THERAPEUTIC PHARMACEUTICAL COMPOSITION FOR BONE AND SOFT TISSUE TUMORS

Abstract

A method for treating a bone and soft tissue tumor, including administering a conditionally replicating adenovirus having a E1A gene under expression control of a Survivin promoter, to a subject in need thereof. A conditionally replicating adenovirus including a E1A gene under expression control of a Survivin promoter and the conditionally replicating adenovirus is not defective in a E3 region.

Claims

1-15. (canceled)

16. A method for treating a bone and soft tissue tumor, comprising: administering a conditionally replicating adenovirus having a E1A gene under expression control of a Survivin promoter, to a subject in need thereof.

17. The method according to claim 16, wherein the bone and soft tissue tumor is a primary bone and soft tissue tumor.

18. The method according to claim 16, wherein the bone and soft tissue tumor is a recurrent primary bone and soft tissue tumor or a recurrent metastatic bone and soft tissue tumor.

19. The method according to claim 16, wherein the bone and soft tissue tumor is a bone tumor.

20. The method according to claim 19, wherein the bone tumor is chordoma.

21. The method according to claim 16, wherein a total dosage of the conditionally replicating adenovirus to the subject per dose is 1?10.sup.10 vp to 1?10.sup.12 vp.

22. The method according to claim 16, wherein the conditionally replicating adenovirus is administered once or repeatedly once every 4 weeks.

23. The method according to claim 16, wherein a dosage volume of the conditionally replicating adenovirus is varied in accordance with a volume of the bone and soft tissue tumor to receive administration.

24. The method according to claim 16, wherein a total dosage of the conditionally replicating adenovirus to the subject per dose is 1?10.sup.11 vp.

25. The method according to claim 16, wherein the bone and soft tissue tumor is present in a plurality of sites, wherein the conditionally replicating adenovirus is administered to at most 3 sites, and wherein a dosage is divided into portions in accordance with volumes of the bone and soft tissue tumors.

26. The method according to claim 16, wherein the bone and soft tissue tumor is present at one site and has a volume of 30 cm.sup.3 or more, and wherein the conditionally replicating adenovirus is administered only to the one site.

27. The method according to claim 23, wherein the dosage volume of the conditionally replicating adenovirus is determined as follows: 1 mL when the volume of the bone and soft tissue tumor to receive administration is less than 5 cm.sup.3; 2 mL when the volume of the bone and soft tissue tumor to receive administration is 5 cm.sup.3 or more and less than 9 cm.sup.3; 3 mL when the volume of the bone and soft tissue tumor to receive administration is 9 cm.sup.3 or more and less than 15 cm.sup.3; 5 mL when the volume of the bone and soft tissue tumor to receive administration is 15 cm.sup.3 or more and less than 21 cm.sup.3; 7 mL when the volume of the bone and soft tissue tumor to receive administration is 21 cm.sup.3 or more and less than 30 cm.sup.3; and 10 mL when the volume of the bone and soft tissue tumor to receive administration is 30 cm.sup.3 or more.

28. The method according to claim 16, wherein the conditionally replicating adenovirus is not defective in a E3 region.

29. The method according to claim 16, wherein a E1B region of the conditionally replicating adenovirus is E1B?55K.

30. The method according to claim 16, wherein a E1B region of the conditionally replicating adenovirus is controlled by a promoter different from a promoter controlling a ETA region.

31. The method according to claim 16, wherein a E1B region of the conditionally replicating adenovirus is controlled by a CMV promoter.

32. A conditionally replicating adenovirus having a ETA gene under expression control of a Survivin promoter, wherein the conditionally replicating adenovirus is not defective in a E3 region.

33. The conditionally replicating adenovirus according to claim 32, wherein a E1B region of the conditionally replicating adenovirus is E1B?55K.

34. The conditionally replicating adenovirus according to claim 32, wherein a E1B region of the conditionally replicating adenovirus is controlled by a promoter different from a promoter controlling a E1A region.

35. The conditionally replicating adenovirus according to claim 32, wherein a E1B region of the conditionally replicating adenovirus is controlled by a CMV promoter.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0109] FIG. 1 The figure shows the therapeutic process of a patient (45 years old, female) having sacral chordoma and treated with a low dose of a drug. The patient developed sacral chordoma in 2007, had a recurrence in 2013 in the right buttock soft tissue, not applicable either chemotherapy or radiation therapy, and was administered with Surv.m-CRA-1 in August of 2016. The middle table shows response evaluations (upper stage) based on the RECIST standard and response evaluations (lower stage) based on the Choi standard from before administration to 114 weeks after administration. The photographs shown in the lowermost part are CT images of a lesion before administration, 12 weeks, 28 weeks and 100 weeks after administration in the order from the left (PR: partial response, SD: stable disease condition).

[0110] FIG. 2 The figure shows the therapeutic process of a patient (61 years old, male) having thoracic chordoma and treated with a low dose of a drug. The patient had a resection surgery of thoracic chordoma from the right mediastinum in 2006 and the 4th recurrence in the adjacent vertebral body (seventh thoracic spine) in 2017. Since a standard treatment was not effective, Surv.m-CRA-1 was administered. The middle table shows the therapeutic process from before administration to 135 weeks after administration. The table shows the major-axis diameter (mm) and a change rate thereof (%), CT value (HU) and a change rate thereof (%), response evaluations based on the RECIST standard, response evaluations based on the Choi standard, non-contrast-enhanced CT value and a change rate thereof (%) in the order from the top. The photographs shown in the lowermost part are CT images of a lesion before administration, 4 weeks, 12 weeks and 56 weeks after administration in the order from the left (PR: partial response, SD: stable disease condition).

[0111] FIG. 3 The figure shows graphs showing the cell viabilities of liver cancer cell line (HepG2) and normal fibroblasts (WI-38) infected with Surv.m-CRA-1 in vitro. The vertical axis shows the ratios (%) of viable cell counts in the cases of Surv.m-CRA-1 (E3 is present) and Surv.m-CRA (E3 is absent) in the same conditions (the same MOI and days) to the viable cell counts (regarded as 100%) of the control, Ad.dE1.3 in individual conditions. The horizontal axis shows the multiplicity of infection (MOI). Reference symbol * indicates statistically significant (P<0.05) to a control.

DESCRIPTION OF EMBODIMENTS

[0112] In an embodiment, the present invention relates to a pharmaceutical composition for treating a bone and soft tissue tumor, containing a conditionally replicating adenovirus having the E1A gene under expression control of a Survivin promoter as an active ingredient.

[0113] The conditionally replicating adenovirus (CRA) refers to a virus proliferating specific to a tumor to damage the tumor. The conditionally replicating adenovirus is also called an oncolytic virus, which is commonly known in the field to which the invention pertains.

[0114] In the specification, having the E1A gene under expression control of a Survivin promoter means that the Survivin promoter directly binds to a site upstream of the E1A gene so as to control the expression of the E1A gene. For example, the Survivin promoter may bind to a site 10 to 200 bp upstream of the transcription initiation region of the E1A gene. Preferably, the E1B region of conditionally replicating adenovirus is E1BA55K. The E1BA55K refers to a protein obtained by removing a p53 binding region from the adenovirus E1B protein (JAMES R. BISCHOFF et al., SCIENCE 18 Oct. 1996: 373-376), and is also called E1B19K. It is known that the virus having ElBA55K can be proliferated in the tumor cells having a deletion of p53 but cannot be proliferated in normal cells having p53.

