Gene therapy for tumors using minus-strand RNA viral vectors encoding immunostimulatory cytokines Number:7,521,043 from the United States Patent and Trademark Office (PTO) owispatent The present invention provides methods for treating tumors, which comprise the step of administering into tumor sites a minus-strand RNA viral vector encoding an immunostimulatory cytokine or cells introduced with the vector. The present invention also provides compositions for treating tumors, which comprise as an active ingredient the minus-strand RNA viral vector encoding an immunostimulatory cytokine or cells introduced with the vector. The present invention also provides kits for treating tumors, which comprise the minus-strand RNA viral vector encoding an immunostimulatory cytokine, and a tumor antigen or a vector expressing the antigen.<H immunostimulatory, cytokines, Gene, therapy, fo, 7521043.html, Patent, pto, 7521043

Title: Gene therapy for tumors using minus-strand RNA viral vectors encoding immunostimulatory cytokines

Abstract: The present invention provides methods for treating tumors, which comprise the step of administering into tumor sites a minus-strand RNA viral vector encoding an immunostimulatory cytokine or cells introduced with the vector. The present invention also provides compositions for treating tumors, which comprise as an active ingredient the minus-strand RNA viral vector encoding an immunostimulatory cytokine or cells introduced with the vector. The present invention also provides kits for treating tumors, which comprise the minus-strand RNA viral vector encoding an immunostimulatory cytokine, and a tumor antigen or a vector expressing the antigen.

Patent Number: 7,521,043 Issued on 04/21/2009 to Iwadate, et al.

Inventors: Iwadate; Yasuo (Chiba, JP), Yamaura; Akira (Chiba, JP), Inoue; Makoto (Ibaraki, JP), Hasegawa; Mamoru (Ibaraki, JP)
Assignee: DNAVEC Research Inc. (Ibaraki, JP)

Appl. No.: 10/585,884

Filed: January 12, 2005

PCT Filed: January 12, 2005

PCT No.: PCT/JP2005/000238

371(c)(1),(2),(4) Date: November 20, 2006

PCT Pub. No.: WO2005/067981

PCT Pub. Date: July 28, 2005

Foreign Application Priority Data


Jan 13, 2004 [JP]			2004-005186

Current U.S. Class: 424/93.2 ; 514/44R

Current International Class: A01N 63/00 (20060101); A01N 43/04 (20060101)

Field of Search: 424/93.2,93.21 514/44

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U.S. Patent Documents


6514728	February 2003	Kai et al.
6645760	November 2003	Nagai et al.
6723532	April 2004	Nagai et al.
7101685	September 2006	Nagai et al.
2002/0012995	January 2002	Fukumura et al.
2002/0169306	November 2002	Kitazato et al.
2003/0022376	January 2003	Kitazato et al.
2003/0166252	September 2003	Kitazato et al.
2003/0170266	September 2003	Kitazato et al.
2004/0053877	March 2004	Fukumura et al.
2004/0101965	May 2004	Griesenbach et al.
2004/0265272	December 2004	Iwamoto et al.
2005/0130123	June 2005	Inoue et al.
2005/0158279	July 2005	Fukumura et al.
2005/0191617	September 2005	Inoue et al.
2005/0266566	December 2005	Nagai et al.
2005/0271628	December 2005	Fukumura et al.
2006/0104950	May 2006	Okano et al.
2006/0128019	June 2006	Kobayashi et al.
2007/0009949	January 2007	Kitazato et al.
2007/0269414	November 2007	Okano et al.
2008/0014183	January 2008	Okano et al.
2008/0031855	February 2008	Okano et al.

Foreign Patent Documents


0 864 645	Sep., 1998	EP
58-157723	Sep., 1983	JP
7-503455	Apr., 1995	JP
2000-253876	Sep., 2000	JP
WO 97/16539	May., 1997	WO
WO 00/01837	Jan., 2000	WO
WO 00/70055	Nov., 2000	WO
WO 00/70070	Nov., 2000	WO
WO 01/04271	Jan., 2001	WO
WO 02/31138	Apr., 2002	WO
WO 02/38726	May., 2002	WO
WO 03/025570	Mar., 2003	WO
WO 03/029475	Apr., 2003	WO
WO 03/102183	Dec., 2003	WO
WO 2004/022731	Mar., 2004	WO
WO 2004/038029	May., 2004	WO

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Primary Examiner: Wilson; Michael C.
Attorney, Agent or Firm: Clark & Elbing LLP

Claims

The invention claimed is:

1. A method of anti-tumor treatment comprising the step of intracerebrally administering to a subject having a brain tumor, an effective amount of a Sendai virus vector which encodes interleukin-2 or a cell into which the vector has been introduced.

2. The method of claim 1, wherein the Sendai virus vector lacks both M and F genes.

3. The method of claim 1 further comprising the step of subcutaneously administering tumor cells from the brain tumor, wherein the tumor cells have lost their growth ability.

4. The method of claim 3, wherein the Sendai virus vector lacks both M and F genes.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Stage of International Application No. PCT/JP2005/000238, filed Jan. 12, 2005, which, in turn, claims the benefit of Japanese Patent Application No. 2004-005186, filed Jan. 13, 2004.

TECHNICAL FIELD

The present invention relates to tumor gene therapy using minus-strand RNA viral vectors that encode immunostimulatory cytokines.

