JPH08127542A - Genetic therapeutic agent for cancer - Google Patents

Abstract

PURPOSE: To obtain the cancer genetic therapeutic agent containing effector cells transduced with cytokine gene and a tumor vaccine prepared by transducing the cytokine gene into tumor cells. CONSTITUTION: A cytokine gene [e.g. interleukin (IL)-2 gene] is transduced into an effector cell as a lymphocyte relating to the decomposition of cancer cells using an Adenovirus vector. The cytokine gene (e.g. granulocytic macrophage colony-stimulating factor gene) is transduced into a tumor cell (e.g. melanoma cell) using a retrovirus vector and subsequently irradiated with X-ray to obtain the tumor vaccine. The cytokinin gene effector cells and the tumor vaccine are used as such of processed with an excipient, etc., into a preparation. The agent is administered at a daily dose of 0.1-100mg (as the weight of the cytokinin gene) to an adult. The agent can be used for men, mice, monkeys, dogs, cats, horses, pigs, etc., and is effective as a therapeutic agent against fine metastasized cells.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a gene therapeutic agent for cancer, and more particularly, to a gene therapeutic agent for cancer containing an effector cell into which a cytokine gene has been introduced and a tumor vaccine into which a cytokine gene has been introduced into a tumor cell.

[0002]

2. Description of the Related Art Cytokines exert antitumor effects, and they are mainly classified into the following three types depending on their mechanism of action.
That is, such as tumor necrosis factor (TNF) -α, β, interferon (IFN) -α, β, γ, interleukin (IL) -1, etc., does not directly inhibit the growth of cancer cells in vitro. Interleukin (IL) -2, which shows impaired activity
4,6, 7, 8, 9, 10, 11, 12, etc., which indirectly show an antitumor effect via the immune effector mechanism of the living body,
IL-3, IL-6, granulocyte-macrophage colony stimulating factor
(GM-CSF), granulocyte colony stimulating factor (G-CSF), stem cell factor (SCF), etc., are used for the prevention and treatment of side effects in the body associated with other anticancer therapies.

Furthermore, in recent years, cancer treatment by introducing a cytokine gene has been attempted. One of these is to introduce cytokine gene into lymphocytes such as lymphokine-activated killer cells (LAK), cytotoxic T cells (CTL), tumor infiltrating lymphocytes (TIL), and Attempts have been made to activate lymphocytes by autocrine or paracrine to enhance passive immunity, or to enhance antitumor properties at tumor sites by directly introducing an antitumor IFN or TNF gene into TIL. (Nishihara et al., Cancer Res., 48, 4730,
1988, Miyatake et al., J. Natl. Cancer Inst., 82, 2
17, 1990, Itoh et al., Jpn. J. Cancer Res., 82, 1
203, 1991). For the introduction of cytokine genes into TIL, retroviruses have been used so far, but the introduction efficiency is very poor, which is an obstacle to this approach.

The other is to induce a tumor-specific immune cell of a host by introducing a cytokine gene into a tumor cell by a retrovirus vector or the like and using it as a tumor vaccine. For example, Fearon et al. Spontaneously regressed after transplantation of mouse colon cancer and malignant melanoma introduced with IL-2 gene into syngeneic mice, and the mouse was resistant to re-implantation of tumor cells not introduced with IL-2 gene. Have been reported to acquire sexual immunity (Fearon et al., Cell, 60, 397, 199).
0). However, none of these results have always been satisfactory in terms of antitumor properties, and no studies have been conducted in terms of their metastasis-suppressing effect, and there are no promising ones for cancer metastasis. Currently not reported.

[0005]

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide a gene therapy for cancer, which can exert higher antitumor activity and is effective in suppressing cancer metastasis in order to solve the above problems. To provide the agent.

[0006]

Means for Solving the Problems As a result of intensive studies on the above-mentioned problems, the present inventors have found that if an effector cell into which a cytokine gene is introduced and a tumor vaccine into which a cytokine gene is introduced into a tumor vaccine are used in combination, The present invention has been completed by finding that systemic immunity is efficiently induced and antitumor effect and metastasis suppressing effect are enhanced.

