WO2004089189A2 - Minimally-invasive imaging of viral gene therapy and presurgical applications thereof - Google Patents

Abstract

Visualization of a tumor in a patient diagnosed with cancer to evaluate the size and location of the tumor is achieved by administering to a patient having a tumor a reproduction-competent virus that specifically infects tumor tissue to express from the virus a detectable marker; and observing expression of the marker by direct observation using a minimally invasive procedure such as laparoscopy. Based on the results of this method, the appropriateness of surgical intervention in the patient can also be determined. The same minimally invasive procedure can also be used to detect the extent and persistence of viral gene therapy by observing, in vivo the expression of the detectable marker, and in addition through the use of an oncolytic virus can provide therapeutic benefits.

Description

Minimally-Invasive Imaging of Viral Gene Therapy and Presurgical Applications Thereof

Statement Regarding Government Funding [001] The research leading to this invention was funded in part by a training grant T32

CA 09501 from the National Institute of Health, and by Grant Nos. R01 CA75416 and R01CA72632 from the US Public Health Service. The US Government may have certain rights in this invention.

Statement of Related Applications [002] The application claims the benefit and priority of US Provisional Application No.

60/460,246, filed April 4, 2003, which is incorporated herein by reference in its entirety in jurisdictions permitting such incorporation.

Background of the Invention

[003] This application relates to a method for minimally invasive imaging of viral gene therapy which results in the selective expression of a detectable gene product, particularly a fluorescent gene product such as a green fluorescent protein (GFP) and to presurgical applications thereof.

[004] Transgene expression has been monitored in the past through the use of fluorescent markers such as GFP. For example, Paquin et al., Hum Gene Therap. 12(1): 13-23 (2001) disclose the use of a fluorescent marker to facilitate in vitro sorting of engineered cells. Fluorescent labels expressed in engineered tumor cells transfected in vitro have also been used to follow the growth and spread of the tumor cells following the introduction of the cells into mouse models. Yang, et al., Proc. Nat'lAcad. Sci. (USA) 99(6): 3824-3829( 2002); Yang, et al, Proc. Nat'lAcad. Sci. (USA) 98(5): 2616-2621 ( 2001); Yang, et al, Proc. Nat'l Acad. Sci. (USA) 97(22): 12278-12282 ( 2000). The procedures are used in a research context, however, and are not described as having direct therapeutic application. Summary of the Invention

[005] The present invention provides a method for visualization of a tumor in a patient diagnosed with cancer to evaluate the size and location of the tumor comprising the steps of (a) administering to a patient having a tumor a reproduction-competent virus that specifically infects tumor tissue to express from the virus a detectable marker; and

(b) observing expression of the marker by direct observation using a minimally invasive procedure such as laparoscopy. In a further aspect of the invention, based on the results of this method, the appropriateness of surgical intervention in the patient can also be determined. The same minimally invasive procedure can also be used to detect the extent and persistence of viral gene therapy by observing, in vivo the expression of the detectable marker.

Brief Description of the Drawings [006] Figs. 1 A-C show cell survival, viral titer and EGFP expression reflecting toxic effect in BE3 cells after infection with NV1066. [007] Figs. 2 A and B show the antitumor effect as reflected in tumor volume and tumor weight of NV1066. [008] Fig. 3 shows a schematic representation of an apparatus that can be used for laparoscopic observation of tumor cells. While the figure shows the subject as a mouse, the invention is fully applicable for use with other subjects including larger animals and humans. [009] Fig. 4 shows a comparison of NV1066 and wild type HSV-1.

[010] Fig. 5 shows that NV1066 lyses OCUM cells in a dose dependent fashion and completely lyses all cells by day 7 at 1 viral particle per 100 tumor cells. Cytotoxicity is expressed as % of cell survival compared to untreated control cells grown under identical conditions. [011] Fig. 6 shows that NV1066 induces GFP expression in OCUM cells.

[012] Fig. 7 shows reduction in weight of peritoneal tumor after treatment with NV1066.

Description of the Invention [013] As used in the specification and claims of this application, the term "visualization" means the creation of a state in which cells infected with the administered virus, and in particular tumor cells, are distinguishable from non-infected cells by visual observation, either as a result of observable color or observable fluorescence.

[014] The term "fluorescence" is used in the general sense to refer to emission of light in response to absorption of light of an excitation wave length, and is not intended to exclude phosphorescence.

[015] The term "administering" refers to any delivery of the virus to a patient that results in infection and the subsequent expression of the detectable marker in the patient.

[016] The term "specifically infects" refers to a virus that preferentially infects tumor tissue, as opposed to non-tumor tissue in situ within a patient with a degree of selectivity that is sufficient to render tumor tissue visually distinguishable from non-tumor tissue as a result of expression of the detectable marker.

[017] The term "minimally invasive procedure" refers to procedures that require little or no surgical invasion of the patients' body. In general such procedures make use of miniaturized instrumentation such that the size of any incision is minimized, and smaller than would be required to insert a surgeon's hands or to avoid a direct view of the interior space being viewed. A particular example of a minimally invasive procedure is a laparoscopy.

[018] The present invention provides for the in vivo use of fluorescent markers for the detection of the tumors, and for the selection of appropriate treatment based on the observed results. Unlike the applications discussed above which use virally-expressed markers in research systems, the present invention uses in vivo infection in the patient suffering from the cancer. In accordance with the invention, a reproduction-competent virus that selectively infects tumor cells and which results in expression of a marker, for example a fluorescent marker such as a GFP, is introduced into a patient suffering from a tumor. After a period of time sufficient to permit expression of the fluorescent marker, direct visualization of tumor location is used to determine whether the tumor is a surgical candidate, or whether some other course of treatment is appropriate. In addition, where surgery is performed, the fluorescent marker can be used as a guide to detect tumor materials and maximize tumor removal.

[019] In the present application, the invention is illustrated using esophageal carcinoma,

A549 lung cancer cells and gastrointestinal cancer but the invention is not limited to these tumor types. Indeed, the herpes simplex- 1 virus used is an oncolytic agent that selectively infects and lyses numerous primary and metastatic human tumors, but not normal tissues. These tumors include: Central nervous system tumors: (Yazaki T, Manz HJ, Rabkin SD, Martuza RL.