BACKGROUND ART

In recent years, cancer immunotherapy using cytokines has received attention. For example, therapeutic strategies using gene introduction have been explored for treatment of glioblastoma (Glioblastoma multiforme; GBM) (Shapiro, W. R., Arch. Neurol., 56: 429-432, 1999), which is one of the malignant brain tumors that has been thought to be untreatable in spite of various approaches including surgery, radiotherapy, and chemotherapy (Ram, Z. et al., Cancer Res., 53: 83-88, 1993; Sampson, J. H. et al., Proc. Natl. Acad. Sci. USA, 93: 10399-10404, 1996; Herrlinger, U. et al., Cancer Gene Ther., 4: 345-352, 1997; Seleh, M. et al., J. Natl. Cancer Inst., 91: 438-445, 1999; Giezeman-Smits, K. M. et al., Cancer Res., 60: 2449-2457, 2000). Some gene therapy strategies are expected to be effective based on in vivo animal model studies. However, low gene introduction efficiency limits their therapeutic effects in almost all cases. Major obstacles to a successful use of such gene therapy strategies are inability of recombinant viral vectors to spread to the whole tumor mass and low efficiency of in vivo introduction (Ram, Z. et al., Nat. Med., 3: 1354-1361, 1997). To promote gene therapy, development of a new vector system that is capable of safely and efficiently introducing genes into target cells is needed. Non-patent Document 1: Shapiro, W. R., Arch. Neurol., 56: 429-432, 1999 Non-patent Document 2: Ram, Z. et al., Cancer Res., 53: 83-88, 1993 Non-patent Document 3: Sampson, J. H. et al., Proc. Natl. Acad. Sci. USA, 93: 10399-10404, 1996 Non-patent Document 4: Herrlinger, U. et al., Cancer Gene Ther., 4: 345-352, 1997 Non-patent Document 5: Seleh, M. et al., J. Natl. Cancer Inst., 91: 438-445, 1999 Non-patent Document 6: Giezeman-Smits, K. M. et al., Cancer Res., 60: 2449-2457, 2000 Non-patent Document 7: Ram, Z. et al., Nat. Med., 3: 1354-1361, 1997

DISCLOSURE OF THE INVENTION

Problems to Be Solved by the Invention

An objective of the present invention is to provide methods for treating tumors using minus-strand RNA viral vectors that encode immunostimulatory cytokines. Another objective of the present invention is to provide compositions and kits for treating tumors which comprise minus-strand RNA viral vectors encoding immunostimulatory cytokines, and methods for producing them.

Means to Solve the Problems

The minus-strand RNA virus is an envelope virus that carries minus-strand RNA (also referred to as negative strand RNA) as genome. The virus exhibits high infectivity and is capable of expressing genes that it carries at high levels in the cytoplasm. In recent years, the advance in handling minus-strand RNA viral genome has enabled additional insertion of non-viral genes into the viral genome and thus the development of a new class of viral vectors for gene introduction approaches (Bitzer, M. et al., J. Gene Med., 5: 543-553, 2003).

The replication cycle of minus-strand RNA virus occurs in the cytoplasm without integration into the genome DNA of infected cells. Thus, this ensures safety in clinical applications of gene therapy, and the virus is thought to be useful as a tool for producing therapeutic recombinant proteins by cell culture, or for producing secretory proteins suitable for application in immunogene therapy that uses cytokines or chemokines. The minus-strand RNA virus has advantages such as: (i) there is no risk of integration into genome DNA because the replication cycle occurs exclusively in the cytoplasm; (ii) the introduction efficiency does not depend on the cell cycle of target cells; (iii) homologous recombination does not take place between the virus and a different viral genome or the wild type virus; (iv) virus incorporation into cells requires only a very short contact time; (v) genes encoded by the virus can be strongly expressed in a broad range of host cells with adjustability.

To test the therapeutic potential of the tumor gene therapy strategy that uses minus-strand RNA viral vectors for introducing cytokine genes, the present inventors introduced into tumors SeV vectors that carry immunostimulatory cytokine genes and evaluated their anti-tumor effects. An SeV carrying the interleukin-2 (IL-2) gene, which is an immunostimulatory cytokine, was constructed. The SeV vector was administered intracerebrally (I.C.) into a brain tumor model rat. As a result, it was found that the introduction of the cytokine gene into tumors by SeV resulted in significant suppression of tumor growth. Moreover, when the IL-2-expressing SeV vector was injected into an established brain tumor after peripheral vaccination with irradiated wild-type 9L cells, it was revealed that tumor growth was drastically reduced and the brain tumors were eliminated in three of the ten rats tested. Immunohistochemical analyses revealed that high levels of CD4.sup.+ and CD8.sup.+ T cells infiltrated into brain tumors that were treated with the IL-2-expressing SeV vector. It was thus discovered that the SeV vector-mediated gene introduction into tumors produces significant therapeutic effects. The introduction of immunostimulatory cytokine-encoding minus-strand RNA viral vectors into tumors is expected to become a new gene therapy strategy against tumors.

Specifically, the present invention relates to methods for treating tumors using minus-strand RNA viral vectors that encode immunostimulatory cytokines, and compositions and kits for treating tumors comprising minus-strand RNA viral vectors that encode immunostimulatory cytokines, and such. More specifically, the present invention relates to the invention of each of the claims. The present invention also relates to inventions comprising a desired combination of one or more (or all) inventions set forth in the claims, in particular, to inventions comprising a desired combination of one or more (or all) inventions set forth in the claims (dependent claims) that cite the same independent claim(s) (claim(s) relating to inventions not encompassed by inventions recited in other claims). An invention set forth in each independent claim is also intended to include any combinations of the inventions set forth in its dependent claims. Specifically, the present invention includes: [1] a method of anti-tumor treatment comprising the step of administering a minus-strand RNA viral vector encoding an immunostimulatory cytokine or a cell into which the vector has been introduced; [2] the method of [1] further comprising the step of immunizing with a tumor antigen or a vector expressing the antigen; [3] the method of [2], wherein the immunization is achieved by subcutaneously inoculating the tumor antigen or the antigen-expressing vector; [4] the method of [2] or [3], wherein the tumor antigen is a tumor cell that has lost growth ability or a tumor cell lysate; [5] the method of any one of [1] to [4], wherein the tumor is a brain tumor; [6] the method of any one of [1] to [5], wherein the immunostimulatory cytokine is interleukin-2; [7] an anti-tumor composition which comprises as an active ingredient a minus-strand RNA viral vector encoding an immunostimulatory cytokine or a cell introduced with the vector; [8] the composition of [7], wherein the immunostimulatory cytokine is interleukin-2; [9] an anti-tumor treatment kit comprising: (a) a minus-strand RNA viral vector encoding an immunostimulatory cytokine and (b) a tumor antigen or a vector expressing the antigen; and [10] the kit of [9], wherein the immunostimulatory cytokine is interleukin-2.