That is, the present invention provides a gene therapeutic agent for cancer, which comprises an effector cell into which a cytokine gene has been introduced and a tumor vaccine into which a cytokine gene has been introduced into a tumor cell. The gene therapy of cancer referred to in the present invention is intended to treat cancer from both the antitumor effect and the metastasis suppressing effect by the cytokine gene.

The present invention will be described in detail below. The cytokine gene used in the present invention includes granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin (IL) -2, IL-3, IL-4, IL-6, IL-7, IL-10. , IL-12, IL-
13, IL-15, interleukin-1α (IL-1α), interleukin receptor 1 antagonist (IL-1RA), tumor necrosis factor (TNF) -α, lymphotoxin (LT) -β, granulocyte colony stimulating factor (G -CSF), macrophage colony stimulating factor (M-CSF), interferon (IFN) -γ, macrophage migration inhibitory factor (MIF), leukemia inhibitory factor (LIF),
T cell activating costimulatory factor B7 (CD80) and B7-2 (CD86),
It refers to genes that encode various cytokines such as kit ligand and oncostatin M. Various cytokine cDNAs have already been cloned [human GM-CSF cD
For NA, Wong et al., Science, 228, 810-815 (1
985), for human IL-2 cDNA, see Taniguchi et al., Na
ture, 302, 305-310 (1983)], and for human IFN-γ cDNA, Gray et al., Nature, 298, 859-863 (1982)].
The cytokine gene used in the present invention may be cDNA obtained by isolating it from a cell using a known technique, or a method such as a polymerase chain reaction (PCR) based on the information disclosed in the above documents. Although it may be chemically synthesized according to the above, it is preferably of human origin in order to minimize immunological rejection and to enhance the therapeutic effect.

In the present invention, the effector cell means a cell population that is in charge of the final stage of destruction of cancer cells and is directly involved in the destruction, and specifically, tumor infiltrating lymphocytes (TIL), lymphokine-activated killer cells (LAK). ), Cytotoxic T cells
(CTL) etc. The introduction of a cytokine gene into these effector cells can be performed very efficiently by using an adenovirus vector. The adenovirus vector used here is not particularly limited as long as it contains an insertion site for a cytokine gene and can express the cytokine in the introduced effector cells.
Adex1 [Saito, I. et al., J. Viol., 54, 711-719 (198
5)] is preferably used.

On the other hand, the tumor vaccine referred to in the present invention does not inhibit cytokine production by introducing the above-mentioned cytokine gene into an isolated (cultured) tumor cell by a retrovirus vector and irradiating it with X-rays. Only the growth is stopped. The tumor-specific immune cells of the host can be induced by administering this tumor vaccine to the host.

The retroviral vector used here is not particularly limited as long as it contains an insertion site for a cytokine gene and is capable of expressing the cytokine in the introduced tumor cells.
MFG, α-SCG, PLJ, pEm and the like disclosed in JP-A-8 are listed. The retrovirus vector containing the cytokine gene is mixed with a plasmid having a neomycin resistance gene as a marker for selecting that the gene of interest has been introduced, and Ψ 2, Ψ-Am, ΨCRIP, ΨCRE are mixed.
[Danos et al., PNAS, 85, 6460-6464 (1988)] and the like are introduced into packaging cells by the calcium coprecipitation method (cotransfection). Furthermore, this is drug G41
By culturing in the presence of 8 and collecting cells that survive and form colonies, only cells into which the gene of interest has been introduced can be collected. Next, using the culture supernatants of these cells, NIH3T3 mouse fibroblasts,
It can be confirmed by Southern hybridization as a stable gene-transfected cell that has been infected with various tumor cells such as B16 mouse melanoma cells and finally introduced into the chromosome of the cell. The amount of cytokine secreted from infected cells can be measured by an immunological assay such as ELISA.

