WO2006115170A1

WO2006115170A1 - Hsv1-hf10 virus therapy for head-and-neck cancer

Info

Publication number: WO2006115170A1
Application number: PCT/JP2006/308317
Authority: WO
Inventors: Yukihiro Nishiyama; Yasushi Fujimoto; Fumi Goshima; Tsutomu Nakashima; Terukazu Mizuno
Original assignee: National University Corporation Nagoya University
Priority date: 2005-04-21
Filing date: 2006-04-20
Publication date: 2006-11-02
Also published as: JPWO2006115170A1

Abstract

Disclosed is a method for treating human head-and-neck cancer by using a herpes simplex virus mutant having inactivated UL56 gene. Also disclosed is a pharmaceutical composition for use in this method. Preferably, the herpes simplex virus mutant is herpes simplex virus type 1 strain HF10. A clinical trial for study purposes is made on a patient with head-and-neck cancer by inoculating the pharmaceutical composition comprising attenuated herpes simplex virus to the patient at a subcutaneous tumor site. As a result, it is confirmed that the virus can produce an anti-tumor effect without adversely affecting the patient.

Description

Specification

HSV1-HF10 virus therapy for head and neck cancer

Technical field

[0001] The present invention relates to treatment of human head and neck cancer using attenuated herpesvirus.

Background art

[0002] Head and neck cancer may involve all of the cicada-camilla nerves, easily impair survival functions such as breathing, swallowing, and vocalization, and may involve deformation of the exposed parts of the face and neck There is a feature. Therefore, in the treatment method selection, while organ preservation and function preservation are emphasized, local control is also the biggest issue. Head and neck cancer is difficult to achieve with chemotherapy alone. In addition, dysfunction and deformation have become problems in the treatment of head and neck cancer by surgery, and radiation therapy has problems such as the limitation of irradiation dose and impaired salivation after swallowing, and dysphagia. There are many examples where the effects are limited or the effects are not fully obtained. In addition, clinical trials for the treatment of head and neck cancer using adenovirus mutants have been carried out, but the results are not satisfactory. Therefore, it is desired to develop a new treatment method for treating head and neck cancer.

[0003] In recent years, methods have been developed that use attenuated viruses to reduce cancer regression, and are also in clinical trials in the West. This method takes advantage of the fact that cancer cells grow more rapidly than normal cells, are insensitive to interferons, and that certain oncogenes have mutations' activation. Cancer cells with these properties are often highly sensitive to HSV, which is often permissive for viral growth. Therefore, when the attenuated virus is directly infected with cancer cells, the virus grows in the cancer cells, and it is considered that the cancer cells can be destroyed and the cancer can be regressed.

[0004] As one of such treatment methods, a method using an attenuated simple herpesvirus (HSV) has been proposed. The virus has the following useful properties: 1) HSV can infect almost all types of cells, 2) HSV has the ability to infect other viruses, eg, adeno Higher than viruses, adeno-associated virus (AAV), etc. 3) All basic sequences of HSV genome have been elucidated, 4) HSV kills infected cells at low doses 5) Antiviral agents against HSV are available.

[0005] It has been disclosed that an attenuated mutant of a simple herpesvirus (HSV) type 1 that incorporates an exogenous suicide gene! 218975). It has also been reported to prolong the survival of nude mice inoculated with herpes simplex virus attenuated mutant tumors in which certain genes are inactivated (Tes higahara, et al, J. Surg. Oncol, 85: 42-47, 2004; However, it was unpredictable whether such a mutant herpesvirus would actually be effective in the treatment of human head and neck cancer.

[0006] References cited in the present specification are as follows. All the contents described in these documents are cited herein as part of this specification.

Patent Document 1: JP 2002-218975

Patent Literature 2: Tokuheihei 2004—535798

Non-Patent Document 1: J. Surg. Oncol, 85: 42-47, 2004

Disclosure of the invention

Problems to be solved by the invention

[0007] An object of the present invention is to provide a novel therapeutic method and therapeutic agent for head and neck cancer.

Means for solving the problem

[0008] The present inventors conducted a research clinical trial in which a patient with head and neck cancer was inoculated with an attenuated simple herpesvirus at the subcutaneous tumor site, and the virus had a harmful effect on the patient. It was found that it showed a tumor suppressive effect.

[0009] The present invention provides a pharmaceutical composition for treating human head and neck cancer, comprising a mutant simple herpesvirus in which the UL56 gene is inactivated. The present invention also provides a pharmaceutical composition for enhancing the effect of radiation therapy for human head and neck cancer, comprising as an active ingredient a mutant herpesvirus in which the UL56 gene is inactivated. . The present invention further provides a pharmaceutical composition for enhancing the effect of chemotherapy for human head and neck cancer, comprising as an active ingredient a mutant herpes simplex virus in which the UL56 gene is inactivated. . In another aspect, the present invention provides a method for treating human head and neck cancer by administering a mutant herpes simplex virus in which the UL56 gene is inactivated to a cancer site, Methods for enhancing the effects of radiation therapy for cervical cancer and methods for enhancing the effects of human head and neck cancer chemotherapy are provided. In the methods and pharmaceutical compositions of the present invention, the variant simple herpesvirus is preferably simple herpesvirus type 1 HF10 strain.