The present invention demonstrates that the minus-strand RNA viral vector efficiently delivered immunostimulatory cytokine genes into intracerebral tumors. The present invention also demonstrates that the i.c. administration of an immunostimulatory cytokine-encoding minus-strand RNA viral vector could produce marked anti-glioma effects and, in particular, completely eliminated established brain tumors when combined with tumor antigen immunization. It has been previously reported that secretion of an appropriate amount of immunostimulatory cytokines in glioma tissues can recruit sufficient cytotoxic T cells for eliminating established brain tumors in animals immunized by s.c. administration of irradiated whole tumor cell vaccine (Iwadate, Y. et al., Cancer Res., 61: 8769-8774, 2001). Even in an immunologically privileged state, brain tumors can become susceptible to systemic immunization if the local expression of chemotactic molecules such as IL-2 effectively enhances the migration of effector cells to tumor tissues. In the present invention, the minus-strand RNA viral vector induced a substantive expression of IL-2 protein in glioma tissues, and the local concentration of the IL-2 protein reached a level required for significantly inducing immunocompetent cells and thereby suppressing the growth of brain tumors. Thus, the method of the present invention is an effective therapeutic means especially for immunologically privileged intracerebral tumors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing the genomic structure of a Sendai virus vector. Wild-type SeV carrying lacZ or the human IL-2 gene, and an SeV vector lacking both the M and F genes are shown. Together with the end and start signals, which are SeV-specific transcriptional regulation signal sequences, the open reading frame of lacZ or the human IL-2 gene is inserted between the leader (ld) and the NP gene.

FIG. 2 is a set of photographs showing X-gal staining patterns of rat brain tissue (upper panel) and 9L brain tumor that had grown for 7 days in the brain (lower panel), into which lacZ-SeV/.DELTA.M.DELTA.F was administered in situ. X-gal staining was carried out 4, 7, and 14 days after vector administration (magnification: .times.200). In both the brain tissue and the brain tumor, the maximal expression or accumulation of .beta.-galactosidase was observed 7 days after vector injection, and the expression level was maintained on day 14.

FIG. 3 shows MRI images of the whole 9L brain tumor treated with intracerebral administration of hIL2-SeV/.DELTA.M.DELTA.F and subcutaneous immunization with irradiated 9L cells (T1-weighted image of a coronal plane after Gd-DTPA injection). The tumor in the Gd-DTPA enhanced T1-weighted image is visualized as a white region. In three of the ten rats tested, established brain tumors, which were detected three weeks after inoculation of tumor cells, were completely eliminated by week 4 with the combination therapy (Rat #3, Rat #5, and Rat #10).

FIG. 4 shows an evaluation of the mean volume of 9L brain tumors based on Gd-DTPA-enhanced MRI three weeks after the inoculation of tumor cells. A combination of the administration of hIL2-SeV/.DELTA.M.DELTA.F and subcutaneous immunization significantly reduced the volume (86.5.+-.63.8 mm.sup.3, n=10) as compared with no treatment (286.+-.51.2 mm.sup.3, n=10), the subcutaneous immunization alone (197.+-.48.9 mm.sup.3, n=10), intracerebral administration of lacZ-SeV/.DELTA.M.DELTA.F in combination with subcutaneous immunization (233.+-.73.2 mm.sup.3, n=6), or intracerebral administration of hIL2-SeV/.DELTA.M.DELTA.F alone (256.+-.53.2 mm.sup.3, n=6). Each bar represents a "mean.+-.S. E.".

FIG. 5 shows Kaplan-Meier survival curves for rats that were intracerebrally inoculated with 9L cells on day 0, and subjected to vector administration and/or immunization with irradiated tumor cells on day 3. Untreated control (open circle); subcutaneous immunization alone (closed circle); intracerebral administration of lacZ-SeV/.DELTA.M.DELTA.F and subcutaneous immunization (closed triangle); intracerebral administration of hIL2-SeV/.DELTA.M.DELTA.F alone (open triangle); intracerebral administration of hIL2-SeV/.DELTA.M.DELTA.F in combination with subcutaneous immunization (closed square). Statistical analyses based on a log-rank test demonstrated that rats treated with intracerebral administration of hIL2-SeV/.DELTA.M.DELTA.F and subcutaneous immunization survived significantly longer than the other treated groups (p<0.05).

FIG. 6 shows an immunohistochemical analysis of the IL-2 expression in 9L brain tumors. 9L brain tumors were intracerebrally administered with hIL2-SeV/.DELTA.M.DELTA.F. Magnification: A, .times.100; B, .times.200. IL-2 protein is expressed diffusely.