Next, the tumor cells into which the cytokine gene has been introduced are usually irradiated with X-rays of 10,000 to 150,000 rad,
This is used as a tumor vaccine. The tumor cells referred to herein are melanoma cells or renal cancer cells, but other tumor cells such as breast cancer cells, squamous cell carcinoma, adenocarcinoma, transitional cell carcinoma, sarcoma, glioma (glioma), etc. may also be used. it can. The cytokine gene-introduced effector cells and tumor vaccine respectively prepared as described above can be used as they are, but as a pharmaceutical composition with a pharmaceutically acceptable excipient, they are formulated into a solution, suspension, gel or the like. It is also possible to administer it in the form of

As the administration form of the gene therapeutic agent of the present invention, in addition to the usual systemic administration such as intravenous and intraarterial administration, it is possible to treat the carcinogenic lesion or to the predicted metastatic site corresponding to the cancer type. Local administration such as local injection and oral administration can be performed. Furthermore, the gene therapy agent of the present invention can be administered in a dosage form combined with a catheter technique, a gene introduction technique, a surgical operation, or the like. The dose of the gene therapeutic agent of the present invention varies depending on age, sex, symptoms, administration route, number of administrations, and dosage form.
A range of 0.1-100 mg is suitable.

[0014]

According to the present invention, human, mouse,
A gene therapeutic agent that can exhibit high antitumor properties in animals such as monkeys, dogs, cats, horses, and pigs, and that is also effective in suppressing cancer metastasis, is provided, and is useful for cancer treatment of micrometastases.

[0015]

The present invention will be described in more detail by the following examples. These examples are illustrative and do not limit the scope of the invention. [Reference Example 1] Mouse IL-2, mouse GM-CSF, and mouse IF
N-γ cDNA was derived from mouse splenic lymphocyte mRNA.
RT-PCR (Reverse transcripiton-polymerase chain rea
ction) method. When performing PCR,
-For Primer # 2, primer # 479, # 480, mouse
Primers # 477 and # 478 shown below for GM-CSF and primers # 485 and # 486 shown below for mouse IFN-γ
Were used respectively.

Primer (# 479): CCGAATTCTAGACACC AT
G TAC AGC ATG CAG CTC GCA TCC TGT G primer (# 480): C TGT CAA AGC ATC ATC TCA ACA A
GC CCT CAA TAA GGATCC CC primer (# 477): CCGAATTCTAGACACC ATG TGG CTG CA
G AAT TTA CTT TTC CTG GGC primer (# 478): C CCC TTT GAA TGC AAA AAA CCA A
GC CAA AAA TGA GGATCC GG primer (# 485): CC GAA TTC TAGA CACC ATG AAC GC
T ACA CAC TGC ATC TTG GC primer (# 486): C AGG AAG CGG AAA AGG AGT CGC T
GC TGA GGATCCGG

[Example 1] Preparation of cytokine gene-introduced effector cells (TIL / IL-2) (1) Preparation of mouse melanoma B16F10-derived TIL Preparation of TIL was performed by Alexander, RB et al., J. Immunol.,
145, 1615-1620 (1990), Matis, LA et al., Method
s Enzymol., 150, 342-351 (1987), Livingstone, A.
The method described in et al., Methods Enzymol., 150, 325-333 (1987) was modified and carried out as follows. 6-10
B16F10 (Whitehead Institute Dr. Glenn), a highly metastatic strain of mouse melanoma B16 (ATCC CRL6322), transplanted to week-old female C57BL / 6 mice (purchased from Charles River Japan)
Fresh tumor mass from Dranoff) was added to complete culture medium (C
5 × 10 ⁷ cells / ml were suspended in M) at 4 ° C. CM is heat inactivated 10% fetal bovine serum, 2 mM L-glutamine, 5 x
10 ^-5 M 2-mercaptoethanol, 100 U / ml penicillin, 100 μg / ml streptomycin, 0.5 μg / ml amphotericin B, 10 mM 3- (N-morpholino) propanesulfonic acid, and 70 U / ml recombinant human IL-2 (Shiono) Yi Pharmaceutical Co., Ltd.
It is RPMI 1640 to which is added. TIL was mixed with an equal volume of anti-CD-8-conjugated immunosorbent beads at 1 x 10 ⁸ / ml and
Incubated at ℃ for 2 hours. The beads with TIL attached were pelletized, washed 3 times with cold CM, and 1 × 10 in CM.
⁷ beads / ml was suspended, seeded on a 24-well tissue culture plate, and incubated at 37 ° C. under 5% CO ₂ . After 1 day of culture, the beads separated from TIL were pelleted and removed. Separated TILs were irradiated with 2 × 10 ⁵ per well (10,
000rad) tumor cells and 1 × 10 ⁶ irradiation (3,000ra)
d) Stimulated with normal splenocytes. In vitro stimulation was repeated every 7 to 14 days. When it becomes confluent
TIL was collected and resuspended in fresh CM at 2 × 10 ⁵ cells / ml.