[0010] FIG. 1 shows an illustration of the structure of HSV HF10 used in the present invention.

FIG. 2 shows the course of treatment with the pharmaceutical composition of the present invention.

FIG. 3 shows the results of HE staining and anti-HSV staining of tissue administered with the pharmaceutical composition of the present invention.

FIG. 4 shows the course of treatment with the pharmaceutical composition of the present invention.

FIG. 5 shows the results of HE staining of the tissue administered with the pharmaceutical composition of the present invention.

FIG. 6 shows the results of anti-HSV staining of tissue administered with the pharmaceutical composition of the present invention.

[Fig. 7] Fig. 7 shows the results of anti-HSV staining of the HSV-uninfected part of the tissue administered with the pharmaceutical composition of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

[0011] The herpes simplex virus UL56 protein is a tail anchor type II membrane protein localized in the Golgi apparatus and endonom. The function of UL56 protein is still well elucidated !, but it is thought to be related to neurotoxicity! /

[0012] Mutant herpesviruses in which the UL56 gene is inactivated are mutated or deleted by a known method, or another gene fragment is inserted into the UL56 gene. It can be produced by insertion. An example of such a method is a homologous gene introduction method in which a gene chain having a target mutation site sandwiched between homologous gene sites is introduced and the gene is introduced. You can also use naturally occurring mutants in which the UL56 gene is inactivated.

[0013] One preferred example of such a mutant is the HF10 strain (eg Arch Virol, 148

(4): See 813-825, 2003). The HF10 strain is a clone found as a naturally occurring attenuated mutant virus that has not been genetically modified, and causes extensive cell membrane fusion in infected cells. The genomic structure of HF10 is shown in Figure 1. HF10 is central Although at least four accessory genes, including the gene for neuroinvasiveness (UL56), are deficient and the pathogenicity is markedly reduced, the proliferation ability in tumor cells is extremely high (over 100 times that of the wild type). ). HF10 has been reported to prolong the survival of nude mice inoculated with tumor cells derived from colon cancer, breast cancer, melanoma and sarcoma (J. Surg. Oncol, 85: 42-47, 2004). HF10 was non-toxic when intraperitoneally inoculated into mice.

[0014] The mutant simple herpesvirus in which the UL56 gene of the present invention is inactivated can be propagated by a known method. For example, it was confirmed that Vero cells derived from African green monkey kidney cells showed a cytopathic effect, collected, and subjected to three consecutive freeze-thaw operations, followed by centrifugation. Precipitate cell components by separation and collect virus-containing supernatant. The grown mutant herpes simplex virus can be formulated as an injection solution by suspending it in sterile water, sterile physiological saline or a suitable buffer. The preparation may be further added with a silkworm, a stabilizer, a preservative and the like.

[0015] The pharmaceutical composition comprising the mutant herpesvirus of the present invention as an active ingredient is useful as a therapeutic agent for head and neck cancer. The pharmaceutical composition of the present invention can be administered by directly inoculating the tumor site. Examples of head and neck cancer that can be treated by the present invention include tongue cancer, oral cavity cancer including oral floor cancer and gingival cancer, lip cancer, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, laryngeal cancer, nasal cavity cancer, thyroid gland. Examples include cancer, parotid gland cancer, submandibular gland cancer and the like. Head and neck cancer is generally characterized by having a lesion near the body surface, or a site that can be easily reached by opening or opening, so that it is easy to administer the virus and the effect of virus therapy is effectively exerted. Expected. Endoscopic injection is useful for the nasopharynx, hypopharynx, and larynx that cannot be reached by opening alone. This is a method of injecting a virus under a laryngeal fiber using a local injection needle under clear vision, and it can be administered repeatedly every day because the burden on the patient and the invasiveness are light. Further, the viral therapy of the present invention may be used in combination with radiation therapy or chemotherapy. Synergistic effects can be obtained in combination with virus therapy, and function-preserving therapy can be developed by improving local control rate. In combination therapy, it is necessary to administer the virus in such a way that the patient is not invaded as much as possible in order to carry out daily radiotherapy without interruption. In that respect, it can be injected only through the opening or under the laryngeal fiber. This treatment method does not require a facility such as an operating room, and is a treatment method that reduces the burden on the patient.