FIG. 7 shows immunohistochemical analyses of the expression of CD4, CD8, and NK cell antigens in rats treated with intracerebral administration of lacZ-SeV/.DELTA.M.DELTA.F and subcutaneous immunization with irradiated 9L cells (A), intracerebral administration of hIL2-SeV/.DELTA.M.DELTA.F alone (B), and intracerebral administration of hIL2-SeV/.DELTA.M.DELTA.F in combination with subcutaneous immunization (C). (Magnification: .times.200). The infiltration of CD4.sup.+ T cells and CD8.sup.+ T cells was more significantly detected in tumors treated with intracerebral administration of hIL2-SeV/.DELTA.M.DELTA.F vector and subcutaneous immunization as compared with tumors that were subjected to the other treatments.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention relates to methods of anti-tumor treatment comprising the step of introducing into tumor sites minus-strand RNA viral vectors that encode immunostimulatory cytokines or cells introduced with such vectors. Minus-strand RNA viral vectors that carry immunostimulatory cytokine genes induce immune response against a target (tumor) that has been introduced with the vectors and thus can significantly suppress tumor growth. Herein, the anti-tumor treatment means suppression of tumor development and/or growth. Specifically, the local administration of immunostimulatory cytokine-encoding minus-strand RNA viral vectors or cells introduced with the vectors to tumor sites, such as tumor tissues, potential sites for tumorigenesis, and tumor excision sites, can induce an anti-tumor immune response at the administration site and thereby suppress tumor development (including recurrence) or growth (including metastasis). The vector introduction can be achieved in vivo or ex vivo. For in-vivo introduction, the vector is injected directly into tumor sites. For ex-vivo introduction, the vector is introduced into cells outside the body and the cells are injected into tumor sites. A tumor site refers to a tumor itself, a tumor excision site, or a region adjacent thereto. Herein, the adjacent region refers to a region from which immunostimulatory cytokines secreted from cells introduced with the vector can reach the tumor or tumor excision site. The region is preferably within 5 mm, for example, within 3 mm, 2 mm, or 1 mm of a tumor or tumor excision site. The vector introduction may be conducted by dissolving or suspending a vector or cells introduced with the vector in a desired carrier (desired physiological aqueous solution, for example, culture medium, physiological saline, blood, plasma, serum, or body fluid), and then directly injecting them into a tumor or a region adjacent thereto. The method of the present invention allows effective treatment and prevention of tumors.

An important advantage of the minus-strand RNA viral vector-mediated gene delivery is that high efficiency gene delivery is achieved by a simple technique. In general, gene delivery mediated by a retroviral vector or the like has low efficiency, and therefore requires the vector be concentrated by centrifugation for optimal gene delivery. However, centrifugation often reduces the viral titer. Furthermore, a toxic agent, polybrene, is sometimes needed for high efficiency infection (Bunnell, B. A. et al., Proc. Natl. Acad. Sci. U S A, 1995, 92: 7739-7743; Chuck, A. S., Hum. Gene Ther., 1996, 7: 743-750; Chinnasamy, D. et al., Blood 2000, 96: 1309-1316; Fehse, B. et al., Br. J. Haematol., 1998, 102: 566-574). Furthermore, ex-vivo administration sometimes requires the step of selecting cells retaining the introduced gene after cell infection. In contrast, a minus-strand RNA viral vector can achieve a more superior gene delivery by merely contacting cells with the virus without a special agent. In addition, the infection efficiency is very high, and it is usually unnecessary to select cells that have been introduced with the gene with agents or such after vector infection. Furthermore, in the minus-strand RNA viral vector-mediated gene delivery, the optimal efficiency can be achieved in very short cell exposure time (30 minutes or shorter). In view of clinical situations, these characteristics enable to simplify the procedure for administration ex vivo, in vivo, and such, and to minimize procedure-dependent adverse effects such as cell damage.

When carrying out vector infection ex vivo, the MOI (multiplicity of infection; the number of infecting viruses per cell) is preferably in the range of 1 to 500, more preferably 2 to 300, even more preferably 3 to 200, still more preferably 5 to 100, yet more preferably 7 to 70. Contact between the vector and target cells needs only a short period of time, which may be, for example, 1 minute or longer, preferably 3 minutes or longer, 5 minutes or longer, 10 minutes or longer, or 20 minutes or longer, about 1 to about 60 minutes, and more specifically about 5 to 30 minutes. Alsoy, the contact time may be longer, for example, several days or longer. Cells derived from a patient to be administered may be used, for example, primary cultured fibroblast cells derived from a patient can be preferably used. Alternatively, xenogeneic cells and allogeneic cells can be used (Iwadate, Y. et al., Cancer Res., 61: 8769-8774, 2001). Xenogeneic or allogeneic cells are expected to be eliminated through host immune response after ex-vivo injection. The cells may be treated by UV, X-ray or gamma-ray irradiation, or such to make their dividing ability defective, and introduced ex vivo into tumors subsequently.

Immunostimulatory cytokines to be carried on the vector may be cytokines that induce differentiation and/or growth of immune cells and have anti-tumor activity. Such cytokines include cytokines that are produced by T cells, NK cells, monocytes, macrophages, or such, and induce differentiation and/or growth of T cells. An immunostimulatory cytokine gene can be isolated, for example, from a T-cell derived cDNA or such by PCR using primers designed based on the sequence of the gene. Cytokines having anti-tumor activity are well known to those skilled in the art. Such cytokine genes can be preferably used in the present invention. Specifically, immunostimulatory cytokines that are particularly preferably used in the present invention include interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-12 (IL-12), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-23 (IL-23), which have been shown to elicit migration and adhesion of immune cells. Fas ligand (Fas-L) can also be used. IL-2 cDNA is described, for example, in Accession number NM.sub.--000586 (protein ID: NP.sub.--000577); IL-4 cDNA, for example, in Accession numbers M13982 (protein ID: AAA59149) and M23442 (protein ID: AAA59150); IL-12 (p 35+p 40), for example, in AF180562 (protein ID: AAD56385) (p 35) and AF180563 (protein ID: AAD56386) (p 40); GM-CSF, for example, in M11220 (protein ID: AAA52578) and A14305 (protein ID: CAA01150); IL-23(p 19+p 40), for example, in AF301620 (protein ID: AAG37232) (p 19) and AF180563 (p 40: identical to IL-12 p 40); and Fas-L, for example, in D38122 (protein ID: BAA07320). Therefore, a desired nucleic acid that encodes any of the amino acid sequences of the immunostimulatory cytokines described above can be incorporated into the vector, and used in the present invention.