Treatment of human malignant meningiomas by G207, a replication-competent multimutated herpes simplex virus 1. Cancer Research 1995; 55(21):4752-4756.); Breast tumors: (Toda M, Rabkin SD, Martuza RL. Treatment of human breast cancer in a brain metastatic model by G207, a replication-competent multimutated herpes simplex virus 1. Human Gene Therapy 1998; 9:2177-2185.); Bladder tumors (Oyama M, Ohigashi T, Hoshi M, Nakashima J, Tachibana M, Murai M et al. Intravesical and intravenous therapy of human bladder cancer by the herpes vector G207. Hum Gene Ther 2000; 11(12): 1683-1693.; Cozzi PJ, Malhotra S, McAuliffe P, Kooby DA, Federoff HJ, Huryk B et al. Intravesical oncolytic viral therapy using attenuated, replication-competent herpes simplex viruses G207 and Nvl020 is effective in the treatment of bladder cancer in an orthotopic syngeneic model. FASEB Journal 2001; 15(7):1306-1308.); Gallbladder (Nakano K, Todo T, Chijiiwa K, Tanaka M. Therapeutic efficacy of G207, a conditionally replicating herpes simplex virus type 1 mutant, for gallbladder carcinoma in immunocompetent hamsters. Mol Ther 2001; 3(4):431-437.); Colorectal tumors (Kooby DA, Carew IF, Halterman MW, Mack JE, Bertino JR, Blumgart LH et al. Oncolytic viral therapy for human colorectal cancer and liver metastases using a multi-mutated herpes simplex virus type-1 (G207). FASEB Journal 1999; 13(11): 1325-1334.; Yoon SS, Nakamura H, Canoll NM, Bode BP, Chiocca EA, Tanabe KK. An oncolytic herpes simplex virus type 1 selectively destroys diffuse liver metastases from colon carcinoma. FASEB J 2000; 14(2):301-311.; Todryk S, McLean C, Ali S, Entwistle C, Boursnell M, Rees R et al. Disabled infectious single-cycle herpes simplex virus as an oncolytic vector for immunotherapy of colorectal cancer. Hum Gene Ther 1999; 10(17):2757-2768.); Gastric (Bennett JJ, Kooby DA, Delman K, McAuliffe P, Halterman MW, Federoff H et al. Antitumor efficacy of regional oncolytic viral therapy for peritoneally disseminated cancer. Journal of Molecular Medicine 2000; 78(3): 166-174.); Melanoma (Randazzo BP, Bhat MG, Kesari S, Fraser NW, Brown SM. Treatment of experimental subcutaneous human melanoma with a replication-restricted herpes simplex virus mutant. J Invest Dermatol 1997; 108(6):933-937.; Todo T, Martuza RL, Rabkin SD, Johnson PA. Oncolytic herpes simplex virus vector with enhanced MHC class I presentation and tumor cell killing. Proc Natl Acad Sci U S A 2001; 98(11):6396-6401.); Ovarian Tumors (Coukos G, Makrigiannakis A, Montas S, Kaiser LR, Toyozumi T, Benjamin I et al. Multi-attenuated herpes simplex virus- 1 mutant G207 exerts cytotoxicity against epithelial ovarian cancer but not normal mesothelium and is suitable for intraperitoneal oncolytic therapy. Cancer Gene Ther 2000; 7(2):275-283.); Prostate (Advani SJ, Chung SM, Yan S Y, Gillespie GY, Markert JM, Whitley RJ et al. Replication-competent, nonneuroinvasive genetically engineered herpes virus is highly effective in the treatment of therapy-resistant experimental human tumors. Cancer Res 1999; 59(9):2055-2058.; Walker JR, McGeagh KG, Sundaresan P, Jorgensen TJ, Rabkin SD, Martuza RL. Local and systemic therapy of human prostate adenocarcinoma with the conditionally replicating herpes simplex virus vector G207. Hum Gene Ther 1999; 10(13):2237-2243.); Neuroblastoma tumors (Todo T, Rabkin SD, Chahlavi A, Martuza RL. Corticosteroid administration does not affect viral oncolytic activity, but inhibits antitumor immunity in replication-competent herpes simplex virus tumor therapy. Hum Gene Ther 1999; 10(17):2869-2878.); Lung (Ebright MI, Zager JS, Malhotra S, Delman KA, Weigel TL, Rusch VW et al. Replication-competent herpes virus NV1020 as direct treatment of pleural cancer in a rat model. J Thorac Cardiovasc Surg 2002; 124(1): 123-129.); Squamous cell head and neck (Chahlavi A, Todo T, Martuza RL, Rabkin SD. Replication-competent herpes simplex virus vector G207 and cisplatin combination therapy for head and neck squamous cell carcinoma. Neoplasia 1999; 1(2): 162-169.; Carew IF, Kooby DA, Halterman MW, Federoff HJ, Fong Y. Selective infection and cytolysis of human head and neck squamous cell carcinoma with sparing of normal mucosa by a cytotoxic herpes simplex virus type 1 (G207). Hum Gene Ther 1999; 10(10): 1599-1606.). Other cancers to which the invention can be applied are hepatoma; cholangiocarcinoma; mesothelioma; and pancreatic cancer. Accordingly, the invention has broad applicability in cancer therapy. The mutant virus used in the study described below is NV1066 obtained from

Medigene, Inc. (San Diego, CA). This mutant includes a gene encoding a transgene for enhanced green fluorescent protein. As map of NV1066 is shown in Fig. 4. Other viral vectors can also be employed, however, provided they provide selective infection of tumor cells. Examples include adenovirus, retrovirus, lentevirus, adeno-associated virus, vaccinia virus, Newcastle virus. A specific alternative virus is herpes viral mutant, is NV1020, which has been used in the treatment of pleural tumors in animal models (32). In addition, other transgenes expressing readily detectable gene products, for example other fluorescent proteins or proteins which can generate a fluorescent product in situ can be used, such as blue fluorescent protein (EBFP), cyan fluorescent protein (ECFP), yellow fluorescent protein (EYFP), fluorescein, red fluorescent protein (RFP).

[022] Introduction of the virus into tumor cells can be achieved by direct localized injection of virus into an identified tumor mass. Virus may also be introduced by other routes for systemic administration, including intraperitoneal injection, intravenous, infra-arterial, intrapleural, intravesical, and oral. The virus is introduced in an amount sufficient to infect tumor cells and provide an observable level of the detectable marker, for example observable levels of fluorescence.

[023] Once the virus is established within the tumor cells and expressing the marker protein (for example eGFP), the tumor may be directly observed using a minimally invasive technique such as laparoscopy, endoscopy, colonoscopy, thoracoscopy, cytoscopy and arthroscopy. Fig. 3 shows a schematic representation of an apparatus for this purpose. The observations may be used for the following purposes:

(1) to determine whether surgical excision of the tumor is an appropriate intervention. In many cases, the size and spread of a tumor may argue against surgical intervention, or in favor of such intervention only after chemotherapy to reduce tumor volume. The ability to directly visualize the tumor gives the surgeon the opportunity to make a more informed

(2) to provide for rapid in vivo assessment of viral-based gene therapy and of therapeutic effects of oncolytic virus.

(3) helping to guide the timing of repeat viral therapy, by tracking with a minimally invasive procedure the persistence of the viral infection.

(4) monitoring the spread of reproduction-competent viral vectors.

(5) diagnosis and localization of metastatic tumors. [024] Thus, in a first embodiment, the invention provides a method for in vivo evaluation of tumor size and location in a patient. The method comprises the steps of administering to a patient having a tumor a reproduction-competent virus that specifically infects tumor tissue in situ in the patient, as opposed to normal tissue, to express from the virus a detectable marker, for example a fluorescent protein such as a GFP and observing expression of the marker using a minimally invasive procedure such as laparoscopy. The virus used in the method is suitably an oncolytic virus, for example a mutant virus based on HSV-1.

[025] In a further embodiment, the invention provides a method for directing the selection of an appropriate treatment regimen for a patient suffering from a tumor, comprising the steps of administering to a patient having a tumor a reproduction- competent virus that specifically infects tumor tissue in situ in the patient, as opposed to normal tissue, to express from the virus a detectable marker, for example a fluorescent protein such as a GFP and observing expression of the marker using a minimally invasive procedure such as laparoscopy, wherein the distribution of the detected marker indicates whether the patient is a candidate for surgical intervention. The virus used in the method is suitably an oncolytic virus, for example a mutant virus based on HSV-1.

[026] In a further embodiment, the invention provides a method for confirming the extent and persistence of viral-based gene therapy comprising the steps of administering to a patient having a tumor a reproduction-competent virus that specifically infects tumor tissue, as opposed to normal tissue, to express from the virus a detectable marker, for example a fluorescent protein such as a GFP and observing expression of the marker using a minimally invasive procedure such as laparoscopy. The virus used in the method is suitably an oncolytic virus, for example a mutant virus based on HSV-1.

[027] The invention will now be further defined with reference to the following non- limiting examples.

Example 1 [028] Esophageal carcinoma carries a grave prognosis, with surgical therapy alone rarely leading to long-term survival. Controversy exists over the best method to treat patients with this aggressive cancer. Several clinical trials have been undertaken to assess the impact of multimodality therapy on outcome. Unfortunately, results of these trials have demonstrated only modest, if any, overall survival benefit in groups of patients receiving chemotherapy and/or radiation therapy as an adjunct to, or without surgery (1). As such, new treatment strategies and therapeutics deserve investigation.

[029] Controversy also exists over the extent of resection necessary in the surgical treatment of esophageal cancer (2-4). En bloc esophagectomy with two-field or three-field lymphadenectomy has been suggested to reduce local recunence, to enhance survival, and to improve staging (3;4). This operation is associated with considerably higher morbidity than other commonly used techniques (5;6). A method to identify tumor-bearing tissue and lymph nodes mtraoperatively may make more selective resections possible, decreasing the morbidity of radical operations. Additionally, such a method incorporated into a minimally invasive surgical (MIS) staging technique could facilitate better staging of esophageal cancer prior to resection. Accurate clinical staging can prevent unwananted surgical resections and may help to determine appropriate preoperative treatment plans for individual patients.