[0115] It is preferable that the E1A region and the E1B region (E1B?55K) are controlled by different promoters. Due to this, it is possible to separately control the expression of E1A and the expression of E1B?55K (Nagano et al., WO2005/012536). In the specification, such an adenovirus is set forth as CRA regulated with multiple tumor-specific factors; m-CRA. A promoter controlling the E1B region is not particularly limited as long as it is different from a promoter controlling the E1A region.

[0116] Examples of the promoter include a CMV promoter, an RSV promoter, a CA promoter, an E2F promoter, an EF1A promoter, an EFS promoter, a CAG promoter, a CBh promoter, a CBA promoter, an SFFV promoter, an MSCV promoter, an SV40 promoter, an mPGK promoter, a hPGK promoter, a UBC promoter, an Nanog promoter, an Nes promoter, a Tuba1a promoter, a Camk2a promoter, an SYN1 promoter, a Hb9 promoter, a Th promoter, an NSE promoter, a GFAP promoter, an iba1 promoter, a ProA1 promoter, a hRHO promoter, a hBEST1 promoter, a Prnp promoter, a Cnp promoter, a K14 promoter, a BK5 promoter, an mTyr promoter, a cTnT promoter, an QMHC promoter, a Myog promoter, an ACTA1 promoter, an MHCK7 promoter, an SM22a promoter, an EnSM22a promoter, a Runx2 promoter, an OC promoter, a Col1a1 promoter, a Col2a1 promoter, an aP2 promoter, an Adipoq promoter, a Tie1 promoter, a Cd144 promoter, a CD68 promoter, a CD11b promoter, an Afp promoter, an A1b promoter, a TBG promoter, an MMTV promoter, a Wap promoter, a HIP promoter, a Pdx1 promoter, an Ins2 promoter, an Hcn4 promoter, an NPHS2 promoter, an SPB promoter, a CD144 promoter, a TERT promoter, a TRE promoter, an FLK-1 promoter, a VEGF promoter, a c-Myc promoter, an SLPI promoter, a PSA promoter, and a tyrosinase promoter.

[0117] Examples of the m-CRA include those disclosed in International Publication No. WO2005/115476, or Junichi Kamizono et al., Cancer Res Jun. 15, 2005 (65) (12) 5284-5291. The adenovirus is a m-CRA obtained by replacing an endogenous promoter of the E1A region in the adenovirus genome and an endogenous promoter of the E1B region (preferably, E1B?55K) with a Survivin promoter and a CMV promoter, respectively.

[0118] The conditionally replicating adenovirus preferably has no insert of an exogenous gene in the E3 region of an adenovirus. In other words, the conditionally replicating adenovirus is a conditionally replicating adenovirus which is not defective in the E3 region (i.e., having the E3 region of a wild-type adenovirus).

[0119] Since the E3 region has a region encoding a gene of Adenovirus Death Protein inducing tumor cell death, it has been generally considered that an adenovirus having the E3 region has a higher therapeutic effect (Fanny Georgi, et al., FEBS Letters 594 (2020) 1861-1878). However, contrary to the common technical knowledge, the present inventors found that a conditionally replicating adenovirus having the E3 region has a higher tumor-specific cytotoxic effect in vitro than that having no E3 region; and confirmed that the conditionally replicating adenovirus having the E3 region (Surv.m-CRA-1) has a sufficient therapeutic effect and safety in the clinical trials set forth in Examples. In short, since a conditionally replicating adenovirus having the E3 region (not defective in E3 region) maintains a tumor-cell specific cytotoxic effect sufficient to clinically produce a therapeutic effect but drastically reduces a cytotoxic effect non-specific to normal cells, the adenovirus can be a safer and more effective therapeutic drug.

[0120] The conditionally replicating adenovirus of the present invention is useful as (a component of) a pharmaceutical composition for treating a bone and soft tissue tumor.

[0121] In the specification, the bone and soft tissue tumor refers to a tumor formed in the bone and soft tissue (e.g., muscle, fat, nerve, blood vessel). Examples of the bone and soft tissue tumor include osteosarcomas such as conventional osteosarcoma, chondroblastic osteosarcoma, fibroblastic osteosarcoma, osteoblastoma, vascular dilatation osteosarcoma, small cell osteosarcoma, secondary osteosarcoma, parosteal osteosarcoma, periosteal osteosarcoma and high-grade surface osteosarcoma; chondrosarcomas such as conventional chondrosarcoma, dedifferentiated chondrosarcoma, mesenchymal chondrosarcoma, clear cell chondrosarcoma and osteomyxochondrosarcoma; Ewing's sarcoma; high-grade pleomorphic undifferentiated sarcoma (so-called MFH); fibrosarcoma of bone; chordoma; malignant giant cell tumor of bone; angiosarcoma of bone; leiomyosarcoma of bone; and liposarcoman of bone. The bone and soft tissue tumor includes a bone tumor and a soft tissue tumor, is preferably, bone tumor and more preferably chordoma.

[0122] The bone and soft tissue tumor is preferably a malignant bone and soft tissue tumor. The bone and soft tissue tumors are classified into a primary bone and soft tissue tumor group and a metastatic bone and soft tissue tumor group. The primary bone and soft tissue tumor refers to a malignant tumor (cancer) derived from the bone and soft tissue itself. The metastatic bone and soft tissue tumor refers to a malignant tumor formed in the bone and soft tissue by metastasis of cancer in other organs such as internal organs.

[0123] The conditionally replicating adenovirus of the present invention is effective for a recurrent primary bone and soft tissue tumor and a metastatic bone and soft tissue tumor. The recurrent primary bone and soft tissue tumor refers to a primary bone and soft tissue tumor, to which a treatment such as radiation therapy and chemotherapy is already applied and/or surgical treatment such as resection is applied, and thereafter exacerbated or becomes metastatic. Also, the recurrent primary bone and soft tissue tumor refers to a metastatic bone and soft tissue tumor, to which a treatment is already applied, and thereafter, exacerbated or becomes metastatic.

[0124] The conditionally replicating adenovirus of the present invention may be used in combination with a therapy for a bone and soft tissue tumor commonly known. The therapy for a bone and soft tissue tumor is disclosed in the clinical practice guidelines for soft tissue tumors at the time of filing the application (Japanese Orthopaedic Association (JOA) Clinical Practice Guidelines on the Management of Soft Tissue Tumors), the NCCN guidelines, the ESMO clinical practice guidelines, the EURACAN guidelines, and the guidelines in other countries (e.g., Cancer Chemother Pharmacol. 2016 January; 77 (1): 133-46). For example, a bone and soft tissue tumor is treated by a chemotherapeutic agent such as vincristine, doxorubicin, cyclophosphamide, ifosfamide, etoposide, pazopanib, trabectedin, eribulin or a combination of these.