The above cytokines have been reported to elicit migration and adhesion of immune cells. In particular, the effectiveness of IL-2, IL-4, and GM-CSF has been demonstrated in brain tumor animal models (IL-2: Iwadate, Y et al., Cancer Res., 61: 8769-8774 (2001); IL-2, IL-4, GM-CSF: Sampson, J. H. et al., Proc. Natl. Acad. Sci. USA 93, 10399-10404 (1996); GM-CSF: Herrlinger, U. et al., Cancer Gene Ther. 4, 345-352 (1997); IL-4: Seleh, M. et al., J. Natl. Cancer Inst. 91, 438-445, (1999); IL-4: Giezeman-Smits, K. M. et al., Cancer Res. 60, 2449-2457 (2000)). IL-23 has been demonstrated to be closely associated with the migration of immune cells in brain autoimmune diseases (Becher B. et al., J Clin Invest. 112(8), 1186-91 (2000)). Meanwhile, Fas-L has been shown to be effective as a chemoattractant (Silvestris F et al., Br J Haematol. 122(1) 39-52. (2003)). For activation of the immune system against tumors by IL-2 or such, see the following documents: Iwadate, Y. et al. (2000) Cancer Gene Ther. 7, 1263-1269; Iwadate, Y. et al. (2001) Cancer Res. 61, 8769-8774; Iwadate, Y. et al. (2002) Int. J. Mol. Med. 10, 741-747; Iwadate, Y. et al. (1997) Oncology (Basel) 54, 329-334; Iwadate, Y. et al. (2003) Int. J. Oncol. 23, 483-488.

Cytokine genes that are used in the present invention may be derived from human or other mammals, for example, mouse, rat, rabbit, pig, and primates, such as monkey. In the present invention, cytokines include variants of naturally occurring cytokines so long as they retain biological activity. Such variants include, for example, polypeptides with a deletion or addition of one to several amino acid residues (for example, 2, 3, 4, 5, or 6 residues) at the N- or C-terminus, and polypeptides with a substitution of one to several amino acid residues (for example, 2, 3, 4, 5, or 6 residues). The biological activity of a cytokine can be determined by known methods for assaying cytokine activity. Alternatively, the activity can be determined by the method for assaying tumor suppression described herein. Genes encoding variants with a biological activity equivalent to that of a naturally occurring cytokine are expected to exhibit an anti-tumor growth effect equivalent to that of the naturally occurring cytokine. Variants of a naturally occurring cytokine include fragments, analogues, and derivatives of a naturally occurring cytokine, and fusion proteins with other polypeptides (for example, a cytokine having a heterologous signal peptide and a polypeptide fused with an antibody fragment). Specifically, cytokines that are used in the present invention include polypeptides that comprise a sequence with a substitution, deletion, and/or addition of one or more amino acids in the amino acid sequence of a naturally occurring cytokine or fragment thereof, and have a biological activity equivalent to that of the naturally occurring cytokine. The fragment refers to a polypeptide comprising a portion of a naturally occurring cytokine polypeptide, which includes, for example, N- or C-terminal truncated forms. Cytokine fragments with the biological activity typically comprise a continuous region of 70% or more, preferably 80% or more, more preferably 90% or more of a naturally occurring polypeptide (in its mature form after secretion).

Amino acid sequence variants can be prepared, for example, by introducing mutations into DNAs that encode naturally occurring polypeptides (Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York); Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods Enzymol. 154:367-382; Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Plainview, N.Y.); U.S. Pat. No. 4,873,192). Guidance for substituting amino acids without affecting the polypeptide's biological activity includes, for example, Dayhoff et al. (1978) in Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.)).

The number of amino acids altered is not specifically limited, but is, for example, 30% or less of the total amino acids in the mature form of a naturally occurring polypeptide, preferably 25% or less, more preferably 20% or less, even more preferably 15% or less, still more preferably 10% or less. It is, for example, 15 amino acids or less, preferably 10 amino acids or less, even more preferably 8 amino acids or less, still more preferably 5 amino acids or less, yet more preferably 3 amino acids or less. In amino acid substitution, substituting amino acids with those that have side chains with similar properties is expected to maintain a protein's original activity. This substitution is referred to as "conservative substitution" in the present invention. Conservative substitutions include substitutions between amino acids within the same group, such as basic amino acids (for example, lysine, arginine, and histidine), acidic amino acids (for example, aspartic acid, and glutamic acid), non-charged polar amino acids (for example, glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), nonpolar amino acids (for example, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), .beta.-branched amino acids (for example, threonine, valine, and isoleucine), and aromatic amino acids (for example, tyrosine, phenylalanine, tryptophan, and histidine). Conservative substitutions also include, for example, substitutions between amino acids that give a positive score in the BLOSUM62 substitution matrix (S. Henikoff and J. G. Henikoff, 1992, Proc. Acad. Natl. Sci. USA 89: 10915-10919).