[030] The herpes simplex virus type-1 (HSV-1) mutant NV1066, offers the intriguing possibility of both treatment and localization of esophageal cancer. Oncolytic HSV-1 mutants are replication-competent viruses that selectively infect and lyse tumor cells, while sparing normal tissues. These viruses have previously been demonstrated to be effective against a number of malignancies in experimental models (7-10). NV1066 is unique from other HSV-1 mutants previously used in our laboratory, in that it carries a transgene for EGFP. In this study we sought to determine cytotoxicity of NV1066 to an esophageal cell line both in vitro and in vivo in murine xenograft tumor models. We also sought to determine the reliability of EGFP expression as a marker of viral infection and whether EGFP expression could be used to monitor viral therapy and to identify tumor deposits in vivo, by minimally invasive means.

MATERIALS AND METHODS [031] Cells and Virus. BE3 cells are a human esophageal adenocarcinoma cell line, kindly provided by Dr. Nasser Altorki (New York Presbyterian Hospital, New York, NY). Cells were grown in RPMI media with 10% FCS, 100 U/ml penicillin, and lOOmg/ml streptomycin. NV1066 is a replication-competent attenuated herpes simplex-1 mutant virus constructed and provided by Medigene, Inc. (San Diego, CA). NV1066 is effective against a wide range of human cancer cell lines (data not reported). The virus lacks single copies of the ICP-4, ICP-0, and gl34.5 genes. NV1066 contains the EGFP sequence under the control of a constitutive cytomegalovirus (CMV) promoter. Viral stocks were generated from Vero cells using standard techniques.

[032] Cytotoxicity assay and viral titering. BE3 cells were plated in 12 well flat-bottom plates (Costar, Corning Inc., Corning, NY) in 1 ml of media and subsequently infected with NV1066 at multiplicities of infection (MOI: number of viral plaque forming units per tumor cell) of 0.1 or 1.0, in a total volume of 50 ml. Every other day after infection, media was removed, cells were washed with PBS, and lysed (1.35% Triton-X solution) to release intracellular lactate dehydrogenase (LDH). LDH was quantified using a Cytotox 96 nonradioactive cytotoxicity assay (Promega, Madison, WI) that measures the conversion of a tetrazolium salt into a red formazan product. The amount of color formed is directly proportional to the number of lysed cells. Absorbance was measured at 450 nm using a microplate reader (EL 312e: Bio-Tek Instruments, Winooski VT.) Results are expressed as surviving fraction, based on the measured absorbance of treated cellular lysates, compared to that of untreated, control cellular lysates. All samples were tested in triplicate. To determine release of viral progeny from cells, media was collected every other day after infection from the triplicate sample wells used in the cytotoxicity assay. Serial dilutions were made of the suspension and viral plaques were grown and counted on confluent Vero cells in a standard viral plaque assay.

[033] Flow cytometry for EGFP. BE3 cells were plated in 6 well plates with 2 ml of media and subsequently infected with NV1066 at MOIs of 0.1 or 1.0 in 100 ml of PBS. Untreated cells served as negative controls. Cells were harvested with 0.25% trypsin in 0.02% EDTA, combined with the supernatant fraction, centrifuged, washed in PBS, and brought up in 100 ml of PBS. Five ml of 7-amino-actinomycin (7-AAD, BD Pharmingen, San Diego, CA) was added as an exclusion dye for cell viability. Data for EGFP expression from 104 cells was acquired on a FACS Calibur machine equipped with Cell Quest software (Becton Dickinson, San Jose, CA). Results are reported as percent of live cells expressing EGFP. Animal experiments. Animal procedures were approved by the Memorial Sloan-Kettering institutional Animal Care and Use Committee (New York, NY). Six-week-old male athymic mice (National Cancer Institute, Bethesda, MD) used for experiments were provided food and water ad libitum.

[034] Flank tumors. To establish flank tumors, 1 x 10⁷ BE3 cells were harvested and resuspended (50% PBS, 50% Matrigel, BD Biosciences, Bedford, MA) in a total of 50 ml. Mice were anesthetized by intraperitoneal injection of ketamine and xylazine in sterile water. The cell suspension was injected into the left flank of mice using a 28 gauge needle. Tumors were measured in two dimensions with Vernier calipers and volume determined using the formula for a prolate spheroid, 4/3(π)ab², with "a" as the radius of the long axis and "b" the radius of the short axis in millimeters. To assess the treatment effect on established tumors, mice were randomized to receive intratumoral injection of either 50 ml of PBS (n=10) or 50 ml of PBS (n=10) solution containing 1 x 10⁷ plaque forming units (pfu) of NV1066 after flank tumors had reached an average volume of 100 mm³. Tumors were followed by weekly volume determination.

[035] In vivo viral replication. BE3 flank tumors were implanted and grown in athymic mice as described above. When tumors reached an average volume of approximately 85 mm3, they were treated with intratumoral injection of 2.5 x 106 pfu of NV1066 in 50 ml of PBS. Tumors (n=3) were harvested at serial time points, 6, 24, 48, and 72 hours, and snap frozen in liquid nitrogen. Genomic DNA was isolated per standard protocols (Wizard Genomic DNA Isolation Kit, Promega, Madison, WI). Real-time quantitative PCR was performed using a Bio-Rad iCycler thermal cycler (Bio-Rad Laboratories, Hercules, CA). Forward (5'-ATGTTTCCCGTCTGGTCCAC-3' (SEQ ID No. 1)) and reverse (5'-CCCTGTCGCCTTACGTGAA-3' (SEQ JD No. 2)) primers and a dual-labeled fluorescent TaqMan probe (5'-6FAM-CCCCGTCTCCATGTCCAGGATGG-TAMRA-3' (SEQ ID No. 3)) were designed for the 111-bp fragment of the HSV-1 ICP0 immediate-early gene. Forward (5'-CGCCTACCACATCCAAGGAA-3' (SEQ ID No. 4)) and reverse (5'-GCTGGAATTACCGCGGCT-3' (SEQ JD No. 5)) primers and a dual-labeled fluorescent TaqMan probe

(5'-VIC-TGCTGGCACCAGCTTGCCCTC-TAMRA-3' (SEQ JD No. 6)) were designed for the 87-bp coding sequence for 18s rRNA to normalize the amount of DNA. The PCR consisted of 50 cycles (step 1: 94°C for 30 sec; step 2: 55°C for 30 sec; and step 3: 72°C for 30 sec). Results are reported as fold increase in ICPO, based upon normalized values, using the 6 hour timepoint as the standard.

[036] Micrometastatic peritoneal carcinomatosis. One x 10⁷ BE3 cells were injected intraperitoneally (IP) into athymic mice in 500 ml of PBS. On the day following tumor cell injection, mice were divided into 2 groups and then treated IP with either 500 ml of PBS containing 1 x 10⁷ pfu of NV1066 (n=8) or with 500 ml of PBS alone (n=10). Four weeks after tumor inoculation, animals were sacrificed and abdominal contents were harvested en bloc. Peritonei and tumor were stripped from abdominal organs and weighed, subtracting the average peritoneal weight of same-age, non-tumor bearing animals (n=4) to approximate tumor weight.

In vivo imaging. For flank tumors, animals (n=3) were treated with 1 x 10⁷ pfu of NV1066 as above after tumors had reached an average size of 95 mm³. Imaging was performed with a stereomicroscope (Olympus America Inc., Melville, NY) in brightfield mode and after placement of both excitation and emission filters to detect EGFP. The excitation filter was fixed passage 470 ± 40 nm wavelength light as GFP has a minor excitation peak at 475 nm. The emission filter was fixed at 500 nm, to accommodate the emission peak of GFP at 509 nm. The image-capture system consisted of a Retiga EX digital CCD camera (Qimaging, Burnaby, BC). The EGFP images were taken in the near-absence of visible light.

[037] For peritoneal tumor EGFP localization studies, mice were treated 2 weeks following BE3 injection. At this time point, gross peritoneal disease is present. Again, 1 x 10⁷ pfu of NV1066 in 500 ml was administered IP. Mice were examined 48 hours later, either by laparotomy (n=2) using the stereomicroscope as above, or by laparoscopy (n=5). Laparoscopy was performed through a 6 mm midline incision in anesthetized nude mice. A 5 mm laparoscope was inserted into the abdomen and secured by sutures sunounding the incision. The laparoscopic system was developed in concert with Olympus America Inc. (Scientific Equipment Division, Melville, NY) to allow for the detection of routine white light as well as EGFP. The light source was an Olympus Visera CLV-U40 model with an adaptable excitation filter set at 470 ± 20 nm. The camera processor was an Olympus Visera OTV-S7V with an emission filter set at 510 nm. All figures were prepared using Adobe Photoshop (Pantone, Inc., Carlstadt, NJ). [038] Viral specificity for tumor. Samples of tissue emitting EGFP under stereomicroscopy were frozen in Tissue-Tek embedding medium (Sakura Finetek, Tonance, CA) and sectioned by cryotome for histological examination. Slides were first examined under fluorescent microscopy for EGFP expression, then stained with hematoxylin and eosin (H & E) to determine whether EGFP expression localized to foci of cancer. A pathologist confirmed the results.