[0125] In another embodiment, the present invention relates to a method for treating a patient with a bone and soft tissue tumor, characterized by administering an effective amount of a conditionally replicating adenovirus having the E1A gene under expression control of a Survivin promoter; a conditionally replicating adenovirus having the E1A gene under expression control of a Survivin promoter for treating a primary bone and soft tissue tumor; or use of a conditionally replicating adenovirus having the E1A gene under expression control of a Survivin promoter for producing a pharmaceutical composition for treating a primary bone and soft tissue tumor. The above conditionally replicating adenovirus is a conditionally replicating adenovirus maintaining a wild-type E3 region and preferably having E1B?55K as the E1B region.

[0126] For one example, the conditionally replicating adenovirus of the present invention or a pharmaceutical composition containing the conditionally replicating adenovirus as an active ingredient is administered in accordance with an administration schedule: for example, 1 to 10 times, 2 to 8 times, 3 to 5 times, or 4 or 5 times at a rate of once every 4 weeks. Administration is made by injection. Administration to the blood vessel or intratumoral administration may be performed, and intratumoral administration is preferred.

[0127] In the case of intratumoral administration, the dosage volume of the pharmaceutical composition of the present invention can be varied in accordance with the tumor volume. For example, the dosage volume of the pharmaceutical composition disclosed herein may be determined as follows: [0128] 1 mL if the volume of a tumor to receive administration is less than 5 cm.sup.3; [0129] 2 mL if the volume of a tumor to receive administration is 5 cm.sup.3 or more and less than 9 cm.sup.3; [0130] 3 mL if the volume of a tumor to receive administration is 9 cm.sup.3 or more and less than 15 cm.sup.3; [0131] 5 mL if the volume of a tumor to receive administration is 15 cm.sup.3 or more and less than 21 cm.sup.3; [0132] 7 mL if the volume of a tumor to receive administration is 21 cm.sup.3 or more and less than 30 cm.sup.3; and [0133] 10 mL if the volume of a tumor to receive administration is 30 cm.sup.3 or more.

[0134] The virus can be more widely spread within the tumor by varying the dosage volume in accordance with the volume of the tumor.

[0135] In the target patient for treatment, if a tumor is present in a plurality of sites, the conditionally replicating adenovirus may be administered, for example, at most three tumor sites (preferably, three large-diameter tumor sites). In this case, the dose can be divided into portions in accordance with the tumor volumes. Alternatively, if the volume of a tumor at one site is 30 cm.sup.3 or more, the conditionally replicating adenovirus may be administered only to the site without being divided. In the case of multiple administrations, if the volume of the tumor to which administration is previously made is found to reduce at the time on and after the second administration time, the conditionally replicating adenovirus may be administered to large three tumors of the lesions to be given at the time on and after the second administration time. The large three tumors herein refer to the largest-diameter tumor, the 2nd largest-diameter tumor and the 3rd largest-diameter tumor. Alternatively, in the case of multiple administrations, if the volume of one of the tumors to which administration is previously made is found to reduce in the administration time on and after the second administration, the conditionally replicating adenovirus may be administered to the 4th largest-diameter tumor of the lesions to be given determined in the first administration time. Similarly, if the volumes of two of the tumors to which administration is previously made are found to reduce, the conditionally replicating adenovirus may be administered to the 4th largest-diameter tumor and the 5th largest-diameter tumor of the lesions to be given determined in the first administration time. If the volumes of three of the tumors to which administration is previously made are found to reduce, the conditionally replicating adenovirus may be administered to 4th largest-diameter tumor, the 5th largest-diameter tumor and 6th largest-diameter tumor of the lesions to be given determined in the first administration time.

[0136] The dosage of the conditionally replicating adenovirus per dose can be 1?10.sup.10 vp to 1?10.sup.12 vp, and preferably, 1?10.sup.11 vp. The conditionally replicating adenovirus may be administered once or repeatedly in a plurality of times. The total dosage can be 1?10.sup.10 vp to 1?10.sup.12 vp, and is preferably, 1?10.sup.11 to 5?10.sup.11 vp or 3?10.sup.11 to 5?10.sup.11 vp.

[0137] The dosage form of the pharmaceutical composition of the present invention is not particularly limited as long as it is suitable for administration to a tumor site. The pharmaceutical composition may contain pharmacologically acceptable carriers and additives. Examples of the carriers and additives include a solvent, a solubilizer, a suspending agent, an emulsifier, a tonicity agent, a stabilizer, a soothing agent, a preservative, an antioxidant and a buffer.

[0138] Examples of the solvent include purified water, physiological saline, and phosphate buffer.

[0139] Examples of the solubilizer include polyethylene glycol, propylene glycol, trehalose, benzyl benzoate, ethanol, sodium carbonate, sodium citrate, sodium salicylate, and sodium acetate.

[0140] Examples of the suspending agent or emulsifier include sodium lauryl sulfate, gum Arabic, gelatin, lecithin, glyceryl monostearate, polyvinyl alcohol, polyvinylpyrrolidone, cellulose such as carboxymethylcellulose sodium, polysorbates, and polyoxyethylene hydrogenated castor oil.

[0141] Examples of the tonicity agent include sodium chloride, potassium chloride, a saccharide, glycerin, and urea.

[0142] Examples of the stabilizer include polyethylene glycol, dextran sodium sulfate and amino acids.

[0143] Examples of the soothing agent include glucose, calcium gluconate, and procaine hydrochloride.

[0144] Examples of the preservative include paraoxybenzoate, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, and sorbic acid.

[0145] Examples of the antioxidant include water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, and sodium sulfite; fat-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, ?-tocopherol; and metal chelating agents such as citric acid, ethylene diamine tetra acetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0146] The pharmaceutical composition of the present invention can be a preparation for injection containing the conditionally replicating adenovirus in, for example, a glycerin-containing tris-buffered saline buffer (containing 2.5% (v/v) glycerin, 25 mM NaCl, 20 mM Tris, pH 8.0). The amount of the conditionally replicating adenovirus included can be appropriately selected depending on the purpose and dosage regimen, and is preferably, 1?10.sup.11 vp/mL.

EXAMPLES

[0147] Now, Examples will be shown to illustrate the present invention in detail but the present invention is not limited to these examples. Note that, all documents cited in the specification are incorporated in their entirety by reference.

Example 1

[0148] The medicinal product (Surv.m-CRA-1) used herein contains m-CRA type 5 which was prepared by modifying the E1 region such that the E1A gene is expressed under the control of the Survivin promoter and the E1B19K gene is expressed under the control of the CMV promoter, as an active ingredient. The adenovirus has m-CRA, which is obtained by removing endogenous promoters in E1A and E1B regions required for viral proliferation from the type 5 adenovirus genome, as a basic backbone and has no therapeutic gene. Since the medicinal product contains an adenovirus having the Survivin promoter (Survpr), which was introduced into a site from which the endogenous E1A promoter was deleted (removed) and the CMV promoter (CMVpr), which was introduced into a site from which the endogenous E1B promoter was deleted (removed) by a recombination technique, the adenovirus of the medicinal product selectively proliferates in tumor cells having survivin activated. In contrast, proliferation of the adenovirus in normal cells low in survivin activity is suppressed. Further, Surv.m-CRA-1 has E3.