Cytokine variants also include polypeptides comprising an amino acid sequence with high homology to the amino acid sequence of a naturally occurring polypeptide. High homology amino acid sequences include those with an identity of, for example, 70% or higher, more preferably 75% or higher, even more preferably 80% or higher, still more preferably 85% or higher, yet more preferably 90% or higher, even still more preferably 93% or higher, yet still more preferably 95% or higher, yet still even more preferably 96% or higher. The amino acid sequence identity can be determined, for example, using the BLASTP program (Altschul, S. F. et al., 1990, J. Mol. Biol. 215: 403-410). For example, search is carried out on the BLAST web page of NCBI (National Center for Biotechnology Information) using default parameters, with all the filters including Low complexity turned off (Altschul, S. F. et al. (1993) Nature Genet. 3:266-272; Madden, T. L. et al. (1996) Meth. Enzymol. 266:131-141; Altschul, S. F. et al. (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J. & Madden, T. L. (1997) Genome Res. 7:649-656). Sequence identity can be determined, for example, by comparing two sequences using the blast2sequences program to prepare an alignment of the two sequences (Tatiana A et al. (1999) FEMS Microbiol Lett. 174:247-250). Gaps are treated in the same way as mismatches. For example, an identity score is calculated in view of the entire amino acid sequence of a naturally occurring cytokine (its mature form after secretion). Specifically, the ratio of the number of identical amino acids to the total number of amino acids in a naturally occurring cytokine (mature form) is calculated.

Preferred variants include polypeptides encoded by nucleic acids that hybridize under stringent conditions with the entire or a portion of the coding region of a naturally occurring cytokine gene, and have a biological activity equivalent to that of the naturally occurring cytokine. When hybridization is used, such a variant can be identified, for example, by preparing a probe either from a nucleic acid that comprises the sequence of the coding region of a naturally occurring cytokine gene or the complementary sequence thereof, or from a nucleic acid to be hybridized, and then detecting whether the probe hybridizes to the other nucleic acid. Stringent hybridization conditions are, for example, hybridization at 60.degree. C., preferably at 65.degree. C., more preferably at 68.degree. C. in a solution containing 5.times.SSC, 7% (WV) SDS, 100 .mu.g/ml denatured salmon sperm DNA, and 5.times. Denhardt's solution (1.times. Denhardt's solution contains 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin, and 0.2% Ficoll); and washing twice at the same temperature as the hybridization while shaking in 2.times.SSC, preferably 1.times.SSC, more preferably 0.5.times.SSC, still more preferably 0.1.times.SSC.

The most preferable cytokine used in the present invention is interleukin (IL)-2. IL-2 is a cytokine that functions as a ligand for IL-2 receptors (IL-2 receptors .alpha., .beta., and .gamma.) and regulates the growth and differentiation of T cells (Kuziel, W. A. and Gree, W. C. (1991), Interleukin-2, in The Cytokine Handbook, A. Thompson (Ed.), San Diego, Calif., Academic Press, pages 83-102; Waldmann, T. A., 1993, Immunol. Today, 14:264). IL-2 is produced mainly by CD4.sup.+ T cells, and functions as an autocrine growth factor. IL-2 also acts on other T lymphocytes including both CD4.sup.+ and CD8.sup.+ cells. IL-2 also induces local inflammatory responses which lead to the activation of both subsets of helper and cytotoxic T cells. In addition, IL-2 stimulates the growth and activity of natural killer (NK) cells. Tumor cells that are altered to express IL-2 stimulate immune response against tumors and thereby suppress tumor growth. The nucleotide sequence of human IL-2 (mature form) cDNA is exemplified in sequence SEQ ID NO: 1, and the amino acid sequence of IL-2 is exemplified in SEQ ID NO: 2. Genes encoding the amino acid sequence of SEQ ID NO: 2 can be preferably used in the present invention.

Meanwhile, many biological active IL-2 variants are known to those skilled in the art. IL-2 variants that can be used in the present invention include, for example, those described in European Patent Application Nos. 136,489, 91,539, 88,195, and 109,748; U.S. Pat. Nos. 4,518,584, 4,588,584, 4,752,585, 4,931,543, and 5,206,344; International Patent Application WO 99/60128; Japanese Patent Application Kokai Publication (JP-A) No. S61-78799 (unexamined, published Japanese patent application); and Wang, et al. Science (1984) 224:1431-1433. Such variants include, for example, an IL-2 fragment lacking the N-terminal Ala, an IL-2 fragment lacking four amino acids (JP-A No. S60-126088), a carboxyl terminal-truncated IL-2 fragment (JP-A No. S60-126088), a polypeptide in which a neutral amino acid, such as serine or alanine, has been substituted for cysteine 125 in the natural occurring polypeptide after secretion (des-ala-1, ser-125 IL-2, or des-ala-1, ala-125 IL-2) (U.S. Patent Nos. 4,518,584 and 4,588,584), and a polypeptide in which a neutral amino acid, such as alanine, has been substituted for methionine 104 (des-ala-1, ala-104). In addition to those described above, desired variants retaining the biological activity of IL-2 may also be used. The IL-2 biological activity of a variant can be confirmed, for example, by testing its ability to stimulate the growth of IL-2-dependent cytotoxic or helper T cells using known methods (Gillis et al., J. immunol. (1978) 120:2027-2032; Watson, J., J. exp. Med. (1979) 1570:1510-1519).

cDNAs that encode the above-described cytokines are used to construct recombinant minus-strand RNA viruses expressing the cytokines. Herein, a minus-strand RNA virus refers to viruses that include a minus strand (an antisense strand corresponding to a sense strand encoding viral proteins) RNA as the genome. The minus-strand RNA is also referred to as negative strand RNA. The minus-strand RNA virus used in the present invention particularly includes single-stranded minus-strand RNA viruses (also referred to as non-segmented minus-strand RNA viruses). The "single-strand negative strand RNA virus" refers to viruses having a single-stranded negative strand [i. e., a minus strand] RNA as the genome. Such viruses include viruses belonging to Paramyxoviridae (including the genera Paramyxovirus, Morbillivirus, Rubulavirus, and Pneumovirus), Rhabdoviridae (including the genera Vesiculovirus, Lyssavirus, and Ephemerovirus), Filoviridae, Orthomyxoviridae, (including Influenza viruses A, B, and C, and Thogoto-like viruses), Bunyaviridae (including the genera Bunyavirus, Hantavirus, Nairovirus, and Phlebovirus), Arenaviridae, and the like.