RESULTS

[039] Figs. 1 A shows the cytotoxic effect of NV1066 on BE3 cells. NV1066 effectively kills BE3 cancer cells at multiplicities of infection (MOI) of 0.1 or 1.0 in vitro. Results are expressed as cell survival compared to untreated control cells grown under identical conditions. Fig. 1 B shows that NV1066 replicates effectively in BE3 cells. Peak viral titers following doses of 1 x 10³ pfu and 1 x 10⁴ pfu were 3.5 x 10⁶ pfu and 1.5 x 10⁶ pfu respectively. Higher titers often occur following lower initial doses during in vitro treatment with oncolytic HSV-1 mutants, as early death in cell populations treated with the higher MOI limits the time for productive cellular replication of virus. Fig 1C shows that following treatment with NV1066, infected BE3 cells express EGFP as determined by flow cytometry. Results are reported as percent of live cells in the population expressing EGFP. While initially dose-dependent, following viral replication EGFP expression incrementally increased to nearly 100% with both MOIs.

[040] NV1066 progressively killed BE3 cells in vitro at both MOIs (Figure la). Although cell kill was initially more pronounced at the higher MOI of 1.0, the MOI of 0.1 also demonstrated significant cytotoxic effects. By day 7, in vitro cell kill at MOIs of 0.1 and 1.0 is 82% and 95% respectively (p < .01, t-test). Significant in vitro viral replication occuned in BE3 cells following infection at both MOIs (Figure lb). Treatment of the cells with the lower MOI led to higher peak viral titers as determined by viral plaque assay, 3.5 x 10⁶ pfu vs. 1.5 x 10⁶ pfu, although this difference was not statistically significant (p = .09, t-test). With an MOI of 0.1, there was an estimated 3500 ± 630 fold increase in viral production and release from BE3 cells over the original infecting viral dose. [041] Flow cytometry for EGFP. The EGFP transgene carried by NV1066 was used as a marker of viral infection in BE3 cells. In vitro, with no barrier to viral replication, EGFP expression in BE3 cell populations increased over time to nearly 100%, following treatment with NV1066 at both MOIs, as determined by flow cytometry (Figure lc, p < .01 vs. untreated control cells, t-test).

[042] In vivo viral replication. NV1066 replicated in vivo following direct intratumoral injection, as determined by real-time quantitative PCR for the HSV-1 gene, ICPO. The amount of viral genome ICPO present in DNA extracted from flank tumors (normalized to 18S rRNA) at 24, 48, and 72 hours increased 2-fold, 97-fold, and 126-fold respectively over the injected amount in tumors at 6 hours. The 72 hour viral genome equivalent was 3.2 x 108 (± 7.6 x 10⁷), compared to the starting dose of 2.5 x 106 (p = .05).

[043] Treatment of in vivo tumors. Direct injection of NV1066 significantly suppressed growth of established subcutaneous BE3 tumors (Figure 2a). Intratumoral NV1066 decreased average flank tumor volumes by 77% compared to PBS treated mice at 4 weeks, 329 ± 91 mm3 vs. 1457 ± 325 mm3, respectively (p = .01, t-test). Mice who showed no evidence of tumor ulceration or other evidence of morbidity were evaluated for duration of response. In the NV1066 treated group, 3 out of 10 mice were tumor-free at 24 weeks following treatment, compared to 0 of 10 mice in the PBS group.

[044] Similarly, in a murine model of micrometastatic peritoneal carcinomatosis, treatment with intraperitoneal NV1066 decreased development of tumor burden by 73% after 4 weeks, compared to treatment with PBS alone (Figure 2b). Four of 8 mice treated with NV1066 had no evidence of gross peritoneal disease, while all 10 mice treated with PBS alone had macroscopic tumor nodules. Nodules were found throughout the abdomen, although they were predominantly located in the left upper quadrant. Average tumor weight for NV1066 treated animals was 70 ± 23 mg, compared to 252 ± 39 mg for animals treated with PBS alone.

[045] In vivo imaging. Following administration of NV1066 in vivo, EGFP expression was easily visualized by fluorescent microscopy, in both the flank and peritoneal tumor models . EGFP expression localized to tumor deposits, sparing normal tissues, even following intraperitoneal administration of virus. EGFP expression could be used to localize intraperitoneal tumor deposits, identifying foci of tumor less than even 2 mm in diameter. Similarly, using the fluorescent laparoscopic system, EGFP expression was demonstrated to localize tumor foci. The abdominal cavity was examined in both white light and GFP modes using the laparoscopic system. Non-tumor bearing organs did not fluoresce strongly when examined through the EGFP filter, although some autofluorescence was apparent from undigested food contents of the stomach and small bowel. [046] Viral specificity for tumor. Tissue specimens were selected by EGFP expression, under stereomicroscopy in intact animals. Serial sectioning was performed and specimens were examined under fluorescent microscopy (5a), then H & E stained (5b) for identification of tumor cells. Representative sections of a nodule taken from the surface of the liver are shown were observed visually for EGFP fluorescence. Following intraperitoneal administration of NV1066, strong EGFP expression was noted on the periphery of the tumor nodule. All sections that expressed EGFP had tumor cell infiltrates.

DISCUSSION [047] Esophageal cancer is characterized by a high rate of treatment failure and by an almost uniformly poor prognosis. Controversy exists over the best means to treat patients with this aggressive cancer. In this study we demonstrate that the oncolytic HSV-1 mutant, NV1066, has significant cytotoxic activity against the BE3 esophageal adenocarcinoma cell line. NV1066 effectively killed BE3 cells in vitro and in vivo in murine xenograft tumor models. The virus was effective against both established tumors and against micrometastic disease. Oncolytic HSV-1 mutants have previously been demonstrated to cause both lysis and apoptosis in infected tumor cells, often targeting pathways of cell death distinct from those induced by chemotherapy and by radiation (11-13). Studies in our laboratory using a ribonucleotide reductase deficient HSV-1 mutant, G207, demonstrated a synergistic effect with chemotherapy and with radiation therapy in the treatment of colorectal cancer cells (30). Additionally, HSV-1 mutants lacking gl34.5 genes have been shown to be efficacious against tumors that are resistant to apoptosis and or chemotherapy (14). As such, NV1066 could have synergistic effects with established therapies and may serve as a useful adjunct in the treatment of esophageal cancer.