[0149] The final formulation of the drug of the invention was designed as an injectable preparation. Viruses (prepared in a concentration of 1?10.sup.11 vp/mL) are contained at a rate of 0.6 mL per vial of a preparation. The base of the preparation is a GTS buffer consisting of 25 mM NaCl containing 2.5% glycerin (v/v) and 20 mM Tris, pH8.0.

<Eligibility of Subject>

[0150] Patients who satisfy all of the following selection criteria and none of the exclusion criteria were determined as eligible and registered.

(Inclusion Criteria)

[0151] 1) Histologically diagnosed as any one of the following tumors [0152] primary malignant bone tumor [0153] metastatic bone tumor [0154] primary malignant soft tissue tumor [0155] metastatic soft tissue tumor

[0156] 2) At the time of registration, 4 weeks or more have passed since the standard treatment if it was applied as a pretreatment

[0157] 3) Not a subject for a standard treatment commonly recognized and possibly providing life extension and symptom relief

[0158] 4) having a (tumor) lesion whose minor axis, major axis and height can be measured and to which an investigational product can be administered.

[0159] 5) The age of a patient at the time of providing informed consent is 10 or more and less than 85 years old.

[0160] 6) ECOG PS is 0 to 2.

[0161] 7) expected to live for 3 months or more.

[0162] 8) having major organ functions (Latest medical data within two weeks before registration) [0163] hemoglobin ?8 g/?L [0164] leucocyte count ?2,000/?L [0165] platelet count ?70,000/?L [0166] AST (GOT) ?100 U/L [0167] ALT (GPT) ?100 U/L [0168] total bilirubin ?1.5 mg/?L [0169] serum creatinine ?2.0 mg/?L

[0170] 9) In the case of a male, one of the following requirements is satisfied. [0171] azoospermia [0172] after surgical contraception such as vasectomy. [0173] already agreed on the prevention of pregnancy by appropriately using condoms or practicing asceticism in the period from the time of providing informed consent to the disappearance of the investigational product, Surv.m-CRA-1 administered from the body.

[0174] The female partner is in a postmenopausal state for more than 1 year or after surgery such as bilateral oophorectomy, or already agreed on prevention of pregnancy by oral contraceptives (contraceptive pill) or intrauterine device (pessary) in the period from the time of providing informed consent to the disappearance of the investigational product, Surv.m-CRA-1 administered from the body.

[0175] 10) In the case of a female, one of the following requirements is satisfied.

[0176] In a postmenopausal state for more than one year

[0177] After surgical contraception such as bilateral oophorectomy [0178] already agreed on the prevention of pregnancy by oral contraceptives (contraceptive pill) or intrauterine device (pessary) in the period from the time of providing informed consent to the disappearance of the investigational product, Surv.m-CRA-1 administered from the body.

[0179] The male partner is azoospermia or after surgical contraception such as vasectomy; or already agreed on prevention of pregnancy by appropriately using condoms in the period from the time of providing informed consent to the disappearance of the investigational product, Surv.m-CRA-1 administered from the body.

[0180] 11) already obtained written informed consent on participation in the clinical test from a subject oneself or a legal representative.

(Exclusion Criteria)

[0181] 1) having one of the following complications. [0182] serious heart disease, respiratory disease, digestive disorder, liver disease [0183] poorly controlled diabetes [0184] infection that requires ongoing treatment

[0185] 2) having a history of allergy to penicillin or a pig/cow (including milk).

[0186] 3) having a disease that requires systemic administration of an immunosuppressant or a steroid (use of a steroid for preventing allergy to a contrast agent is acceptable. If used, about a week interval must be set until the administration of an investigational product and measure is presumably taken for preventing infection).

[0187] 4) having active double cancer (basal cell carcinoma, carcinoma endoepidermale, superficial bladder cancer, or malignant tumor from which no metastasis or recurrence occurred for more than 5 years is not ineligible)

[0188] 5) having uncontrolled primary-disease-associated fever/pain

[0189] 6) a pregnant female, a breastfeeding female, and a premenopausal female or a female within a year of menopause being positive pregnancy test

[0190] 7) taking an unapproved drug within 4 weeks before providing informed consent.

[0191] 8) Other females who are determined as inappropriate to participate in the clinical study by an investigator/sub-investigator.

[0192] A phase-I study with ascending doses for open-label (Surv.m.CRA-1-001 study) was carried out to evaluate safety/tolerability and preliminary effectiveness of Surv.m.CRA-1 for a progressive solid cancer (primary malignant bone tumor, metastatic bone tumor, primary malignant soft tissue tumor, metastatic soft tissue tumor) by a single administration to a local site of a tumor. A low dose (1?10.sup.10 viral particle hereinafter referred to as vp), a medium dose (1?10.sup.11 vp) and a high dose (1?10.sup.12 vp) of Surv.m-CRA-1 were each administered to 3-eligible subject each described above.

[0193] As the tumor site to receive administration, a single site was selected from anatomical positions, based on the following standards. It does not matter if the site is a primary lesion or a metastatic lesion. If 2 or more tumor sites are present, a site to receive administration is selected in consideration of which one is technically easier to administer. [0194] 1) A tumor is large in size and to which administration is technically easily made [0195] 2) A site not adjacent to major blood vessel and thoracic cavity [0196] 3) A site not presumed to be responsible for symptoms such as pain.

[0197] Based on a tumor image evaluation finding of a subject at the time of registration, a tumor volume was calculated in accordance with the following formula (rounded down to the second decimal place).

<Calculation of Tumor Volume>

[0198] [00001]$\mathrm{Tumor}\mathrm{volume}\left({\mathrm{cm}}^3\right)=\mathrm{Minor}\mathrm{axis}\mathrm{diameter}\left(\mathrm{cm}\right)?\mathrm{Major}\mathrm{axis}\mathrm{diameter}\left(\mathrm{cm}\right)?\mathrm{Height}\left(\mathrm{cm}\right)?1/2$

[0199] The liquid volume of a medicinal product to be administered to a single lesion is determined as follows: if a tumor volume is 33.3 cm.sup.3 or larger in a low-dose cohort, a medium-dose cohort and a high-dose cohort, the medicinal product is diluted so as to be 10 mL and administered. In a low-dose cohort and a medium-dose cohort, if a tumor volume is 3.4 cm.sup.3 or less, the medicinal product was administered without dilution; if the tumor volume is 3.5 cm.sup.3 to 33.3 cm.sup.3 or less, the medicinal product was diluted so as to become 30% of the tumor volume and then administered. In a high-dose cohort, if the tumor volume is 33.3 cm.sup.3 or less, a whole amount of the medicinal product was administered without dilution.

[0200] In a treatment room, the dosage volume for a single-lesion to receive administration was administered uniformly within the same tumor by changing the direction of a needle tip so as to distribute 5 to 10 sites, under direct visual guidance, X-ray fluoroscopy, CT guidance, or ultrasound guidance.