(2) Introduction of mouse IL-2 gene into TIL by adenovirus A method for preparing a recombinant adenovirus is described in Saito, I. e.
t al., J. Viol., 54,711-719 (1985). That is, the cytomegalovirus enhancer,
Expression unit consisting of chicken β-actin promoter, mouse IL-2 cDNA sequence prepared in Reference Example 1, and rabbit-β-globin poly (A) signal sequence [Niwa, H. et.
al., J. Gene 108, 193-200 (1991)] is a 42 kb cosmid containing 31 kb of adenovirus type 5 lacking E1A, E1B, and E3 genes, pAdex1w (Hiromi Kanegae, Shizuko Harada, Izumi Saito, Experimental). Biomedical Manual 4, 189-204,
In 1994, Yodosha), a cosmid cassette for expression was constructed by inserting it into the SwaI restriction enzyme site (Fig. 1). This expression cosmid cassette and adenovirus DNA-terminal protein complex (DNA-TPC) were added to 293 cells (ATCC CRL1573).
Were co-transfected by the calcium coprecipitation method. The recombinant virus containing the expression cassette was confirmed by digestion with the appropriate restriction enzymes. The recombinant virus was subsequently propagated in 293 cells and the virus solution was stored at -80 ° C. Viral stock titer was determined by plaque assay on 293 cells. For in vitro infection of TIL prepared in (1) with adenovirus,
-TIL cells seeded in well culture plates were removed and 150 μl of virus stock was added to each well. After incubating at 37 ℃ for 1 hour, the growth medium was added and the TIL cells were cultured for 2 to 3 days.
(TIL / IL-2) was obtained.

[Example 2] Tumor vaccine (B16F10 / IL-
2 + GM-CSF vaccine) (1) Preparation of mouse IL-2, mouse GM-CSF high titer recombinant retrovirus producing clone Retrovirus vector MFG (Dranoff, G. et al.,
Proc. Natl. Acad. Sci. USA, 90, 3539-3543, 1993).
EagI / BamHI fragment (5200bp) including two LTRs (Long Terminal Repeat), EagI / XbaI fragment (1000bp),
And mouse IL-2 and mouse GM-CSF prepared in Reference Example 1.
3 of the XbaI / BamHI fragments of each cDNA of T4 DNA
It was ligated with ligase and integrated into a plasmid (Fig. 2). The resulting plasmid and pPGKneo [H. Takeshima et
al., Nature, 369, 556-559 (1994)] by the calcium coprecipitation method with ΨCRIP [Danos et al., Proc. Natl. Acad. S.
ci. USA, 85, 6460 (1988)], and introduced this into G-418 (GI
The cells were cultured in a medium containing BCO (1 mg / ml) for 1 week, and cells forming colonies were collected. Next, the culture supernatants of these cells were infected with NIH3T3 mouse fibroblasts (ATCC CRL1658) in the presence of polybrene (Sigma, 8 μg / ml). Genomic DNA of infected cells was isolated and the viral titer was determined by Southern blotting and evaluated as the copy number of the integrated provirus. Transfection efficiency into NIH3T3 was usually 1 to 3 copies per cell of provirus integration. In addition, cytokines secreted from infected cells are
48 hours after seeding 1 × 10 ⁶ cells in a 10-cm dish containing 10 ml of medium, assay was performed by ELISA (Endogen) (Table 1).

[0020]

[Table 1] ─────────────────────────────── cDNA titer (copy number) Expression level ─────── ──────────────────────── Mouse IL-2 2.0 6350 IU / ml Mouse GM-CSF 2.0 13.5 ng / ml ──────── ───────────────────────

(2) Tumor vaccine (B16F10 / IL-2 ＋ GM-SCF
Vaccine) The high-titer recombinant retrovirus-producing clones of mouse IL-2 and mouse GM-CSF selected in (1) were cloned into mouse melanoma B16 (ATCC CRL 6322), which is a highly metastatic strain B16F10.
(Obtained from Whitehead Institute Dr. Glenn Dranoff). The transgenic cells were maintained in Dulbecco's Eagle's medium supplemented with 10% fetal bovine serum and 2 mM glutamine, treated with trypsin / EDTA and treated with HITACHI MBR-15.
The 05R X-ray generator was used to irradiate 10,000 rad X-rays. Irradiated cells are HBSS (Hank's Balanced Salt Solutio
The cells were washed twice with n) and resuspended in HBSS at a concentration of 5 × 10 ⁶ cells / ml to obtain a tumor vaccine (B16F10 / IL-2 + GM-CSF vaccine).