[0016] As shown in the following examples, HSV for locally advanced recurrent head and neck squamous cell carcinoma

-1 After local administration of HF10, mild fever may have occurred due to the administration of HF10, but other side effects were obvious. Infection with HF10 was confirmed in line with the necrotic part of the cancer cells, and the antitumor effect of HF10 was confirmed in humans.

[0017] The pharmaceutical composition comprising the mutant herpes simplex virus of the present invention as an active ingredient is very attenuated and its neurotoxicity is very low, so there are almost no side effects in patients. It is thought that it will not occur. Even if a few adverse events occur, the attenuated herpesvirus is highly sensitive to anti-herpes drugs such as acyclovir and ganciclovir, It is possible to administer various drugs.

[0018] The contents of all patents and references explicitly cited herein are hereby incorporated by reference as part of the present specification. In addition, the contents described in the specification and drawings of Japanese Patent Application No. 2005-123776, which is the application on which the priority of the present application is based, are cited herein as part of this specification.

[0019] The present invention will be described in more detail with reference to the following examples, but these examples do not limit the scope of the present invention.

Example

[0020] Creation of HSV-1 HF10

SPF chicken fertilized eggs for vaccine production were used to infect the virus using primary embryo cells of chicken embryos as host cells, and cultured in a culture solution supplemented with bovine serum and kanamycin, which have been denied the presence of stray viruses. The virus was infected with the master virus bank with MOIO.Olpfo / cell, cultured for 24 hours under serum-free condition, and the culture supernatant was collected. The cultured virus was subjected to sterility testing by taking over the sterility test method of the Japanese Pharmacopoeia General Test Method and the Mycoplasma Negative Test Method. In addition, HIV, EBV, CMV, and HBV were tested by PCR to rule out foreign viruses, and a febrile test and endotoxin test were also conducted. lxl0 ⁶ pfo / ml virus solution was prepared and stored at -80 ° C. At the time of administration was used by diluting ©-virus solution lxl0 ⁴ pfo / ml.

[0021] Throw method A single dose of 10 ⁴ -10 ⁵ / ml HF10 was injected at several sites into the primary lesion or lymph node metastasis lesion. The same site was injected 3 times for 3 consecutive days. Subject's quantitative globulins, leukocyte fractions, platelets, clotting ability, general biochemical tests, anti-HSV antibody titers, tumor markers, creatine clearance, and immunological tests were performed twice a week for 2 months. The effect was judged based on the evaluation index with reference to “Solid Cancer Chemotherapy Direct Effect Judgment Criteria”. O Identification of infiltrating mononuclear cells and immunohistochemical examination of the isolated tissue

[0022] Case 1

58-year-old male with recurrence of hypopharyngeal cancer rT4N2cMl

Medical history: None to be noted.

Current medical history: Difficult swallowing, pain, hoarseness appeared from around February 2003. Recurrence after chemotherapy / radiotherapy at the previous doctor, but after 6 surgeries, he did not heal and a pharyngeal cutaneous fistula formed. Metastasis has also increased in the left supraclavicular skin. On July 13, 2004, treatment with HSV HF10 was started.

[0023] 1 X 10 ⁴ PFU of HF10 was administered locally to the supraclavicular skin metastases (40x33x28mm) for 3 consecutive days. On the second day after administration, fever continued at 37 ° C for 10 days, but then calmed down. In addition, the adverse events that should be noted were conspicuous. Figure 2 shows the course of treatment.

[0024] The tumor size increased 45x40x38mm after 7 days, 55x44x34mm after 15 days, and 55x50x33mm after 21 days, and was evaluated as PD (progressive) in the clinical efficacy assessment. Figure 3 shows the pathological examination. 1: HE staining, 2: HSV-1 immunostaining, 3: Negative control. Although the virus antigen has proved powerful, pathologically, the tumor cell necrosis is found in the local injection part, which is more conspicuous than the non-local injection part, which may be effective by HF10. It is considered high.

[0025] Case 2

A 79-year-old woman. Tongue cancer recurrence rT0N2bM0

Tongue pain from early November 2003. In January 2004, she was diagnosed with tongue cancer T2N1M0 and underwent arterial chemotherapy and radiotherapy. In August 2004, her neck relapsed. Because it was inoperable He received radiation therapy combined with intra-arterial chemotherapy but still has tumors.

[0026] In November 2004, treatment with HSV HF10 was started. At this time, left upper internal deep neck lymph node metastasis invaded the carotid artery and skin, and 19 x 10 mm skin metastasis was observed in the armpit skin. HF10 was administered topically at 1 x 10 ⁵ PFU once a day for 3 consecutive days to the armpit skin metastasis (total 3 x 10 ⁵ PFU).

