
administration of the GLV-1h99 viruses was examined in
immunocompetent animals. C57BL/6 female mice were injected i.v.
with either 1.times.10.sup.7pfu or 1.times.10.sup.8pfu of GLV-1h68
or GLV-1h99 viruses. Body weights were monitored once a week and
calculated ate percentage body weigh over time. Neither the
GLV-1h68 or GLV-1h99 viruses caused any significant decrease in
body weight over the course of the study for either dosage of virus
tested. All mice exhibited comparable increases in body weight over
the 60 day period, with the GLV-1h68 infected mice show slightly
higher increases in body weight, which may be reflective of the
lower replication rate of the virus.
Since modifications will be apparent to those of skill in this art,
it is intended that this invention be limited only by the scope of
the appended claims.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090117034A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090117034A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References