There is provided a novel therapeutic agent for pancreatic cancer,
metastatic breast cancer and recurrent breast cancer in human. The
present invention provides a method for treating a patient having
pancreatic cancer, metastatic breast cancer or recurrent breast
cancer comprising administrating to the patient a herpes simplex
virus mutant where UL56 gene Is inactivated. Preferably, the herpes
simplex virus mutant is a herpes simplex virus type 1 HF10 strain.
1. A method for treating a patient having pancreatic cancer, metastatic
breast cancer or recurrent breast cancer comprising administrating
to the patient a herpes simplex virus mutant where UL56 gene is
2. The method as claimed in claim 1 wherein the herpes simplex
virus mutant is a herpes simplex virus type 1 HF 10 strain.
BACKGROUND OF THE INVENTION
 1. Field of the Invention
 The present invention relates to treatment of human breast
cancer and pancreatic cancer, particularly metastatic breast cancer
and recurrent breast cancer using attenuated virus.
 2. Description of the Related Art
 Breast cancer is one of the most common and feared cancers.
Although many methods for treatment such as novel chemotherapeutic
agents and hormone preparations have been developed, prognosis of
the patients with advanced or metastatic/recurrent breast cancer
remains poor. In Japan, the number of breast cancer patient is in
an increasing due to the changes of food, life style, stress and
so on. Accordingly, there has been a demand for a novel strategy
for the treatment of metastatic breast cancer and recurrent breast
 Pancreatic cancer is one of the most lethal of human malignancies,
and Is difficult to detect at early stage. Common strategy in the
treatment of pancreatic cancer is surgery, radiotherapy and chemotherapy,
and combinations thereof. Currently, the most common strategy is
treatment with gemcitabine. However, the prognosis of pancreatic
cancer is still poor, with the five-year survival rate of 9.5%.
Accordingly, a novel strategy for effective treatment of pancreatic
cancer is required.
 Attenuated herpes simplex virus (HSV) have been proposed
for use in the treatment of cancer. This virus has the following
useful characteristics: 1) HSV is able to infect to nearly all kinds
of cells; 2) infecting ability of HSV is higher than other viruses
such as adenovirus and adeno-accompanied virus (AAV); 3) all fundamental
sequences of HSV genome have already been determined: 4) HSV is
able to kill infected cells at a low dose; and 5) antiviral agent
to HSV is available.
 Many HSV mutants have been modified according to genetic
engineering throughout the world and evaluated for their effect
as agents for treatment in the therapy of cancer. Examples of such
HSV mutants are G207, NV1020 and MGH1. G207 has deficiency of gamma
34.5 (.gamma..sub.1 34.5) of both alleles and insertion of Escherichia
coil lacz gene at UL39, while NV1020 has deficiency at one of the
gamma 34.5 allele and also has deficiency in an internal repetitive
region and deficiency of UL23. All of them have been prepared for
attenuation of virus by inactivating the gamma 34.5 allele. However,
those viruses are too much attenuated and are unable to sufficiently
kill malignant cells and, therefore, nearly all clinical tests using
NV1020 and G207 in the United States failed to achieve a sufficient
effect in terms of shrinking effect for cancer except safety issues.
 It is disclosed that an attenuated mutant of herpes simplex
virus (HSV) type 1 in which exogenous suicide gene is incorporated
is effective for the treatment of cancer (Japanese Patent Laid-Open
No. 2002/218,975). There is another report that an attenuated mutant
of herpes simplex virus in which a certain type of gene is inactivated
can elongate the survival period of nude mice into which tumor cells
are inoculated (Teshigahara, et al., J. Surg. Oncol., 85: 42-47,
2004; WO 02/092826). However, it has not been possible to predict
whether herpes simplex virus mutants as such actually have efficacy
in the treatment of human cancer and whether they are able to be
used for the treatment of metastatic or recurrent cancer.