Apart from its therapeutic effect, NV1066 could also potentially be used in the localization of metastatic or locally advanced esophageal cancer. NV1066 carries a transgene for EGFP, which is constitutively expressed 4-6 hours following viral entry into cells. In this study, we demonstrated the feasibility of using EGFP expression to localize the virus, and secondarily to localize tumor deposits in vivo due to the inherent specificity of NV1066 for cancer cells. Following intraperitoneal administration of NV1066, in situ foci of tumor could be easily distinguished by green fluorescence. This may facilitate identification of tumor bearing tissue and lymph nodes intraoperatively with fluorescent imaging systems, enabling more limited and less morbid resections. EGFP-based localization also presents the opportunity to improve staging methods, preventing unnecessary resections. Clinical trials have been undertaken to determine the best means of staging patients with esophageal cancer. Several investigators have focused on the use of combined thoracoscopic and laparoscopic (TS/LS) staging, also known as MIS staging. This technique has been found to be feasible and safe in patients with esophageal cancer and has been suggested to improve accuracy of clinical staging when compared to conventional imaging studies, including computed tomography and esophageal ultrasound. In this study, using a laparoscopic system with appropriate fluorescent filters, EGFP expression in infected tumor deposits could be identified in vivo by MIS means. Fluorescent laparoscopy has been reported previously using exogenously administered photosensitizers to aid in the detection of peritoneal tumor deposits. This technique has been reported to increase the visualization of tumors by 17-35% compared to conventional white-light laparoscopy alone (15;16). Because of its strong induction of EGFP expression in infected cells, NV1066 has the potential to improve detection of tumor deposits using fluorescent laparoscopy. Identification of metastatic lymph nodes is critical in the staging of esophageal cancer (17; 18). Unfortunately, a consistent animal model of esophageal carcinoma lymph node metastasis is not available at present (19;20). We did not address this issue in the current study, although it is an area of obvious importance in the treatment of esophageal cancer with viral therapy. Whether metastatic lymph nodes can accumulate enough NV1066 to visibly express EGFP through their external capsule remains to be proven. A previous study from our laboratory did demonstrate the ability of oncolytic HSV-1 mutants, including NV1066, to transit from the tumor bed to draining regional lymph nodes in a murine squamous cell carcinoma model (21). Such treatment led to reduced primary site recunences and regional nodal metastases. With regard to esophageal carcinoma, identification of tumor-bearing lymph nodes provides the most complete prognostic information. Due to extensive submucosal lymphatic spread and the probability of skip nodal metastases, classic sentinel node techniques are likely not applicable to esophageal carcinoma. A tumor-targeted replication-competent virus with a marker gene, in this case EGFP, could overcome this problem by trafficking to all involved lymph nodes. Oncolytic viruses such as NV1066 have the added benefit of potentially treating residual micrometastatic nodal disease. A previous study has demonstrated the malignant potential of such cells from patients with esophageal cancer. [049] In conclusion, the HSV-1 virus NV1066 has significant oncolytic activity against the BE3 esophageal adenocarcinoma cell line. NV1066 is effective in the treatment of established tumors and in limiting the progression of micrometastatic disease in xenograft tumor models. Expression of EGFP in infected cells can be used to localize the virus and may help to localize tumor deposits in vivo. By using fluorescent laparoscopic and thoracoscopic systems, oncolytic therapy with NV1066 may potentially be tracked by endoscopy.

Example 2

[050] NV1066 has significant oncolytic activity against A549 human lung cancer cells in vitro and in vivo. Expression of EGFP in infected cells was used to localize the virus and helped to identify tumor foci. By incorporating fluorescent filters into thoracoscopes, laparoscopes, or other endoscopic systems, a minimally invasive means of detection and localization of viral therapy is provided

[051] Cells and Virus. A549 human NSCLC purchased from the ATCC (Rockville,

MD) were used for experiments. Cells were grown in Ham's F-12 media with 10% FCS, 100 U/ml penicillin, and 100 mg/ml streptomycin. NV1066 is a replication-competent, attenuated herpes simplex-1 mutant virus described in detail previously (21). A sequence containing the gene for enhanced green fluorescent protein (EGFP) under the control of a constitutive cytomegalovirus (CMV) promoter was inserted into the internal repeat sequence of the parent virus. This resulted in loss of single copies of the viral genes, ICP-4, ICP-0, and γ^.S. These deletions increase the tumor specificity of viral replication and also serve to attenuate the potential neurovirulence of the parent strain. Viral stocks were propagated on Vero cells, harvested by freeze-thaw lysis and sonication, and titered by standard plaque assay.

[052] Cytotoxicity assay and viral titering. A549 cells were plated in 12 well flat-bottom plates (Costar, Corning Inc., Corning, NY) in 1 ml of media and subsequently infected with NV1066 at multiplicities of infection (MOI: number of viral plaque forming units per tumor cell) of 0.1 or 1.0, in a total volume of 50 1 of saline. Every other day after infection, media was removed, cells were washed with PBS, and lysed (1.35% Triton-X solution) to release intracellular lactate dehydrogenase (LDH). LDH was quantified using a Cytotox 96 nonradioactive cytotoxicity assay (Promega, Madison, WI) that measures the conversion of a tetrazolium salt into a red formazan product. The amount of color formed is directly proportional to the number of lysed cells. Absorbance was measured at 450 nm using a microplate reader (EL 312e: Bio-Tek instruments, Winooski VT.) Results are expressed as surviving fraction, based on the measured absorbance of treated cellular lysates, compared to that of untreated, control cellular lysates. All samples were tested in triplicate. Experiments were repeated at least twice to ensure reproducibility. To determine release of viral progeny from cells, media was collected on days 3-8 after infection from sample wells infected with NV1066 at MOIs of 0.1 and 1.0, prepared as in the cytotoxicity assay. Serial dilutions were made of the suspension and used to infect confluent Vero cells in a standard viral plaque assay. Plaques are fixed in agar, grown for 48 hours, and counted after staining with a neutral red solution.

[053] Flow cytometry for EGFP. A549 cells were plated in 6 well plates with 2 ml of media and subsequently infected with NV1066 at MOIs of 0.1 or 1.0 in 100 l of PBS. Untreated cells served as negative controls. Cells were harvested with 0.25% trypsin in 0.02% EDTA, combined with the supernatant fraction, centrifuged, washed in PBS, and brought up in 100 1 of PBS. Five μl of 7-amino-actinomycin (7-AAD, BD Pharmingen, San Diego, CA) was added as an exclusion dye for cell viability. Data for EGFP expression from 104 cells was acquired on a FACS Calibur machine equipped with Cell Quest software (Becton Dickinson, San Jose, CA). Results are reported as percent of live cells expressing EGFP.

[054] Fluorimetry for EGFP. A549 cells were plated in a T-75 flask and subsequently infected with NV1066 or mock infected with PBS. When 95% or greater of the infected cell population demonstrated EGFP expression by flow cytometry (as described above), cells were harvested with 0.25% trypsin in 0.02% EDTA and serially diluted between 1 x 10⁷ and 1 x 10⁴ cells in 100 μl of PBS. Cell suspensions of both infected and control cells were plated in quadruplicate on 96-well white fluorimeter plates and read on a Fluoroskan Ascent FL fluorimeter (Thermo Electron Corporation, Vantaa, Finland) at an excitation wavelength of 475 nm and an emission wavelength of 510 nm, taking the average of 6 readings per well. Results are reported as fluorescent intensity (RFU) per well and as a ratio of RFU of infected cells to noninfected cells.

[055] Animal experiments. Animal procedures were approved by the Memorial

Sloan-Kettering Institutional Animal Care and Use Committee (New York, NY). Six-week-old male athymic mice (National Cancer Institute, Bethesda, MD) used for experiments were provided food and water ad libitum. Prior to experiments, anesthesia was induced by means of a single intraperitoneal injection of a ketamine/xylazine mixture in sterile water.

[056] EGFP expression over time. To establish flank tumors, 2 x 10⁶ A549 cells were harvested and resuspended (50% PBS, 50% Matrigel, BD Biosciences, Bedford, MA) in a total of 50 μl. Mice were anesthetized by intraperitoneal injection of ketamine and xylazine in sterile water. The cell suspension was injected into the flanks of mice using a 28 gauge needle. Tumors were measured in two dimensions with Vernier calipers and volume determined using the formula for a prolate spheroid, 4/3(π)ab², with "a" as the radius of the long axis and "b" the radius of the short axis in millimeters. Tumors were followed by weekly volume determination until they had reached an average volume of 100 mm³. To determine EGFP expression following viral treatment, 1 x 10⁶ pfu of NV1066 (in 25μl of PBS) was administered by direct intratumoral injection. Tumors were harvested at 24, 48, and 96 hours time points and homogenized in 4 ml/g tissue of Passive Lysis Buffer (Promega, Madison, WI). Samples underwent 3 cycles of freeze-thaw lysis, followed by centrifugation at 14,000 rpm for 15 minutes. Fluorescent activity was assessed by fluorimetry using 100 μl of supernatant from each sample,with excitation and emission parameters as described for the in vitro studies. Results are reported as RFU / mg total protein. Micrometastatic pleural carcinomatosis. One x 10⁶ A549 cells were injected percutaneously into the pleural cavity of athymic mice in 100 μl of PBS as described previously (31). On the day following tumor cell injection, mice were divided into 2 groups and then treated, again by percutaneous injection into the pleural cavity with either 50 1 of PBS containing 1 x 107 pfu of NV1066 (n=9) or with 50 1 of PBS alone (n=5). Three weeks after tumor inoculation, animals were sacrificed and the heart, lungs, and mediastinum were harvested en bloc and weighed. Organ weights taken from same-age, non-tumor bearing animals (n=4) were also obtained. For both PBS and NV1066 treated groups of animals, chest wall nodules were counted after removal of organs as a measure of treatment effect on pleural carcinomatosis.