In addition, the minus-strand RNA viral vector refers to a minus-strand RNA virus-based vehicle for introducing genes into cells. Herein, "infectivity" refers to the capability of a minus-strand RNA viral vector to maintain cell-adhesion ability and introduce a gene carried by the vector to the inside of the cell to which the vector has adhered. The minus-strand RNA viral vector of this invention may be transmissible or may be a nontransmissible defective-type vector. "Transmissible" means that when a viral vector infects a host cell, the virus is replicated in the cell to produce infectious virions.

"Recombinant virus" refers to a virus produced through a recombinant polynucleotide, or an amplification product thereof. "Recombinant polynucleotide" refers to a polynucleotide in which nucleotides are not linked at one or both ends as in the natural condition. Specifically, a recombinant polynucleotide is a polynucleotide in which the linkage of the polynucleotide chain has been artificially modified (cleaved and/or linked). Recombinant polynucleotides can be produced by using gene recombination methods known in the art in combination with polynucleotide synthesis, nuclease treatment, ligase treatment, etc. A recombinant virus can be produced by expressing a polynucleotide encoding a viral genome constructed through gene manipulation and reconstructing the virus. For example, methods for reconstructing a virus from cDNA that encodes the viral genome are known (Y. Nagai, A. Kato, Microbiol. Immunol., 43, 613-624 (1999)).

In the present invention, "gene" refers to a genetic substance, a nucleic acid encoding a transcriptional unit. Genes may be RNAs or DNAs. In this invention, a nucleic acid encoding a protein is referred to as a gene of that protein. Further, in general, a gene may not encode a protein. For example, a gene may encode a functional RNA, such as a ribozyme or antisense RNA. Generally, a gene may be a naturally-occurring or artificially designed sequence. Furthermore, in the present invention, "DNA" includes both single-stranded and double-stranded DNAs. Moreover, "encoding a protein" means that a polynucleotide includes an ORF that encodes an amino acid sequence of the protein in a sense or antisense strand, so that the protein can be expressed under appropriate conditions.

A minus-strand RNA virus particularly preferably used in the context of the present invention includes, for example, Sendai virus, Newcastle disease virus, mumps virus, measles virus, respiratory syncytial virus (RS virus), rinderpest virus, distemper virus, simian parainfluenza virus (SV5), and human parainfluenza viruses 1, 2, and 3 belonging to Paramyxoviridae; influenza virus belonging to Orthomyxoviridae; and vesicular stomatitis virus and rabies virus belonging to Rhabdoviridae.

Further examples of virus that may be used in the context of the present invention include those selected from the group consisting of: Sendai virus (SeV), human parainfluenza virus-1 (HPIV-1), human parainfluenza virus-3 (HPIV-3), phocine distemper virus (PDV), canine distemper virus (CDV), dolphin molbillivirus (DMV), peste-des-petits-ruminants virus (PDPR), measles virus (MV), rinderpest virus (RPV), Hendra virus (Hendra), Nipah virus (Nipah), human parainfluenza virus-2 (HPIV-2), simian parainfluenza virus 5 (SV5), human parainfluenza virus-4a (HPIV-4a), human parainfluenza virus-4b (HPIV-4b), mumps virus (Mumps), and Newcastle disease virus (NDV). A more preferred example is a virus selected from the group consisting of Sendai virus (SeV), human parainfluenza virus-1 (HPIV-1), human parainfluenza virus-3 (HPIV-3), phocine distemper virus (PDV), canine distemper virus (CDV), dolphin molbillivirus (DMV), peste-des-petits-ruminants virus (PDPR), measles virus (MV), rinderpest virus (RPV), Hendra virus (Hendra), and Nipah virus (Nipah).

More preferably, viruses of the present invention are preferably those belonging to Paramyxoviridae (including Respirovirus, Rubulavirus, and Morbillivirus) or derivatives thereof, and more preferably those belonging to the genus Respirovirus (also referred to as Paramyxovirus) or derivatives thereof. The derivatives include viruses that are genetically-modified or chemically-modified in a manner not to impair their gene-transferring ability. Examples of viruses of the genus Respirovirus applicable to this invention are human parainfluenza virus-1 (HPIV-1), human parainfluenza virus-3 (HPIV-3), bovine parainfluenza virus-3 (BPIV-3), Sendai virus (also referred to as murine parainfluenza virus-1), and simian parainfluenza virus-10 (SPIV-10). A more preferred paramyxovirus in this invention is the Sendai virus. These viruses may be derived from natural strains, wild strains, mutant strains, laboratory-passaged strains, artificially constructed strains, or the like.

Genes harbored on a minus-strand RNA viral vector are situated in the antisense direction in the viral genomic RNA. Viral genomic RNA refers to RNA that has the finction to form a ribonucleoprotein (RNP) with the viral proteins of a minus-strand RNA virus. Genes contained in the genome are expressed by the RNP, genomic RNA is replicated, and daughter RNPs are formed. In general, in the minus-strand RNA viral genome, viral genes are arranged as antisense sequences between the 3'--leader region and the 5'--trailer region. Between the ORFs of respective genes are a transcription ending sequence (E sequence)--intervening sequence (I sequence)--transcription starting sequence (S sequence), such that RNA encoding the ORF of each gene is transcribed as an individual cistron. Genomic RNAs in a virus of this invention comprise the antisense RNA sequences encoding N (nucleocapsid)--, P (phospho)--, and L (large)--proteins, which are viral proteins essential for the expression of the group of genes encoded by an RNA, and for the autonomous replication of the RNA itself. The genomic RNAs may or may not encode M (matrix) proteins, which is essential for virion formation. Further, the RNAs may or may not encode envelope proteins essential for virion infection. Minus-strand RNA viral envelope proteins include F (fusion) protein that causes cell membrane fusion, and HN (hemagglutinin-neuraminidase) protein which is essential for viral adhesion to cells. However, HN protein is not required for the infection of certain types of cells (Markwell, M. A. et al., Proc. Natl. Acad. Sci. USA 82(4): 978-982 (1985)), and infection is achieved with F protein only. The RNAs may encode envelope proteins other than F protein and/or HN protein. Thus, the genomic RNAs may be naturally-occurring viral genomes that are appropriately modified (WO 00/70055 and WO 00/70070).