SUMMARY OF THE INVENTION
 An object of the present invention is to provide a novel
agent for the treatment of breast cancer and pancreatic cancer,
in particular metastatic breast cancer or recurrent breast cancer.
 The present inventors have carried out a phase I clinical
test in which an attenuated herpes simplex virus was inoculated
to subcutaneous tumor site of recurrent breast cancer for the patients
having metastatic breast cancer and found that the virus reduced
the metastatic focus without affecting harmful influence to the
patients. Thus, in one aspect, the present invention provides a
method for treating human metastatic breast cancer or recurrent
breast cancer comprising a herpes simplex virus mutant where UL56
gene is inactivated. Preferably, the herpes simplex virus mutant
is a herpes simplex virus type 1 HF 10 strain.
BRIEF DESCRIPTION OF DRAWINGS
 FIG. 1 illustrates the structure of HSV HF10 used in the
 FIGS. 2A and 2B show an ultrasonic picture of subcutaneous
metastasis of breast cancer of the patient No. 3.
 FIGS. 3A, 3B, 3C and 3D show specimens of tumor sites of
patients stained with hematoxylin-eosin.
 FIG. 4 shows immunostaining with an anti-HSV antibody of
a specimen of tumor site to which HSV HF10 was injected.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
 UL56 protein of herpes simplex virus is a membrane protein
of a tail-anchored type II localized in Golgi apparatus and endosome.
Although function of UL56 protein has not been well clarified yet,
it is likely to play a role in the vesicle transportation in virus-infected
cells (J. Virol 76: 6718-6728, 2002), suggesting that UL56 may possibly
be involved in antegrade axone transportation of virus envelope
 A herpes simplex virus mutant in which UL56 gene is inactivated
is able to be prepared either by mutation or deletion of a part
of UL56 gene or by insertion of another gene fragment into UL56
gene by way of a known method. An example of such a method is a
homologous gene introduction method using a gene fragment in which
the sequence to be mutated is flanked by homogeneous sequences.
It is also possible to use a naturally generated mutant in which
UL56 gene is inactivated.
 One of the preferred examples of such a mutant is an HF10
strain (see, for example, Arch. Virol., 148 (4); 813-825, 2003).
HF10 is derived from an HF strain of HSV-1 and is a clone which
causes an extensive cell membrane fusion in infected cells. Genome
structure of HF10 is shown in FIG. 1. Its characteristics are as
follows: (i) a 3,832-base pair (bp) between nucleotides 116,514
and 120,347 is deleted which indicates that HF10 contains a complete
open reading frame (ORF) of ICP0 but has a partial deletion in the
UL56 ORF and promoter is completely deleted; and (ii) sequences
between 6,025 and 8,319 are deleted from the TRL and the 6,027 bp
DNA from nucleotide 110,488 to 116,514 is inserted in an inverted
orientation. As a result, the HF10 contains two complete copies
of UL53, UL54 and UL55, one complete copy and one partial copy of
UL52 and two incomplete copies of UL56 containing no promoter (Arch.
Virol., 148(4): 813-825, 2003). HF10 has been reported to elongate
the survival period of nude mice in which tumor cells derived from
colon cancer, breast cancer, melanoma and sarcoma are inoculated
(J. Surg. Oncol., 85: 42-47, 2004). Further, HF10 was nontoxic when
intraperitoneally inoculated to mice.
 Herpes simplex virus mutant of the present invention in
which UL56 gene is inactivated can be grown by a known method. For
example, Vero cells derived from renal cells of African green monkey
are infected with virus. Round form cells containing virus are collected
and subjected to three cycles of freeze-thaw. Cell debris are precipitated
by centrifugation to obtain a supernatant containing the virus.
The proliferated herpes simplex virus mutant is suspended in sterilized
water, sterilized physiological saline solution or any other appropriate
buffer to prepare a pharmaceutical preparation of an injectable
solution. Further, stabilizers, preservatives or the like may be
added to the preparation.