In vivo imaging. Two x 10⁶ A549 cells were injected into the pleural cavities of mice as described previously. Animals (n=4) were treated with 1 x 10⁷ pfu of intrapleural NV1066 as above at separate time points. For imaging of micrometastatic disease, mice were treated day 2 after cell injection. For imaging of gross pleural disease, mice were treated 3 weeks after cell injection. Mice were examined 48 hours later in both cases, using a fluorescent thoracoscopic system and by fluorescent stereomicroscopy. The thoracoscopic system was developed in concert with Olympus America, Inc. (Scientific Equipment Division, Melville, NY) to allow for the detection of EGFP as well as routine white light. The light source was an Olympus Visera CLV-U40 model with an adaptable excitation filter set at 470 ± 20 nm. The camera processor was an Olympus Visera OTV-S7V with an emission filter set at 510 nm. Imaging was performed in both brightfield and fluorescent modes. With the stereomicroscope (Olympus America Inc., Melville, NY), imaging was also performed in brightfield mode and after placement of both excitation and emission filters to detect EGFP. The excitation filter was fixed passage 470 ± 40 nm wavelength light as GFP has a minor excitation peak at 475 nm. The emission filter was fixed at 500 nm, to accommodate the emission peak of GFP at 509 nm. The image-capture system consisted of a Retiga EX digital CCD camera (Qimaging, Burnaby, BC). For generation of topography maps, images were presented in Zeiss LSM510 software (v3.2) using the "pseudo 3D" tool. Individual pixel intensity values of the image are mapped upon a height grid to quantify fluorescent intensity. Pixels of high intensity are represented by the grid peaks, while pixels of low intensity are represented by grid valleys. [058] Viral specificity for tumor. Samples of tissue emitting EGFP under stereomicroscopy were frozen in Tissue-Tek embedding medium (Sakura Finetek, Tonance, CA) and sectioned by cryotome for histological examination. Following paraformaldehyde fixation, slides were first examined under fluorescent microscopy for EGFP expression, then stained with hematoxylin and eosin (H & E) to determine whether EGFP expression localized to foci of cancer. To confirm that EGFP expression was localizing virus, serial sections that expressed EGFP were stained with rabbit polyclonal HSV-1 antibody using a Histomouse-SP Bulk Staining Kit (Zymed, ) and compared for EGFP expression and viral antibody binding. An institutional animal pathologist confirmed all results.

RESULTS [059] Cytotoxicity assay and viral titering. NV1066 progressively killed A549 cells in vitro at both MOIs. Although cell kill was initially more pronounced at the higher MOI of 1.0, an MOI of 0.1 also demonstrated significant cytotoxic effects over time. By day 9, in vitro cell kill at MOIs of 0.1 and 1.0 is 83 2.4% and 96 0.4%) respectively (p < .01, t-test). Significant in vitro viral replication occuned in A549 cells following infection at MOIs of 0.1 and 1.0. Viral titers increase steadily following infection, then subsequently decline as fewer live cells remain to support further replication. Treatment of the cells with the lower MOI led to higher peak viral titers as determined by viral plaque assay, 5.3 x 106 pfu/ml vs. 2.6 x 106 pfu/ml (p < .001, t-test). This phenomenon often occurs following in vitro treatment with oncolytic HSV-1 mutants, as early cytotoxic death in cell populations treated with the higher MOI limits the cell number and time for productive cellular replication of virus. Peak viral titers at the lower MOI represent an approximate 5300-fold increase over the initial dose of virus. [060] Flow cytometry for EGFP. The EGFP transgene carried by NV1066 was used as a marker of viral infection in A549 cells. This technique has been used previously to assess transduction efficiency of HSV-1 based amplicon vectors (20). EGFP expression was determined by FACS analysis. Control populations of cells did not express significant amounts of EGFP. In contrast, over 60% of cells infected with NV1066 at an MOI of 1.0 expressed EGFP after 48 hours. Looking at the results over time, expression in A549 cell populations was initially dose-dependent after treatment at MOIs of 0.1 and 1.0. Following viral replication, EGFP expression in live cells increased over time to nearly 100% at both MOIs (p < .001 vs. untreated control cells, t-test).

[061] Fluorimetry for EGFP. In order to establish the cell number needed to differentiate infected from unifected cells by standard fluorescence detection, fluorimetry was performed on populations of cells. Significant differences in fluorescent intensity were found with as few as 1000 cells (p < .005, t-test). The fluorescent signal from NV1066 infected cells increased linearly with infected cell number (not shown). By comparing the RFU of equal numbers of infected cells to uninfected tumor cells, a fluorescence intensity ratio could be calculated (Figure 3A). Ratios that have been previously demonstrated to allow for clinical detection of tumors using minimally-invasive techniques (Gahlen, J photochem and photobio, 199, 52:131-135, 46,47) were obtained in vitro at approximately 1 x 104 infected cells. At that number of cells, measured fluorescent intensity was 3.4-fold (± 0.1) higher in cells infected with NV1066 than in control cells. Ratios increased progressively with higher numbers of infected cells.

[062] Time course of in vivo EGFP expression. Viral titers increase over time as measured by real-time PCR for viral genomic DNA, with a large replication burst evident between 24-48 hours. In the present study, we assessed virus-induced EGFP expression over time, as measured by fluorimetry of protein extracts from tumors. At 24 hours following infection, EGFP expression (RFU / mg tissue) in NV1066 infected tumors was higher than in control tumors, although the difference was not statistically significant. By 48 hours however, EGFP expression in infected tumors was significantly higher than in control tumors, 4.3 x 10⁵ vs. 1.2 x 105 RFU/ mg, a 3.6-fold increase in fluorescent intensity (p = .04, t-test). Fluorescence increased to 7.1 x 105 RFU / mg by 96 hours following infection, a 5.9-fold increase over control (p = .02, t-test). We therefore decided to use time points of 48 hours or later to assess for EGFP expression in the pleural tumor model, as these time points were more likely to allow for virus detection.

[063] Treatment of in vivo tumors. Direct injection of NV1066 significantly suppressed growth of pleural A549 tumors. Organ and tumor weights of PBS treated, NV1066 treated, and matched control animals were 536 mg, 351 mg, and 373 mg respectively. There was no statistically significant difference between NV1066 treated and matched control animal organ weights, while the PBS treated animals had significantly higher organ weights, reflecting increased tumor burden (p < .01, vs. NV1066 treated animals, t-test). Similarly, PBS treated animals had an average of 71 7 chest wall nodules compared to only 7.7 7.5 nodules in the NV1066 treated group. Seven out of 9 animals in the NV1066 treated group had no evidence of macroscopic chest wall nodules or other tumor burden, compared to 0 of 5 mice in the PBS group.

[064] In vivo imaging. Following administration of NV1066 in vivo, EGFP expression was easily visualized by fluorescent microscopy in the pleural tumor model. EGFP expression was visualized in all NV1066 treated animals and localized to tumor deposits, sparing normal tissues. Green fluorescence could be used to identify tumor deposits, localizing even microscopic collections of tumor cells not apparent under brightfield microscopy. Similarly, using the fluorescent thoracoscopic system, EGFP expression also localized tumor foci. The pleural cavity was examined in both white light and GFP modes. The thoracoscopic device can be easily changed between the two modes without pausing the procedure. Non-tumor bearing thoracic organs did not fluoresce strongly in vivo when examined through the EGFP filter and were easily distinguished from infected tumor deposits. Of note, microscopic tumor deposits not identified on brightfield microscopy or thoracoscopy could be identified in the fluorescent modes of each. Expression of EGFP on the lung or chest wall identified tumor deposits that would otherwise have been missed with standard imaging. Presence of tumor cells was confirmed by histology. Using computer software, topography maps of EGFP expression could be generated from the digital images obtained with the fluorescent microscopic or thoracoscopic systems. This technology enables quantification of in vivo fluorescent intensity. [065] Viral specificity for tumor. Following pleural administration of NV1066, strong

EGFP expression was noted in tumor nodules when examining serial pathological sections. Some autofluorescence was apparent in the bronchial system, but this was easily distinguished from virus-induced EGFP expression. All sections that expressed EGFP were found to have tumor cell infiltrates conesponding to areas of expression. H&E staining confirmed that EGFP expression localized to foci of cancer. Staining for polyclonal HSV-1 antibody conesponded to areas of EGFP expression by histology. No viral staining was evident in tissues that did not express EGFP in all samples sectioned.