[Example 3] Preparation of cytokine gene-introduced effector cells (TIL / IFN-γ) (1) Preparation of mouse melanoma B16F10-derived TIL was prepared by the same method as in Example 1 (1). (2) Introduction of mouse IFN-γ gene into TIL by adenovirus A method for preparing a recombinant adenovirus is described in Saito, I. e.
t al., J. Viol., 54,711-719 (1985). That is, the cytomegalovirus enhancer,
Expression unit consisting of chicken β-actin promoter, mouse IFN-γ cDNA sequence prepared in Reference Example 1 and rabbit-β-globin poly (A) signal sequence [Niwa, H. et.
al., J. Gene 108, 193-200 (1991)], pAdex1cw, a 42 kb cosmid containing 31 kb of adenovirus type 5 lacking E1A, E1B, and E3 genes (Hiromi Kanegae, Shizuko Harada, Izumi Saito, experiments). Biomedical Manual 4, 189-204,
In 1994, Yodosha) was inserted into the ClaI restriction enzyme site to construct a cosmid cassette for expression (Fig. 1). This expression cosmid cassette and adenovirus DNA-terminal protein complex (DNA-TPC) were added to 293 cells (ATCC CRL1573).
Were co-transfected by the calcium coprecipitation method. The recombinant virus containing the expression cassette was confirmed by digestion with the appropriate restriction enzymes. The recombinant virus was subsequently propagated in 293 cells and the virus solution was stored at -80 ° C. Viral stock titer was determined by plaque assay on 293 cells. For in vitro infection of TIL prepared in (1) with adenovirus,
-TIL cells seeded in well culture plates were removed and 150 μl of virus stock was added to each well. After incubating at 37 ℃ for 1 hour, the growth medium was added and the TIL cells were cultured for 2 to 3 days.
(TIL / IFN-γ) was obtained.

[Example 4] Tumor vaccine (B16F10 / GM-
Preparation of CSF vaccine) (1) Preparation of mouse GM-CSF high titer recombinant retrovirus producing clone It was prepared by the same method as in Example 2 (1). (2) Preparation of tumor vaccine (B16F10 / GM-SCF vaccine) The high-titer recombinant retrovirus producing clone of mouse GM-CSF selected in (1) was cloned into mouse melanoma B16 (ATCC C
B16F10 (Whitehead Institute), a highly metastatic strain of RL 6322)
(Obtained from Dr. Glenn Dranoff). The transgenic cells were maintained in Dulbecco's Eagle medium supplemented with 10% fetal bovine serum and 2 mM glutamine and treated with trypsin.
/ EDTA, HITACHI MBR-1505R X-ray generator
Was irradiated with X-rays of 10,000 rad. Irradiated cells are HBSS
The cells were washed twice with HBSS and resuspended in HBSS at a concentration of 5 × 10 ⁶ cells / ml to obtain a tumor vaccine (B16F10 / GM-CSF vaccine).