Results

[0201] The therapeutic effects of subjects in the phase-I study are shown in Table 1. Of the subjects, a patient (45 years old, female) with sacral chordoma of a low-dose cohort is a case that sacral chordoma metastasized to the liver and lung 6 years after the onset and has a recurrence in the right buttock soft tissue. A chemotherapeutic agent and radiation therapy had no effect and the recurrent tumor was not able to resect. The medicinal product of the invention can achieve a partial response (PR) only by a single administration even in the recurrence case (non-response case by either a chemotherapeutic agent or radiation therapy). In particular, the therapeutic effect and safety were both extraordinarily excellent. In clinical trials for conditionally replicating adenoviruses so far performed, despite repeated administration, it is not reported that a therapeutic effect is obtained at such a low dose. In clinical trials of conditionally replicating adenoviruses, it is not reported that a therapeutic effect was obtained over 2 years or more after administration. The course of treatment is shown in FIG. 1.

[0202] In the case of a patient with thoracic chordoma (61 years old, male) in a low-dose cohort, 11 years after a thoracic chordoma was surgically resected from the right mediastinum, the patient had a 4-th time recurrence in the adjacent vertebral body. This is a case that no effect was produced by a standard treatment. The medicinal product of the present invention was able to achieve PR in such a recurrence case. It should be noted that a therapeutic effect was produced 4 weeks after a treatment and further bone remodeling was observed 12 weeks later. Bone remodeling is a phenomenon of regeneration of bone induced for the first time after a bone tumor disappears. The phenomenon clinically demonstrates a high therapeutic effect. This is a case of a tumor formed within the bone. Even if the tumor leads to cell death, the site where the tumor has been formed cannot be completely filled up by regeneration of bone. Because of this, it is impossible to evaluate a reduction effect on a tumor based on the RECIST, which evaluates tumor diameters. In this sense, it is estimated that a further dramatic therapeutic effect might be obtained. Further, in the Choi, in which evaluation was made by contrast-enhanced CT, the CT value increases. In short, an extremely satisfactory result where bone modeling (regenerative healing) significantly occurs, was shown. In consideration of these results comprehensively, the patient is the case actually obtaining an almost complete response, that is, a strong therapeutic effect. As set forth in the first case, in the clinical trials so far reported, despite the repeated administration of conditionally replicating adenoviruses, it is not reported that a therapeutic effect was observed at such a low dose. From this, the above excellent results obtained at a single administration in the clinical trial are extremely prominent. In clinical trials for conditionally replicating adenoviruses, a case where a therapeutic effect was obtained over 135 weeks (2 years and 7 months (or more)) after administration, was not reported. This is a drastic therapeutic effect. The course of treatment is shown in FIG. 2.

[0203] Table 1 summarizes the therapeutic effects of 9 cases in total tested in the clinical trial. The therapeutic effects were evaluated with reference to tumor reduction effects based on the Choi standard and the RECIST standard. PD is progressive disease, SD is a stable disease and PR is a partial response. Safety (adverse event and side effect) was analyzed for all adverse events identified from the day of administration of an investigational product to the end of the observation period. The adverse events were evaluated using the Common Terminology Criteria for Adverse Events (CTCAE) ver.4.0 translated in Japanese and edited by the Japanese clinical oncology group (JCOG) (CTCAE ver.4.0).

[0204] In a single administration low-dose cohort (1/100 dose of the maximum dose at which no therapeutic effect was obtained even if it is repeatedly administered by competing technology in the world), a long-term/persistent tumor therapeutic effect (tumor shrinkage progresses in 2 years or more and decreases by half) was found. An innovative therapeutic action/effect: 6/9 cases in all dose cohorts being effective (PR or more), was obtained in the clinical trial. Such a high efficacy has not been reported in the conditionally replicating adenovirus clinical trials carried out in the world, to date (listed in Table 2). Thus, it was demonstrated that the drug of the invention has a performance that greatly exceeds the competing technology.

TABLE-US-00001 TABLE 1 Patient Dose Therapeutic effect Safety (serious # (viral load) Type of cancer Target lesion Choi (RECIST) adverse effect) 1 Low-dose Chordoma Soft tissue PR (PR) High (none) 2 (1 ? 10.sup.10) Chordoma Thoracic vertebra PR (PR) High (none) 3 Epithelioid sarcoma Soft tissue PR (SD) High (none) 4 Middle-dose Liposarcoma Soft tissue SD (SD) High (none) 5 (1 ? 10.sup.11) Myxofibrosarcoma Soft tissue PR (PD) High (none) 6 Rectal cancer Sacral bone SD (SD) High (none) 7 High-dose Myxofibrosarcoma Soft tissue PR (SD) High (none) 8 (1 ? 10.sup.12) Uterus cancer Pubic bone PR (SD) High (none) 9 Liposarcoma Soft tissue PD (SD) High (none)

[0205] The medicinal product of the invention administered has no dose-dependent tendency to safety. No significant problems were not recognized. As a serious adverse event, Grade-4 reduction in lymphocyte count was observed in a single case. A patient in this case recovered without symptoms. Reduction in lymphocyte count is commonly observed in oncolytic viruses and a temporary event that possibly occurs in amplification of the recombinant virus, and reported in past clinical trials for a conditionally replicating adenovirus. This occurs in a different mechanism from myelosuppression (extremely significant side effect) by conventional chemotherapy (anticancer drug). The number of lymphocytes in the peripheral blood is presumed to reduce by temporarily recruiting lymphocytes to a tumor and/or lymph node in accordance with viral proliferation/cell lysis within a tumor (the action mechanism of the medicinal product of the invention). Since this is not myelosuppression, a reduction in lymphocyte count is recovered early. This phenomenon, in some sense, is deemed to be a ground for demonstrating that the medicinal product of the invention steadily acts. There were no subjects who died within 28 days from the administration of an investigational product. Note that, there were no adverse events and DLT (dose-limiting toxicity) that led to the cancellation of the clinical study. No variation in laboratory test values and vital signs clinically concerned were observed except for the abnormalities reported as adverse events.

[0206] As a whole, no variation in adenovirus in blood, adenovirus excretion (saliva, urine), anti-adenovirus antibody titer, cytokine (level), and 12-lead electrocardiogram clinically concerned were observed in any one of the cohorts. An increase in cytokine level is minor. It is considered that the minor increase in the cytokine level is collateral evidence supporting that excessive immune response to viral infection is not induced.

[0207] Patients complained of mild symptoms such as temporal fever but no serious symptoms. From this, it was demonstrated that the drug is extremely safe. As set forth in the above, Surv.m-CRA-1, even though it has E3, maintains a strong therapeutic effect on tumor cells, whereas non-specific viral proliferation in normal cells is suppressed. From this, it was demonstrated that a highly safe adenovirus, even though it has E3 region, is successfully constructed.

[0208] The viral load in the body fluid after administration of the virus was measured. The results are shown in Table 2. In a single case of the low-dose cohort, the saliva was virus-positive on the day of administration (Day 1 alone) but negative in the blood and urine. In the other cases, all of the blood, saliva and urine were virus-negative. In the medium-dose cohort, the 2-case blood samples were virus-positive up to 7 days later (Day 8), one of the cases was virus-positive in the saliva up to 7 days later (Day 8), the other case was virus-positive up to 21 days later (Day 22) but on and after 14 days (on and after Day 1), the viral load was as low as 20 copies/?L or less. In the high-dose cohort, all 3-case blood samples were virus-positive up to 7 days later (Day 8); 2-case saliva samples were virus-positive up to 7 days later (Day 8); all 3-case urine samples were virus-negative. Although subjects became temporarily virus-positive after administration of an investigational product but became virus-negative by the end of the study treatment/evaluation period (Day 29).