 A therapeutic agent comprising herpes simplex virus mutant
of the present invention is useful as an agent for the treatment
of cancer. The therapeutic agent of the present invention is able
to be administered by a direct inoculation to tumor site. The therapeutic
agent of the present invention shows a high efficacy particularly
as an agent for the treatment of metastatic breast cancer or recurrent
breast cancer. As will be shown in the following Examples, cancer
cells of the administered site died. Histopathological study showed
death of cancer cells to an extent of 30% to 100%.
 In addition to breast cancer and pancreatic cancer, the
therapeutic agent of the present invention may be used for the treatment
of, for example, stomach cancer, esophagus cancer, large bowel cancer,
liver cancer, and skin cancer.
 The herpes simplex virus mutant used in the therapeutic
agent of the present invention is considerably attenuated and its
neurotoxicity is significantly reduced, thus it is expected to cause
little or no side effect in patients. In the case that some adverse
event is observed, it is possible to administer an anti-herpes agent,
such as acyclovir and ganciclovir, to the patients for the sake
of safety, because attenuated herpes virus has a high sensitivity
to such an anti-herpes agent.
 The present invention will be illustrated in more detail
by way of Examples below, although the present invention is not
limited by those Examples.
Treatment of Breast Cancer
 Patients and Methods
 All six patients were female, aged 48 to 76 and diagnosed
as breast cancer. While all of them had mastectomy, they recurred
and had adjuvant chemotherapy and/or endocrine therapy and/or radiation
therapy, The recurrent foci were progressive and metastasized to
cutaneous or subcutaneous region and that was pathologically proven
to be recurrent breast cancer. The profiles of the patients are
shown in Table 1.
1TABLE 1 Amount of Administered Histological Histopathological
No. Age Sex Virus (pfu) Times Response Observation Toxicity 1 61
female 1 .times. 10.sup.4 1 1b invasive ductal -- carcinoma 2 62
female 1 .times. 10.sup.5 1 1a invasive ductal -- carcinoma 3 48
female 1 .times. 10.sup.5 3 2 invasive ductal -- carcinoma 4 66
female 5 .times. 10.sup.5 1 1b invasive ductal -- carcinoma 5 72
female 5 .times. 10.sup.5 3 2 to 3 mucinous -- carcinoma 6 76 female
5 .times. 10.sup.5 3 judgment scirrhous -- impossible carcinoma
 In order to evaluate a therapeutic possibility of HF10,
which is a herpes simplex virus mutant of the present invention,
in human diseases, toxicity and efficacy of HF10 were tested in
six patients with subcutaneous recurrent breast cancer as a phase
1 test. For each patient, HF10 diluent in various doses was injected
to a test tumor site. For patient No. 1: 1.times.10.sup.4 pfu/0.5
ml; for patient No. 2: 1.times.10.sup.5 pfu/0.5 ml; for patient
No. 3: 1.times.10.sup.5 pfu/0.5 ml for three days; for patient No.
4: 5.times.10.sup.5 pfu/0.5 ml; for patient Nos. 5 and 6: 5.times.10.sup.5
pfu/0.5 ml for three days. As a control, 0.5 ml of sterile physiological
saline solution was injected to the second tumor site. All patients
were monitored for local and systemic adverse effects, and body
temperature, local heat, size of tumor site, reddishness and degree
of inflammation were checked.
 Assessment of Therapeutic Response
 Assessment for the therapeutic response followed the histopathological
criteria for therapeutic effect prepared by the Japanese Breast
 All six patients showed good tolerability in the clinical
test. Cancer cells were killed to an extent of 30 to 100% and histopathologically
denatured. In all patients, local and systemic adverse side effect
was not observed.