[Example 5] Preparation of cytokine gene-transfected effector cells (TIL / IL-2, TIL / IFN-γ) (1) Preparation of mouse colon cancer Colon 26-derived TIL Preparation of TIL was performed by Alexander, RB et al. ., J. Immunol.,
145, 1615-1620 (1990), Matis, LA et al., Method
s Enzymol., 150, 342-351 (1987), Livingstone, A.
The method described in et al., Methods Enzymol., 150, 325-333 (1987) was modified and carried out as follows. 6-10
Week-old female BALB / C mouse (purchased from Charles River Japan)
A fresh tumor mass of mouse colon cancer Colon 26 transplanted in the above was suspended in complete culture medium (CM) at 4 ° C. at 5 × 10 ⁷ cells / ml. CM is heat inactivated 10% fetal bovine serum, 2 mM L-glutamine, 5 × 10 ^-5 M 2-mercaptoethanol, 100 U / m
l Penicillin, 100 μg / ml streptomycin, 0.5 μg
RPMI 1640 supplemented with / ml amphotericin B, 10 mM 3- (N-morpholino) propanesulfonic acid, and 70 U / ml recombinant human IL-2 (obtained from Shionogi Pharmaceutical Co., Ltd.). TI
L was mixed with an equal volume of anti-CD-8-conjugated immunoadsorbent beads at 1 x 10 ⁸ / ml and incubated for 2 hours at 4 ° C. Beads with TIL attached are pelletized, washed 3 times with cold CM, and CM
1 × 10 ⁷ beads / ml were suspended therein, seeded on a 24-well tissue culture plate, and incubated at 37 ° C. under 5% CO ₂ . After 1 day of culture, the beads separated from TIL were pelleted and removed. Irradiate separated TILs with 2 × 10 ⁵ per well
(10,000 rad) tumor cells, as well as 1 × 10 ⁶ irradiation (3,0
(00 rad) Stimulated with normal splenocytes. 7 in vitro stimulation
Repeated every 14 days from day. When it became confluent, TIL was collected and resuspended in fresh CM at 2 × 10 ⁵ cells / ml.

(2) Introduction of mouse IL-2 gene into TIL by adenovirus TI prepared in (1) above in the same manner as in Example 1 (2)
The mouse IL-2 cDNA prepared in Reference Example 1 was introduced into L to obtain mouse IL-2 gene-introduced TIL cells (TIL / IL-2). (3) Introduction of mouse IFN-γ gene into TIL by adenovirus TI prepared in (1) above by the same method as in Example 3 (2)
The mouse IFN-γ cDNA prepared in Reference Example 1 was introduced into L to obtain mouse IFN-γ gene-introduced TIL cells (TIL / IFN-γ).

[Example 6] Tumor vaccine (Colon26 / IL
-2 vaccine) (1) Preparation of mouse IL-2 high titer recombinant retrovirus producing clone It was prepared by the same method as in Example 2 (1). (2) Preparation of tumor vaccine (Colon26 / IL-2 vaccine) The IL-2 high titer recombinant retrovirus producing clone selected in (1) was introduced into mouse colon cancer Colon26. The transgenic cells were maintained in RPMI1640 medium supplemented with 10% fetal bovine serum and 2 mM glutamine to obtain HITACHI MBR-1505.
The X-ray of 10,000 rad was irradiated using the R X-ray generator. The irradiated cells were washed twice with HBSS and resuspended in HBSS at a concentration of 5 × 10 ⁶ cells / ml to obtain a tumor vaccine (Colon26 / IL-2 vaccine).

[Test Example 1] Effect test in lung metastasis system (1) Female C57BL / 6 mice aged 6 to 10 weeks (Charles River Japan
4 × 10 ⁵ mouse melanoma B16F10 cells were inoculated into the tail vein to induce lung metastasis. 2 days later TIL
TIL / IL-2 prepared in Example 1 alone or 4 × 10
^Six veins were administered [E / T ratio = 10: E / T represents the number of effector cells (TIL / IL-2) / the number of tumor cells (B16F10)]. At the same time, 5 × 10 ⁵ B16F10 / IL-2 + GM-CSF vaccines prepared in Example 2 were subcutaneously administered. Sixteen days after the induction of lung metastasis, the tumor was dissected and the number of metastatic nodules in the lung was counted. As a comparison
We also tested TIL alone or TIL / IL-2 alone but not vaccinated. The results are shown in Table 2 and FIG.

[0028]

[Table 2]

In the group administered with TIL / IL-2 and B16F10 / IL-2 + GM-CSF vaccine in combination, the number of metastatic nodules was remarkably decreased, and the effect of suppressing cancer metastasis to the lung was maximized. Was confirmed.

[Test Example 2] Effect test on lung metastasis system (2) Female C57BL / 6 mice aged 6 to 10 weeks (Charles River Japan
3 × 10 ⁵ mouse melanoma B16F10 cells were inoculated into the tail vein to induce lung metastasis. 2 days later TIL
TIL / IFN-γ prepared alone or in Example 3 was added to 4.5
× 10 ⁶ veins were administered (E / T ratio = 15). At the same time, 5 × 10 ⁵ B16F10 / GM-CSF vaccines prepared in Example 4 were subcutaneously administered. Sixteen days after the induction of lung metastasis, the tumor was dissected and the number of metastatic nodules in the lung was counted. For comparison, TIL alone, TIL /
Studies were also conducted with IFN-γ administration only without vaccination, or with B16F10 / GM-CSF vaccine administration only. The results are shown in Table 3 and FIG.