TABLE-US-00002 TABLE 2 Process of viral excretion load (in blood, urine and saliva) after treatment Before After administration Type of cancer Tissue administration 3H Day 1 Day 2 Day 7 Day 14 Day 21 Day 22 Day 23 Day 28 S-01 Chordoma Blood ? ? ? ? ? ? ? (Low Saliva/ ?/? 20/? ?/? ?/? ?/? ?/? dose) Urine S-02 Chordoma Blood ? ? ? ? ? ? ? (Low Saliva/ ?/? ?/? ?/? ?/? ?/? ?/? dose) Urine S-03 Epithelioid Blood ? ? ? ? ? ? ? (Low sarcoma Saliva/ ?/? ?/? ?/? ?/? ?/? ?/? dose) Urine S-04 Liposarcoma Blood ? 21 ? 88 74 ? ? (Middle Saliva/ ?/? ?/? ?/? ?/? 14/? ?/? dose) Urine S-05 Myxofibrosarcoma Blood ? ? ? ? ? ? ? (Middle Saliva/ ?/? ?/? ?/? ?/? ?/? ?/? dose) Urine S-06 Metastasis of Blood ? ? ? ? 18 ? ? (Middle rectal cancer to Saliva/ ?/? 320/? 80/40 ?/? 20,000/18 15/? 13/* ?/* ?/* dose) sacral bone Urine S-07 Myxofibrosarcoma Blood ? ? 11 ? 22 ? ? (High Saliva/ ?/? ?/? 36/? ?/? 134/? ?/? dose) Urine 5-08 Metastasis of Blood ? 28 ? 383 94 ? ? (High uterus cancer to Saliva/ ?/? 534/? 42/? ?/? ?/? ?/? dose) pubic bone Urine S-09 Liposarcoma Blood ? 127 10 19 23 ? ? (High Saliva/ ?/? 174/? 31/? ?/? 766/? ?/? dose) Urine Dose (VP/mL) Viral (Surv.m-CRA-1) load Low (1 ? 10.sup.10) Numerical value: copies/?L Middle (1 ? 10.sup.11)?: < 10 copies/?L High (1 ? 10.sup.12) *: Not performed since negative value was confirmed

[0209] Effectiveness was as follows: at the end of the study treatment/evaluation period (Day 29), local response (effectiveness) was observed in two cases of the low-dose cohort, in a single case of the middle-dose cohort and two cases in the high-dose cohort. In the two cases (primary malignant bone tumor: chordoma) of the low-dose cohort, the local response was maintained from Month 9 to 24 (2 years) after initiation of the observation (in one of the two cases, maintained up to the maximum observation period: 2 years and 7 months). From the above, it was found that the (drug) product of the invention has extremely high safety without any problem in tolerability and provides a greater therapeutic effect (tumor reduction effect) over (the products using) conditionally replicating adenoviruses already reported. The results of clinical trials for the conditionally replicating adenovirus already reported and Surv.m-CRA-1 are shown in comparison in the following Table 3.

TABLE-US-00003 TABLE 3 Therapeutic effect Investigational Choi(RECIST) Subject product Dose PD SD PR Document Gliocytoma ONYX-015 1 ? 10.sup.7 ? 23 1 Mol Ther, 2004 1 ? 10.sup.10 pfu Solid cancer Telomelysin 1 ? 10.sup.10 ? 3 11 1 Mol Ther, 2010 1 ? 10.sup.12 vp Hepatobiliary ONYX-015 6 ? 10.sup.9 ? 1 12 1 Clin Cancer Res. 2003 cancer 3 ? 10.sup.10 pfu Bone and soft Surv.m-CRA-1 1 ? 10.sup.10 ? 1 2 6 The present invention tissue tumor 1 ? 10.sup.12 vp pfu: Plaque-forming unit vp: Viral particles

[0210] In cohort 3 (high dose) of the phase I study of Surv.m-CRA-1, 1?10.sup.12 vp of the investigational product was administered and the safety of the product was confirmed. The virus was detected in the body fluid up to Day 7 after administration of the investigational product in almost all cases. In one case of cohort 2 (medium dose: 1?10.sup.11 vp), the virus was detected in the saliva at longest up to Day 21. In contrast, there were no cases of exhibiting a symptom such as upper respiratory inflammation during the period when the virus was detected in the body fluid. From the above, it was demonstrated that the virus administered disappears from the body fluid 4 weeks after administration of an investigational product and thus, a risk by re-administration is the same as or close to that by the initial administration. From this, it was suggested that the treatment with Surv.m-CRA-1 is suitably made in accordance with a dose-cycling schedule designed so as to administer Surv.m-CRA-1 once every 4 weeks. If the frequency of administration was set as 5 times, it is possible to determine 1?10.sup.11 vp as a single administration dose, with the result that the total dosage (1?10.sup.11 vp?5 times=5?10.sup.11 vp) does not exceed the dosage of cohort 3 of Surv.m-CRA-1 phase I study (high dose: 1?10.sup.12 vp) and a therapeutic effect can be expected.

[0211] It is known that as tumor diameter of bone tumor increases, the risk of pathological fracture increases (Mirel's score: evaluation of pathological fracture at the tumor diameter) and the tumor diameter is used for determining whether surgical operation is appropriate or not. It has been reported that the total volume (Total Tumor Volume) of a primary lesion and metastasis lesion (e.g., lymph node metastasis) is inversely correlated with the prognosis in various types of cancers. Also in osteosarcoma, the total volume of multiple lung metastatic cancers is reported to be a poor prognostic factor. It is expected to reduce the total tumor volume of a patient by treating tumors in descending order from the tumor with a large tumor diameter. Thus, in consideration of the possibility of starting administration from a tumor lesion with a large tumor diameter, the lesion to be treated is selected. The larger the number of target lesions to receive administration, the smaller the dose per lesion and the larger the patient's load. In consideration of this, at most three sites are conceived to be a lesion to receive administration. If the volume of a tumor first to be treated is as large as 30 cm.sup.3 or more, and the dose is divided into 2 or more portions, the dose per tumor can be reduced (in phase-I test of Surv.m-CRA-1, a drug was administered so as to cover 30% of a tumor volume as a target). For this reason, administration was made to only one portion.

[0212] In this study, the tumor lesion to which an investigational product can be administered refers to a lesion satisfying all of the following conditions: [0213] a main part is bone or soft tissue (metastatic lesion of bone tumor) [0214] injection can be made from the surface of a body into a tumor [0215] not a lesion of the lymph node [0216] the minimum diameter of a tumor is 10 mm or more [0217] the entire tumor is not a cystic lesion.

[0218] The liquid volume of a medicinal product to a single (tumor) lesion is determined by calculating the volume of a tumor to receive administration and determining the dosage volume in accordance with the following standard. If dilution is required, dilution is made with saline in accordance with the dosage volume.