Example 3

[066] NV1066 expressing EGFP was used to visually localize human peritoneal carcinomatosis to provide information to assist in surgical planning and the prevention of unnecessary procedures.

[067] Cell lines: OCUM-2MD3 human gastric cancer cells were a gift of Dr.

Masakazu Yashiro (Osaka City University Medical School) and were maintained in DMEM with high glucose, 2 mM L-glutamine and 0.5 mM Na-pyruvate. Vero cells were grown in Minimal Essential Media. All cell lines were maintained in 10% FCS and 100 ?g/ml penicillin and 100 μg/ml streptomycin.

[068] Virus: NV1066 is a replication-competent, attenuated herpes simplex-1 mutant virus (22) derived from HSV-1 virus (F strain) as previously described (21). Briefly, the virus is deficient for UL23 and the internal repeat region (there is only one copy of the immediate early genes ICP-0 and ICP-4 as well as the 7,34.5 gene). The enhanced green fluorescent protein gene (EGFP) under control of a constituitively expressed cytomegalovirus (CMV) promoter was inserted into the deleted internal repeat sequence region. NV1066 was propagated and titered on Vero cells (23). GFP absorbs blue light (major peak at 395 nm) and emits green light (major peak at 509nm) (24). EGFP is a red-shift variant of GFP, fluorescing 35 times brighter (25).

[069] Cytotoxicity assay: 1.0 x 10⁴ OCUM cells were grown in 24 well flat-bottom plates and subsequently infected with NV1066 at MOIs of 1, 0.1 and 0.01 and incubated at 37°C for 1 to 7 days. Each day after infection, cells were washed in PBS and lysed with a 1.35% Triton-X solution (% volume/PBS) to release intracellular lactate dehydrogenase (LDH). The cytotoxic effect was determined by comparing release of intracellular LDH from virally-infected tumor cells to release in untreated, control cells grown under identical conditions. LDH was quantified using a Cytotox 96 nonradioactive cytotoxicity assay (Promega, Madison, WI) that measures the conversion of a tetrazolium salt into a formazan product. Absorbance was measured at 450 nm using a microplate reader (EL 312e: Bio-Tek Instruments, Winooski VT). Results are expressed as the surviving fraction compared to untreated control cells. All samples were tested in triplicate and the experiment was repeated at least three times.

[070] Viral Titering: 1.0 xlO⁴ OCUM cells were grown in 24-well flat-bottom plates and were infected with NV1066 at MOIs of 1, 0.1, and 0.01. Culture well supematants were collected each day for 7 days after infection. Serial dilutions were made of the supematants, cultured on confluent Vero cells, and viral plaques were counted in a standard viral plaque assay. All samples were tested in triplicate and the experiment was repeated at least three times.

[071] Flow cytometry for EGFP: 5 x 10⁴ OCUM cells were grown in 6 well plates and infected with NV1066 at MOIs (ie, virus to tumor ratio) 1, 0.5, 0.1, 0.05 and 0.01. Cells were harvested with 0.25% trypsin in 0.02% EDTA, washed in PBS, and brought up in 100 1 PBS. Five μl of 7-amino-actinomycin (7-AAD, BD Pharmingen) was added as an exclusion dye for cell viability. Data for EGFP expression in 10⁴ cells was acquired on a FACS Calibur machine and analyzed with Cell Quest software. A representative experiment is shown.

[072] Abdominal Carcinomatosis Tumor Model: Athymic male mice from the

National Cancer Institute (Bethesda, MD) were provided food and water ad libitum. All animal work was approved by the Memorial Sloan-Kettering Institutional Animal Care and Use Committee and performed under strict guidelines. Mice were injected intraperitoneally (IP) with 1.5 x 10⁷ OCUM human gastric cancer cells in 1 ml PBS and developed disseminated peritoneal cancer 16 days after infection (26, 27). Mice reliably develop severe carcinomatosis including cachexia and ascites. To determine the effect of NV1066 therapy on development of carcinomatosis, mice were randomized to one of the following treatment groups (n=8/group): 1) a single-treatment 1.5 x 10⁶ pfu JJP NV1066, 2) a single treatment 1.5 x 10⁷ pfu IP NV1066, 3) multiple-treatment 1.5 x 10⁶ pfu IP NV1066, 4) multiple-treatment 1.5 x 10⁷ pfu IP NV1066, or 5) untreated. Single-treatment was administered on day 1 and multiple-treatment was administered each day on days 1, 2 and 3. Mice were examined daily. Fifteen days after tumor inoculation, animals were sacrificed and abdominal contents were harvested en bloc. Peritonei and tumor were stripped from abdominal organs leaving tumor, omentum, gonadal fat and mesentery and weighed. Similar harvesting was performed on 8 mice that were inoculated with 1 ml PBS without OCUM cells to establish baseline weights. Additionally, nomial mice not subjected OCUM inoculation underwent viral treatment. NV1066 Peritoneal weights reported have mean peritoneal weight of PBS-treated animals group subtracted out. Results were confirmed in a second multi-animal study.

[073] In Vivo Imaging: Gastric carcinomatosis was established with 1.0 x 10⁷

OCUM cells. Twenty-one days after tumor inoculation, mice were treated with 1.0 x 10⁷ pfu NV1066. Each day for six days, animals were sacrificed and imaged via open laparotomy and/or laparoscopy.

[074] Open Imaging: Open imaging was performed with a stereomicroscope containing both excitation and emission filters to detect EGFP and standard visible light. The excitation filter had a fixed passage at 470 ± 40 nm as GFP has a minor excitation peak at 475 nm. The emission filter was also fixed at 500nm light, accommodating the emission peak of GFP at 509nm. The image-capture system consisted of a digital Kodak EOS BCS5 camera with shutter speed adjusted to 20X longer for EGFP imaging than for visible light image capture. The GFP images were taken in the near-absence of visible light. Images were superimposed to conelate the findings seen with GFP filtering and with visible light.

[075] Laparoscopy: Direct laparoscopy was performed through a 3 mm incision in anesthetized nude mice. The laparoscopic system allowed for the detection of routine white light as well as GFP. The light source was an Olympus Visera CLV-U40 model with an adaptable excitation filter set at 470±20nm. The camera processor was a Olympus Visera OTV-S7V with an emission filter set at 510nm. A 3mm laparoscope was inserted into the abdomen and secured by sutures sunounding the incision. Without altering the microscopic field, the fluorescent filter was changed to a GFP filter. Images were also superimposed. [076] Real Time Polymerase Chain Reaction (PCR): Organs and tumors from animals with established carcinomatosis treated with either PBS or with 1.0 x 10⁷ pfu NV1066 were harvested and snap-frozen in liquid nitrogen each day for seven days. Genomic DNA was isolated (Wizard Genomic DNA Isolation Kit, Promega) and standard curves were made by spiking uninfected samples with known quantities of NV1066 prior to extraction. Real-time quantitative PCR was performed using an ABI Prism 7700 Sequence Detector (PE Biosystems) as previously reported (28, 29). Forward (5'-ATGTTTCCCGTCTGGTCCAC-3\ SEQ JD No. 1) and reverse (5'-CCCTGTCGCCTTACGTGAA-3', SEQ ID No. 2) primers and a dual-labeled fluorescent TaqMan probe (5'-6FAM-CCCCGTCTCCATGTCCAGGATGG-TAMRA-3' SEQ ID No. 3) were designed for the 111-bp fragment of herpes ICPO immediate-early gene. Forward (5'-CGCCTACCACATCCAAGGAA-3', SEQ ID No. 4) and reverse (5'-GCTGGAATTACCGCGGCT-3', SEQ ID No. 5) primers and a dual-labeled fluorescent TaqMan probe (5'-VIC-TGCTGGCACCAGCTTGCCCTC-TAMRA-3', SEQ JD No. 6) were designed for the 87-bp coding sequence for 18s rRNA to normalize the amount of DNA. The PCR conditions included 40 cycles of PCR (stage 1 : 50°C for 2 minutes; stage 2: 95°C for 10 minutes; stage 3: 95°C for 15 seconds and 60°C for 1 minute; and stage 4, 25°C soak).