[0027] From 3rd day, a fever of 37.5-38 ° C was observed for 7 days. In this case, edema of the floor of the mouth and larynx due to cervical metastasis of the tongue cancer was highly observed, resulting in dysphagia, and aspiration pneumonia occurred on the 11th day of the study. Therefore, an emergency tracheotomy was performed on the 14th day of the test, and at the same time the armpit skin metastasis was removed. Figure 4 shows the course of treatment.

[0028] The tumor diameter at the time of excision was 20 X 12 mm. The clinical efficacy assessment was evaluated as NC (unchanged). A large number of necrotic parts were observed in the pathological specimen, where eosin-favored nuclei were obscured or disappeared. In addition, the appearance of multinucleated cells and infiltration of inflammatory cells were observed (Fig. 5). Furthermore, immunostaining with anti-HSV-1 antibody proved the presence of HSV consistent with the necrotic area (Fig. 6). On the other hand, in the uninfected part of HF10, a large number of dividing cells were recognized, and actively proliferating cancer tissues were observed (Fig. 7).

[0029] At the same time during the clinical study, other metastatic foci showed a clear tendency to increase, but growth was suppressed at the site of local injection. From the pathological examination of the excised specimen, HSV antibody was found in the site of local injection consistent with the necrotic part, and it was confirmed that the cancer cells were necrotized by HF10 in human head and neck squamous cell carcinoma. In other words, the anti-tumor effect of HF10 could be confirmed in humans.

[0030] Case 3

64-year-old man. Hypopharyngeal squamous cell carcinoma (rT0N2cMl)

A part of the hypopharynx was removed through a cervical incision, followed by chemotherapy and radiation therapy. Later, recurrence occurred and metastasis to the skin of the cervical lymph nodes and the cervical, precordial, and supraclavicular areas was observed (rT0N3Ml). The size of the skin metastasis at the start of treatment was 0.9 x 0.9 cm. HF10 (1 × 10 ⁵ PFU / 0.5 ml) was injected into the skin metastases once a day for 3 days. There was a mild fever for several days after injection. No other clinical findings changed during the study. The size of the nodule at the injection site was measured and excised 15 days after inoculation. Excision The size of the skin metastasis at the time was 0.8 x 0.9 cm. On the other hand, nodules in the non-injected area increased rapidly. The specimens were fixed with 10% buffered paraformaldehyde, embedded in paraffin blocks, and sections were prepared. Immunostaining was performed using a polyclonal antibody against HSV-1 and a monoclonal antibody against CD4 or CD8. Anti-HSV IgG and Ig M titers did not change. Pathological examination of the resected skin metastasis revealed tumor cell necrosis and fibrous tissue formation. Interestingly, most HF10-infected tumor cells did not have a well-defined nucleus. In nodules injected with HF10, CD4 positive or CD8 positive cell infiltration was much more potent than untreated nodules. Furthermore, immunohistochemical staining using anti-HSV-1 confirmed that the squamous cell carcinoma was infected with the virus. That is, in human head and neck squamous cell carcinoma, it was confirmed that cancer cells were necrotized, consistent with HF10 infection.

[0031] In all cases 1 to 3, slight fever continued for 1 week after HF10 administration. Although it cannot be denied that it was an effect of HF10 administration, the patient did not complain of pain. In addition, there was no side effect in terms of changes in the virus antibody titer, etc., which are biochemically related to blood biochemistry! Industrial applicability

[0032] The pharmaceutical composition comprising the attenuated mutant herpes simplex virus of the present invention as an active ingredient is effective for the treatment of human head and neck cancer.

Claims

The scope of the claims

[1] A pharmaceutical composition for treating human head and neck cancer comprising, as an active ingredient, a mutant herpesvirus in which the UL56 gene is inactivated.

[2] A pharmaceutical composition for enhancing the effect of radiation therapy for human head and neck cancer, comprising a mutant herpes simplex virus in which the UL56 gene is inactivated as an active ingredient.

[3] A pharmaceutical composition for enhancing the effect of chemotherapy for human head and neck cancer, comprising a mutant herpes simplex virus in which the UL56 gene is inactivated as an active ingredient.

[4] The pharmaceutical composition according to any one of claims 13 to 13, wherein the mutant herpes simplex virus is a simple herpesvirus type 1 HF10 strain.

[5] A method for treating human head and neck cancer by administering to a cancer site a mutant simple herpesvirus obtained by inactivating the UL56 gene.

[6] A method of enhancing the effect of radiation therapy for human head and neck cancer by administering a mutant herpesvirus, which is caused by inactivation of UL56 gene, to the cancer site.

[7] A method for enhancing the effect of chemotherapy for human head and neck cancer by administering to a cancer site a mutant herpesvirus obtained by inactivating the UL56 gene.

[8] The method according to any one of [5] to [7] above, wherein the mutant herpes simplex virus is simple herpesvirus type 1 HF10 strain.