Tumor Volume and the Dosage Volume (after Dilution)

TABLE-US-00004 Administration liquid Tumor volume (cm.sup.3) volume (mL) less than 5 1 5 or more and less than 9 2 9 or more and less than 15 3 15 or more and less than 21 5 21 or more and less than 30 7 30 or more 10

[0219] If there are a plurality of lesions that can be administered, the dosage volume was calculated based on the total of tumor volumes and in accordance with the above standard. At most three sites of tumors can be administered in every administration time. When administration is made to a plurality of tumors, the dosage volume is divided in accordance with the tumor volumes. The total dosage is determined as 1?10.sup.11 vp. Note that if the volume of a single tumor is 30 cm.sup.3 or more, administration is made only the single tumor and administration to another tumor is not performed. If a plurality of lesions that can be administered are present, target lesions are selected based on the following standards by the investigator or a sub-investigator.

The Tumor Diameter is Large

[0220] The approach can be made from the surface of a body

[0221] Administration can be safely made without the presence of an important nerve and a blood vessel in the neighborhood.

[0222] Based on a tumor image evaluation finding of a subject at the time of registration, a tumor volume was calculated in accordance with the following formula (rounded down to the second decimal place).

<Calculation of Tumor Volume>

[0223] [00002]$\mathrm{Tumor}\mathrm{volume}\left({\mathrm{cm}}^3\right)=\mathrm{Minor}\mathrm{axis}\mathrm{diameter}\left(\mathrm{cm}\right)?\mathrm{Major}\mathrm{axis}\mathrm{diameter}\left(\mathrm{cm}\right)?\mathrm{Height}\left(\mathrm{cm}\right)?1/2$

Example 2

[0224] Surv.m-CRA-1 was prepared by an improved method based on the m-CRA preparation technology (Patent Literature 2, Non Patent Literature 7) as follows. The structure of Surv.m-CRA-1 has not been reported. First, pSurv.E1A-CMV.19K plasmid was prepared by inserting, to a vector plasmid (P1 plasmid: Replication-controllable plasmid) containing a proliferation control unit, a mouse Survivin promoter (?173 to ?19) upstream of E1A, and a CMV promoter upstream of E1B?55K in accordance with the method disclosed in Patent Literature 2. Next, the pSurv.E1A-CMV.19K plasmid was digested with restriction enzyme I-Ceul, and then a restriction enzyme PI-Seel, and a DNA sequence (fragment) having a Survivin promoter/E1A/CMV promoter/E1B?55K in the order from the upstream was isolated and purified. In the meantime, a fragment of 342-3523 bp was deleted from the sequence of human adenovirus type 5 containing the E1 region. To the site, I-Ceul and PI-Seel recognition sequences were provided (inserted) in order to sub-clone a gene of P1 plasmid. In the meantime, the adenovirus genome plasmid pAd.HM3 (provided by Dr. Mark A. Kay, Stanford University) having the E3 region was digested with restriction enzyme I-Ceul, and then, a restriction enzyme PI-Seel. In each of the steps, purification was made with phenol/chloroform and followed by ethanol precipitation. The DNA fragment of the Survivin promoter/E1A/CMV promoter/E1B?55K was inserted into the pAd.HM3 site treated with I-Ceul/PI-Seel by use of T4DNA ligase to obtain conditionally replicating adenoviral vector plasmid, pAd.HM4-Surv.E1A-CMV.19K. Then, 293 cells were transfected with the plasmid pAd.HM4-Surv.E1A-CMV.19K. The virus plaque that emerged was extracted, amplified and purified to obtain Surv.m-CRA-1, which is a m-CRA having the E1A gene whose expression is controlled by the Survivin promoter, E1B?55K whose expression is controlled by the CMV promoter and E3 region.

Example 3

[0225] Malignant tumor cells, i.e., liver cancer cell line HepG2 and normal cells, i.e., fibroblasts WI-38, were each infected in vitro with Surv.m-CRA-1 with individual given multiplicity of infections (MOI). On Day 3 and Day 5 after infection, cell viability was evaluated by WST-8 (Nacalai Tesque Inc., catalog number 07553-44). For comparison of properties, E3 region-defective Surv.m-CRA (E3 is absent), which is a m-CRA having the E1A gene whose expression is controlled by the Survivin promoter, E1B?55K whose expression is controlled by the CMV promoter and defective in the E3 region, and a non-replicating adenovirus, Ad.dE1.3 serving as a control for evaluating death by virus proliferation were used in experiments performed in the same conditions as comparative experiments. The conditionally replicating adenovirus rapidly proliferates in a malignant tumor, thereby producing a cytotoxic effect. The MOI of malignant tumor HepG2 was 0.3, 1 and 3. In contrast, the level of cytotoxic effect of viral proliferation on normal cells WI-38 in any one of the groups is low. To clearly find the difference between the groups, MOI was 1, 3 and 10, each of which is three times as large as MOI of HepG2.

[0226] The 3 groups were compared each MOI and each Day (day after infection). The results are shown in FIG. 3. In individual conditions, the viable cell count of control Ad.dE1.3 was regarded as 100%. The viable cell counts of Surv.m-CRA-1 (E3 is present) and Surv.m-CRA (E3 is absent) were compared in the same conditions (each MOI and Day) and expressed in percentages based on the viable cell count of control Ad.dE1.3. In FIG. 3, the vertical axes of the graphs represent the viability (%) of cells for comparing 3 groups in the same condition (MOI and Day) and not the viability of cells for comparison in different MOIs or different Days. In all groups, N=5. An average value and a standard error are also shown in FIG. 3. The statistical significance between the 2 groups in the same condition (each MOI and Day) was calculated by the Student t-test. P<0.05 indicates a significant difference. If Surv.m-CRA-1 (E3 is present) and Surv.m-CRA (E3 is absent) each had a significant difference compared to the control Ad.dE1.3, reference symbol * was attached on the bars of them.

[0227] It was confirmed that a cytotoxic effect by virus proliferation is produced on malignant tumor cell line HepG2 in the experimental conditions at an MOI of 1 or more. A significant cytotoxic effect by virus proliferation was observed only in Surv.m-CRA (E3 is absent) at an MOI of 1 and observed in both Surv.m-CRA (E3 is absent) and Surv.m-CRA-1 (E3 is present) at an MOI of 3. In short, it is shown that finally the same therapeutic effect can be obtained. In contrast, in normal cells, WI-38, a cytotoxic effect was not observed at any one of the MOIs on Day 3 and Day 5 in Surv.m-CRA-1 (E3 is present). However, in Surv.m-CRA (E3 is absent), a significant cytotoxic effect was observed only at MOI 10 on both Day 3 and Day 5. In short, in Surv.m-CRA (E3 is absent), significant cell death was not induced just like in normal cells WI-38 at an MOI of 3, at which significant cell death is induced in tumor cells HepG2. These two types of survivin-responsive m-CRAs both have a tumor-specific cytotoxic effect, that is, an excellent and distinct performance as a cancer drug but Surv.m-CRA-1 (E3 is present) is further excellent in tumor specificity (safety).