Minus-strand RNA viruses of this invention may be, for example, complexes of minus-strand RNA viral genomic RNAs and viral proteins, that is, ribonucleoproteins (RNPs). RNPs can be introduced into cells, for example, in combination with desired transfection reagents. Specifically, such RNPs are complexes comprising a minus-strand RNA viral genomic RNA, N protein, P protein, and L protein. On introducing an RNP into cells, cistrons encoding the viral proteins are transcribed from the genomic RNA by the action of viral proteins, and, at the same time, the genome itself is replicated to form daughter RNPs. Replication of a genomic RNA can be confirmed by using RT-PCR, Northern blot hybridization, or the like to detect an increase in the copy number of the RNA.

Further, minus-strand RNA viruses of this invention are preferably infectious minus-strand RNA viral virions. "Virion" means a microparticle comprising a nucleic acid released from a cell by the action of viral proteins. Infectivity refers to the ability of a minus-strand RNA virus, which retain cell adhesion and membrane-fusion abilities, to introduce nucleic acids inside the virus into cells to which the virion has adhered. The virion of a minus-strand RNA virus has a structure, in which the above-described RNP comprising genomic RNA and viral proteins is enclosed in a lipid membrane (referred to as an envelope) derived from cell membrane. Minus-strand RNA viruses of this invention may be transmissible or may be a nontransmissible defective-type virus. "Transmissible" means that when a virus is introduced into a host cell, the virus can replicate itself within the cell to produce infectious virions.

Genes of Paramyxovirinae viruses are commonly listed as follows. In general, NP gene is also listed as "N gene."

TABLE-US-00001 Respirovirus NP P/C/V M F HN -- L Rubulavirus NP P/V M F HN (SH) L Morbillivirus NP P/C/V M F H -- L

For example, the database accession numbers for the nucleotide sequences of each of the Sendai virus genes are: M29343, M30202, M30203, M30204, M51331, M55565, M69046, and X17218 for NP gene; M30202, M30203, M30204, M55565, M69046, X00583, X17007, and X17008 for P gene; D11446, K02742, M30202, M30203, M30204, M69046, U31956, X00584, and X53056 for M gene; D00152, D11446, D17334, D17335, M30202, M30203, M30204, M69046, X00152, and X02131 for F gene; D26475, M12397, M30202, M30203, M30204, M69046, X00586, X02808, and X56131 for HN gene; and D00053, M30202, M30203, M30204, M69040, X00587, and X58886 for L gene. Examples of viral genes encoded by other viruses are: CDV, AF014953; DMV, X75961; HPIV-1, D01070; HPIV-2, M55320; HPIV-3, D10025; Mapuera, X85128; Mumps, D86172; MV, K01711; NDV, AF064091; PDPR, X74443; PDV, X75717; RPV, X68311; SeV, X00087; SV5, M81442; and Tupaia, AF079780 forN gene; CDV, X51869; DMV, Z47758; HPIV-1, M74081; HPIV-3, X04721; HPIV-4a, M55975; HPIV-4b, M55976; Mumps, D86173; MV, M89920; NDV, M20302; PDV, X75960; RPV, X68311; SeV, M30202; SV5, AF052755; and Tupaia, AF079780 for P gene; CDV, AF014953; DMV, Z47758; HPIV-1, M74081; HPIV-3, D00047; MV, ABO16162; RPV, X68311; SeV,AB005796; and Tupaia, AF079780 for C gene; CDV, M12669; DMV, Z30087; HPIV-1, S38067; HPIV-2, M62734; HPIV-3, D00130; HPIV-4a, D10241; HPIV-4b, D10242; Mumps, D86171; MV, AB012948; NDV, AF089819; PDPR, Z47977; PDV, X75717; RPV, M34018; SeV, U31956; and SV5, M32248 for M gene; CDV, M21849; DMV, AJ224704; HPN-1, M22347; HPIV-2, M60182; HPIV-3, X05303; HPIV-4a, D49821; HPIV-4b, D49822; Mumps, D86169; MV, AB003178; NDV, AF048763; PDPR, Z37017; PDV, AJ224706; RPV, M21514; SeV, D17334; and SV5, AB021962 for F gene; and, CDV, AF112189; DMV, AJ224705; HPIV-1, U709498; HPIV-2, D000865; HPIV-3, AB012132; HPIV-4A, M34033; HPIV-4B, AB006954; Mumps, X99040; MV, K01711; NDV, AF204872; PDPR, Z81358; PDV, Z36979; RPV, AF132934; SeV, U06433; and SV-5, S76876 for HN (H or G) gene. However, multiple strains are known for each virus, and there exist genes that comprise sequences other than those cited above as a result of strain variation.

ORFs encoding these viral proteins and ORFs of foreign genes are arranged in the antisense direction in the genomic RNA via the above-described E-I-S sequence. The ORF closest to the 3'--end of the genomic RNA requires only an S sequence between the 3'--leader region and the ORF, and does not require an E or I sequence. Further, the ORF closest to the 5'--end of the genomic RNA requires only an E sequence between the 5'--trailer region and the ORF, and does not require an I or S sequence. Furthermore, two ORFs can be transcribed as a single cistron, for example, by using an internal ribosome entry site (IRES) sequence. In such a case, an E-I-S sequence is not required between these two ORFs. For example, in wild type paramyxoviruses, a typical RNA geno