 Macroscopic Findings
 Microscopically, in the recurrent tumor site of the patient
No. 3 to whom 1.times.10.sup.5 pfu/0.5 ml of HF10 was injected for
three days, the height of the tumor site decreased. FIG. 2 shows
the image of the ultrasonic test of subcutaneous metastasis of breast
cancer in the patient No. 3. FIG. 2A shows the image of ultrasonic
test for the same focus before injection of HF10 while FIG. 2B shows
the same after 10 days from the injection. As compared with FIG.
2A, the height of the tumor site in FIG. 25 decreased about 30%.
In other patients, macroscopic sizes of the tumor site did not show
 Blood Tests
 Blood tests of the patients were carried out after 0, 1,
3, 7, 14 and 21 day(s) from injection of the virus to measure white
blood cells, HSV IgG, NK, IL10, IL12, IFN.alpha., IFN.beta., etc.
No change was observed in any of those values.
 Histopathological Observation
 After 14 days from injection of the virus, the tumor site
was excised and a histopathologically tested by hematoxylin-eosin
(HE) staining. FIG. 3A shows an HE-staining of the specimen before
injection of HF10 in the patient No. 3 presenting invasive ductal
carcinoma as a result of subcutaneous metastasis of breast cancer.
FIG. 3B shows a tissue excised after 14 days from injection of HF10,
showing that about two thirds of malignant cells died. Inclusion
bodies are noted in breast cancer cells. FIG. 3C shows HE-staining
of the specimen before injection of HF10 in the patient No. 5 who
has mucinous carcinoma as a result of subcutaneous metastasis of
breast cancer. Tumor cells are present around the mucin. FIG. 3D
shows that malignant cells completely died after 14 days from injection
 As a whole, about 30 to 100% of malignant cells died out
by injection of herpes simplex virus mutant and, in the patient
Nos. 1 to 5, no cell death was noted in the tumor site to which
a physiological saline solution was injected. In the patient No.
6, there was nearly no cancer cells in the recurrent tumor site
and, therefore, the effect of the virus was not able to be judged.
 Immunostaining Method
 The tumor site to which the virus was injected was excised
and immunostained with anti-herpes simplex virus type 1 antibody
(DAKO Corporation, Glostrup, Denmark). In all of the patients, presence
of viral infection was found to be limited to breast cancer cells
(FIG. 4). In addition, in all of the six patients, virus was noted
in the breast cancer cells from virus-treated tumor site, while
antigen-staining was not observed in the adjacent normal tissues.
Since each patient was positive for anti-HSV antibody in blood before
injection of the virus, it is noted that the ability of herpes simplex
virus mutant HF10 to replicate in tumor cells is not blocked by
the previous exposure to HSV.
Treatment of Pancreatic Cancer
 All two patients were male, aged 60 and 68 diagnosed as
pancreatic cancer by intra operative histopathological findings.
Virus (HF10) injection was performed into tumor foci after the decision
of inapplicability for respectable tumor due to peritoneum metastasis,
and catheter was inserted into the tumor foci. Consecutive virus
injection was performed on POD1 and POD2 using the catheter. The
profiles of the patients and result of the test are shown in the
2 Amount of Virus Administered Histological Histopathological No
Age Sex (pfu) Time Response Observation Toxicity 1 68 Male 1 .times.
10.sup.5 3 Unknown Adenocarcinoma -- 2 60 Male 1 .times. 10.sup.5
3 Unknown Adenocarcinoma --
 Until the time point of 2 months after the first injection,
no progression of disease was observed.
 This application claims priority to Japanese Patent Application
No. 2004-156475, filed on May 26, 2004. This application is hereby
incorporated by reference herein in its entirety, including the
 All patents and publications mentioned in the specification
are indicative of the levels of skill of those skilled in the art
to which the invention pertains. All references cited in this disclosure
are incorporated by reference to the same extent as if each reference
had been incorporated by reference in its entirety individually.
 It will be readily apparent to one skilled in the art that
varying substitutions and modification may be made to the invention
disclosed herein without departing from the scope and spirit of
the invention. Thus, such additional embodiments are within the
scope of the present invention and the following claims.