[0228] An in vitro experimental system in which a long-term (several ten days, month, year) therapeutic effect having a clinical significance in vivo (human patient) can be completely accurately evaluated and reflected, and an animal model by which the detailed differences can be accurately evaluated, are not present. However, the following scientific conclusion can be sufficiently obtained by the in vitro practicable experimental system of the present invention. That is, a conditionally replicating adenovirus whose proliferation is controlled by the Survivin promoter induces a sufficient tumor cell death and to a tumor specificity (safety). In the respect that Surv.m-CRA-1 (E3 is present) does not damage normal cells, further higher safety (normal cells are not damaged in the in vitro experimental system) is provided. From this, it can be said that Surv.m-CRA-1 (E3 is present) is a further excellent conditionally replicating adenovirus. In view of the therapeutic effect on a tumor, Surv.m-CRA-1 (E3 is present) has a lower cytotoxicity than Surv.m-CRA (E3 is absent) in some in vitro experimental conditions (it is important that the finding is completely contrary to the theory established in the art). However, when Surv.m-CRA-1 (E3 is present) is actually used in clinical sites as a therapeutic drug for a long term (neither several hours nor a few days as in vitro) to provide a clinically beneficial therapeutic effect to a patient in an appropriate amount, Surv.m-CRA-1 (E3 is present) brings a sufficiently strong therapeutic effect equivalent to Surv.m-CRA (E3 is absent). This is consistent with the results that a strong (exceeding conventional conditionally replicating adenoviruses) therapeutic effect and extreme safety (having no serious side effects) were obtained in the clinical trial for a bone and soft tissue malignant tumor of human patients, as shown in Example 1. In short, the technology (technical idea) of the invention, which was found to exceed prior art from a scientific point of view, for the first time, in Example 2 of the present invention, is simultaneously demonstrated for the first time in the human clinical trial in Example 1 of the present invention. Thus, Surv.m-CRA-1 of the present invention is the first conditionally replicating adenovirus greatly exceeding conventional conditionally replicating adenoviruses in performances of both therapeutic effect and safety and having a potential as a cancer drug by itself.

Example 4

Repeat-Dose Study

[Case 1]

[0229] The course of treatment of a patient (60 years old, male) having sacral recurrent chordoma with Surv.m-CRA-1 is shown. The patient developed a sacral chordoma in 2014 and started to receive particle beam therapy. In 2019, metastasis to the left kidney was found and nephrectomy was performed. December in 2020, an increase in sacral chordoma was found and administration of Surv.m-CRA-1 was started in May, 2021. The patient satisfies the inclusion criteria shown in Example 1 and does not satisfy the exclusion criteria shown in Example 1. Since the tumor volume was 315 cm.sup.3 (30 cm.sup.3 or more), the dose (1?10.sup.11 vp) of Surv.m-CRA-1 was divided into 9 portions and a total 5 times (total 5?1 every 4 weeks).

[0230] Table 4 shows the response evaluations of the administration site (sacrum) based on the RECIST standard and the Choi standard before administration to 32 weeks after administration and the response evaluations of the non-administration site (right iliac wing) based on the RECIST standard before administration to 32 weeks after administration.

TABLE-US-00005 TABLE 4 Before administration 4 w 12 w 20 w 32 w Administration site (sacral bone) RECIST 1.1 Major axis (mm) 110 110 110 109 107 Change rate (%) 0.0 0.0 ?0.9 ?2.7 Evaluation SD SD SD SD Choi CT value (HU) 36.9 36.4 35.0 38.7 36.0 Change rate (%) ?1.4 ?5.1 5.1 ?2.4 Evaluation SD SD SD SD Non-administration site (right wing of Ilium) RECIST 1.1 Major axis (mm) 14 17 22 25 27 Change rate (%) 21.4 57.1 78.5 92.8 Evaluation SD* PD PD PD SD: Stable disease, PD: Progressive disease

[0231] As shown in Table 4, a tumor increased at the non-administration site, whereas, a lesion was suppressed and tumor volume decreased at the administration site. The tumor was significantly large but the SD effect was found at a time point of the 8.sup.th month (32 weeks) after initiation of administration. No adverse events were observed during administration.

[Case 2]

[0232] The course of treatment of a patient having recurrent sacral chordoma (73 years old, male) with Surv.m-CRA-1 is shown. The patient developed sacral chordoma in 2016 and a tumor (S3 level) was resected. In 2018, heavy ion radiation therapy (twice) was carried out. In October, 2020, right gastrocnemius muscle metastasis was resected. In 2021, 3 courses of chemotherapy (ADR) were applied. In 2021, a left femoral trochanteric pathologic fracture occurred. In October 2021, tumor growth (recurrent sacral chordoma) in the perineum and sacrum subcutaneous portion was found. In November 2021, the administration of Surv.m-CRA-1 was started. The patient satisfies the inclusion criteria shown in Example 1 and does not satisfy the exclusion criteria shown in Example 1.

[0233] The volume of the tumor in the perineum was 22.8 cm.sup.3 and the volume of the tumor in the sacrum subcutaneous was 14.95 cm.sup.3. From the 1st to 3rd administration times, 5?10.sup.11 vp of Surv.m-CRA-1 was divided as follows and administered. After the 3rd administration time, the dose of Surv.m-CRA-1 was adjusted to 3.5?10.sup.11 vp (7 ml) in accordance with the volume of the tumor reduced in size and the 4th and 5th administrations were carried out.

[0234] Administration was carried out every 4 weeks.

TABLE-US-00006 1-3 times 4 and 5 times Perineum: 7 ml, 4 sites 5 ml, 3 sites Sacrum subcutaneous 3 ml, 2 sites 2 ml, 1 site portion: by injection

[0235] Table 5 shows the response evaluations of the administration sites (perineum, sacrum subcutaneous portion) based on the RECIST standard and the Choi standard before administration to 140 days after administration and the response evaluations of non-administration sites (left rectum, right rectum, in the left gluteus muscle, right ilium) based on the RECIST standard before administration to 84 days after administration.

TABLE-US-00007 TABLE 5 Before administration Day 28 Day 84 Administration site (perineum, sacrum subcutaneous) RECIST (MRI) Sum of Major axes (mm) 86 84 81 Change rate (%) ?2.3 ?5.8 Evaluation SD SD Choi (CT) Perineum CT value (HU) 31 37 25 Change rate (%) 19.3 ?16.7 Evaluation PD PR sacrum subcutaneous CT value (HU) 36 32 25 Change rate (%) ?11.1 ?30.5 Evaluation SD PR Non-administration site (left rectum, right rectum, in the left gluteus muscle, right ilium) RECIST (MRI) Sum of Major axes (mm) 221 230 242 Change rate (%) 4 9.5 Evaluation SD SD PR: Partial response, SD: Stable disease, PD: Progressive disease

[0236] As shown in Table 5, a tumor increased at the non-administration sites, whereas a lesion was suppressed at administration sites. An SD effect at the major axis (RECIST) and a PR effect based on the Choi standard (contrast effect) were confirmed. The volume of a tumor was found to distinguishably reduce. No adverse events except a decrease in lymphocytes (750/?l) were confirmed during administration.

[0237] As set forth in the above, the safety and effectiveness of Surv.m-CRA-1 were demonstrated even in the repeated dose (studies). A safer and more effective treatment can be made by controlling the dosage volume in accordance with variation of tumor volume, as performed in Case 2.