[0031]

[Table 3]

In the group administered with TIL / IFN-γ and B16F10 / GM-CSF vaccine in combination, the number of metastatic nodules was remarkably reduced, and it was confirmed that the effect of suppressing cancer metastasis to the lung was maximized. It was

[Test Example 3] Effect test in lung metastasis system (3) Female BALB / C mice of 6 to 10 weeks of age (purchased from Charles River Japan) had 2 × 10 ⁴ mouse melanoma colon cancer colons.
Twenty-six cells were inoculated into the tail vein to induce lung metastasis. Two days later, 1 × 10 ⁶ TIL alone or TIL / IL-2 or TIL / IFN-γ prepared in Example 5 was intravenously administered (E / T rat
io = 50). At the same time, 5 × 10 ^{5 of} Colon 26 / IL-2 vaccine prepared in Example 6 was subcutaneously administered. Eighteen days after the induction of lung metastasis, the tumor was dissected and the number of metastatic nodules in the lung was counted. For comparison, TIL alone, TIL / IFN-γ alone without vaccination, or Colon26 / IL-2 vaccination alone was also tested. The results are shown in Table 4 and FIG.

[0034]

[Table 4]

In the group administered with TIL / IFN-γ and Colon26 / IL-2 vaccine or TIL / IL-2 and Colon26 / IL-2 vaccine in combination, a particularly remarkable decrease in the number of metastatic nodules was observed, and It was confirmed that the cancer metastasis-suppressing effect on A.

[Brief description of drawings]

FIG. 1 shows the construction of an expression cassette used for IL-2 gene transfer.

FIG. 2 shows the construction of a recombinant retrovirus vector used for IL-2 or GM-CSF gene transfer.

[Fig. 3] Cancer metastasis suppressive effect of mouse melanoma (B16F10) -derived effector cells (TIL) derived from cytokine gene (IL-2) and mouse melanoma (B16F10) vaccine into which cytokine gene (IL-2 + GM-CSF) was introduced The result is shown.

FIG. 4 shows the results of the cancer metastasis-suppressing effect of a mouse melanoma (B16F10) -derived effector cells (TIL) derived from a cytokine gene (IFN-γ) -introduced mouse melanoma (B16F10) vaccine. Show.

FIG. 5: Effector cells derived from mouse colon cancer (Colon26) into which a cytokine gene (IL-2 or IFN-γ) has been introduced
The result of the cancer metastasis suppression effect by the mouse colon cancer (Colon26) vaccine in which (TIL) and the cytokine gene (IL-2) were introduced is shown.

Claims (8)

[Claims] 1. A gene therapeutic agent for cancer comprising an effector cell into which a cytokine gene has been introduced and a tumor vaccine into which a cytokine gene has been introduced into a tumor cell. 2. The gene therapeutic agent according to claim 1, wherein the effector cells are lymphocytes involved in the destruction of cancer cells. 3. The gene therapeutic agent according to claim 2, wherein the lymphocyte is a tumor infiltrating lymphocyte (TIL), a lymphokine-activated killer cell (LAK), or a cytotoxic T cell (CTL). 4. The tumor cells are melanoma cells, renal cancer cells,
The gene therapeutic agent according to claim 1, which is a breast cancer cell, squamous cell carcinoma, adenocarcinoma, transitional cell carcinoma, sarcoma, or glioma (glucoma). 5. The gene therapeutic agent according to claim 1, wherein the cytokine gene introduced into the effector cell is an interleukin (IL) -2 gene. 6. The gene therapeutic agent according to claim 1, wherein the cytokine gene introduced into the tumor cells is at least one of granulocyte macrophage colony stimulating factor (GM-CSF) gene and interleukin (IL) -2 gene. . 7. The gene therapeutic agent according to claim 1, wherein the cytokine gene is introduced into effector cells by an adenovirus vector. 8. The gene therapeutic agent according to claim 1, wherein the cytokine gene has been introduced into tumor cells by a retroviral vector.