RESULTS

[077] In vitro cytotoxicity of NV1066: NV1066 kills OCUM cells over the range of

MOIs tested (Figure 5). Cell killing is dependent on viral load. By day 4 after infection, 5% of cells infected at MOI of 1 survived, 62% of cells infected at an MOI of 0.1 survived and 99% of cells infected at MOI of 0.01 survived. At all three MOIs, cytotoxicity increases progressively over the 7 day period, resulting in near complete tumor cell lysis for MOIs of 1, 0.1, and 0.01 by day 4, 6, and 7, respectively. NV1066 has a cytotoxic effect similar to its parental virus without the EGFP construct, demonstrating that the insertion of the EGFP gene does not significantly alter oncolytic potential.

[078] Viral replication of NV1066: NV1066 replicates within OCUM cells, increasing viral titers at each MOI tested. Supematants from cells infected at three MOIs were assayed for viral titer by routine plaque assay on successive days after infection. In Table 1, viral production at day 4 and peak viral production over the 7-day experiment are compared to input viral titer.

Table 1.

[079] GFP expression by NV1066 in vitro: EGFP was quantified by flow cytometry in cells infected with NV1066 in vitro. Infected cells demonstrate a progressive increase in EGFP expression over time at MOIs from 0.01 to 1 (Figure 6). Each MOI ultimately induces EGFP expression in nearly 100% of cells, demonstrating spread of virus throughout the cell culture well. Greater initial viral inoculation results in a more robust expression of EGFP. For example, at MOI of 1, 50% of the cells express EGFP 1 day after infection, while 50% of those infected at MOI of 0.1 express EGFP 3.5 days after infection.

[080] NV1066 treats peritoneal carcinomatosis: Intraperitoneal administration of

NV1066 effectively treats OCUM carcinomatosis in athymic mice. As described previously, OCUM carcinomatosis was established and treated with either low-dose (1.5 x 10⁶ pfu) or high-dose (1.5 x 10⁷ pfu) NV1066 administered intraperitoneally in a single or multiple treatment regimen (n= 8 per group). Peritoneal weights measured 15 days later demonstrated a statistically significant (p<.05, paired t-test) reduction in tumor burden in all treatment groups (Figure 7), with the exception of the low-dose cohort receiving a single-treatment regimen (p=.22). Dose-dependence was demonstrated as high-dose therapy decreased tumor burden compared to low-dose in both single and multiple treatment groups (p= .04 and .01, respectively). Of note, the high-dose, multiple-treatment group showed the greatest anti-tumor response as there was a complete return to baseline peritoneal weight.

[081] In vivo detection of NV1066: Intraperitoneal inoculation with NV1066 appreciably facilitates detection and visualization of intraabdominal carcinomatosis. Gastric carcinomatosis was established with 1.0 x 10⁷ OCUM cells. Twenty-one days after tumor inoculation, mice were treated with 1.0 x 10⁷ pfu NV1066. Animals were anesthetized and explored under direct white light and then GFP excitation and emission filters. All areas of gross tumor seen under white light fluoresce with EGFP when seen under the GFP filter. Internal organs such as stomach, small bowel and liver do not fluoresce. Additionally, smaller foci of tumor not initially obvious by examination of the abdomen under direct white light expressed EGFP, facilitating detection. In fact, determination of the number of intraperitoneal metastasis under white-light and fluorescent light by three independent observers demonstrated that 25% of the total number of metastasis seen with fluorescent imaging was missed by routine white-light inspection. Animals without established carcinomatosis treated with intraperitoneal NV1066 did not express EGFP and were alive at the termination of the experiment. No toxicity was noted.

[082] Real-Time PCR for Intraperitoneal HSV-1: EGFP fluorescence of tumors in carcinomatosis-bearing mice was brightest 3 days after IP NV1066 treatment. This conesponded to the greatest intratumoral viral copy number. For each of 6 days after infection, abdomen of carcinomatosis-bearing mice were opened and imaged with subsequent harvesting of tumor and organs. Quantitative PCR for HSV DNA in genomic DNA extracts on day 3 demonstrated 142.5 HSV copies per nanogram of isolated genomic DNA. This was roughly 2X greater than the HSV copy number on day 1 after infection (69.1 copies/ng of DNA). By day 6, NV1066 titer drops to 8.8 copies/ng. For the representative experiment shown, there were 3 animals per group.

[083] Laparoscopic Detection of NV1066: To demonstrate the feasibility of evaluating NV1066-induced EGFP expression in a minimally-invasive model, we developed a direct laparoscope. As described previously, OCUM carcinomatosis in mice was established and a 5 mm abdominal incision was made. The laparoscope was inserted and the abdomen was examined under white light. The light detection of the direct laparoscope was changed to GFP mode by the channeling of light through the appropriate excitation and emission filters. The images demonstrated selective infection and fluorescence of tumor in the left paracolic gutter. Abdominal wall, peritoneum and liver are seen under routing lighting conditions, but do not fluoresce under the GFP filters. No non-cancerous organ fluoresced EGFP when examined through the laparoscope. The following references are cited herein by reference numeral. These and all other publications cited herein are incorporated by reference as though fully set forth.

(1) Entwistle JW, JU, Goldberg M. Multimodality therapy for resectable cancer of the thoracic esophagus. Ann Thorac Surg 2002; 73(3): 1009-1015.

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Claims

CLALMS

1. A method for in vivo evaluation of tumor size and location comprising the steps of:

(a) administering to a patient having a tumor a reproduction-competent virus that specifically infects tumor tissue to express from the virus a detectable marker; and

(b) observing expression of the marker by direct observation using a minimally invasive procedure.

2. The method of claim 1, wherein the marker is a fluorescent protein.

3. The method of claim 1, wherein the marker is a green fluorescent protein.

4. The method of claim 1, wherein the minimally invasive procedure is laparoscopy.

5. The method of claim 4, wherein the marker is a fluorescent protein.

6. The method of claim 5, wherein the marker is a green fluorescent protein.

7. The method of any of claims 1 to 5, wherein the virus is an oncolytic virus.

8. The method of claim 7, wherein the virus is a mutant vims based on herpes simplex- 1 vims.

9. The method of claim 7, wherein the minimally invasive procedure is laparoscopy.

10. A method for directing the selection of an appropriate treatment regimen for a patient suffering from a tumor, comprising the steps of evaluating the size and location of the tumor using a method in accordance with any of claims 1 to 9, wherein the distribution of the detected marker indicates whether the patient is a candidate for surgical intervention.

11. A method for confirming the extent and persistence of viral-based gene therapy comprising the steps of

(a) administering to a patient in need of viral-based gene therapy a reproduction-competent virus that specifically infects tissue targeted for gene therapy to express from the vims a detectable marker; and

(b) observing expression of the marker using a minimally invasive procedure such as laparoscopy, wherein the observation of the detectable marker is indicative of the extent and persistence of the viral based gene therapy.

12. The method of claim 11 , wherein the marker is a fluorescent protein.

13. The method of claim 12, wherein the marker is a green fluorescent protein.

14. The method of claim 11 , wherein the minimally invasive procedure is laparoscopy.

15. The method of claim 14, wherein the marker is a fluorescent protein.

16. The method of claim 15, wherein the marker is a green fluorescent protein.

17. The method of claim 15, wherein the virus is an oncolytic virus.

18. The method of claim 17, wherein the viras is a mutant vims based on herpes simplex- 1 viras.

19. The method of claim 17, wherein the marker is a fluorescent protein.

20. The method of claim 19, wherein the marker is a green fluorescent protein.

21 The method of claim 19, wherein the minimally invasive procedure is laparoscopy.

22. Use of a viras having an insert encoding a detectable marker in manufacture of an imaging agent for in vivo evaluation of tumor size and location in patients having a tumor, said oncolytic virus being a reproduction-competent virus that specifically infects tumor tissue to express from the vims the detectable marker.

23. Use of claim 22, wherein the viras is an oncolytic virus.

24. Use of claim 22 or 23, wherein the marker is a fluorescent marker.

25. Use of claim 24, wherein the marker is a green fluorescent protein. .