WO1996007433A1

WO1996007433A1 - Gene therapy drug for cancer, medicinal composition, and therapeutic method

Info

Publication number: WO1996007433A1
Application number: PCT/JP1995/001785
Authority: WO
Inventors: Hirofumi Hamada; Haruo Sugano
Original assignee: Japanese Foundation For Cancer Research
Priority date: 1994-09-09
Filing date: 1995-09-08
Publication date: 1996-03-14
Also published as: JP3822261B2; JPH08127542A

Abstract

A gene therapy drug for cancer containing effector cells having cytokine genes introduced thereinto and tumor vaccines comprising tumor cells having cytokine genes introduced thereinto; a method of gene therapy for cancers by administering a pharmaceutical preparation containing the effector cells and a pharmaceutical preparation containing the tumor vaccines to a patient with cancer; and a medicinal composition containing a combination of the effector cells and the tumor vaccines. The drug, composition and method produce a high antitumor effect, have the effect of inhibiting cancer metastasis, and are useful for treating micrometastatic cancer.

Description

Specification

TECHNICAL FIELD The present invention relates to a gene therapy agent and a pharmaceutical composition for cancer, and a treatment method.

The present invention relates to a gene therapy agent and a pharmaceutical composition for cancer, and a method for treating cancer. More specifically, the present invention relates to a gene therapy agent for cancer, comprising an effector cell into which a cytokin gene has been introduced and a tumor vaccine having a cytokine gene introduced into a tumor cell; a preparation comprising the effector cell A gene therapy method for cancer characterized by administering a preparation containing the tumor vaccine to a cancer patient; and a pharmaceutical composition comprising a combination of the effector cell and the tumor vaccine. Cytokines exhibit antitumor effects, but are roughly classified into the following three types according to their mechanism of action. That is, ① tumor necrosis factor (TNF) _a, β interferon (IFN) -α, β, α, interleukin (ID-1, etc.) directly inhibits growth or damages cancer cells in vitro in vitro (2) Interleukins (such as I-2, 4, 6, 7, 8, 9, 10, 11, 12) which have an antitumor effect indirectly through a mechanism of immune effectors in the living body , ③ IL-3, I-6, granulocyte macrophage colony stimulating factor (GM-CSF :), granulocyte colony stimulating factor (G-CSF), stem cell factor (SCF) It has been used in the prevention and treatment of side effects in living organisms, and in recent years, cancer treatment by introducing cytokine genes has been attempted, one of which is to transfer cytokine genes to lymphocytes such as lymphokines. Activated killer cells

(LAK), cytotoxic T cells (CTL), tumor-infiltrating lymphocytes (TIL), etc., and passive immunization by activating lymphocytes with autocrine or paracrine by cytokines secreted from the transfected cells Attempts have been made to enhance the antitumor activity at the tumor site by enhancing the activity of T1L or by directly introducing the antitumor 1FN or TNF gene into T1L (Nishihara et al., Cancer Res. , 48, 4730, 1988, iyatake et al., J. Natl. Cancer Inst., 82, 217, 1990, Itoh et al., Jpn. J. Cancer Res., 82, 1203, 1991). Retroviruses have been used to introduce cytokine genes into TILs, but the introduction efficiency is very poor. Is an obstacle.

The other is to introduce cytokine genes into tumor cells using a retrovirus vector or the like and use them as a tumor vaccine to induce tumor-specific immune cells in the host. For example, Fearon et al. Transplanted a mouse colon cancer or malignant melanoma transfected with the 1L-2 gene into a syngeneic mouse and then spontaneously regressed after engraftment. The authors have reported that they acquire immune competence that is resistant to the disease (Fear on et al., Cell, 60, 397, 1990).

Neither of these methods can provide satisfactory results for antitumor properties, nor have they been studied in terms of their metastatic effect. Nothing promising has been reported at this time. Disclosure of the invention

Accordingly, an object of the present invention is to provide a gene therapy agent for cancer that can exhibit higher antitumor activity and is also effective in suppressing cancer metastasis in order to solve the above-mentioned problems. Oh O ο

The present inventors have conducted intensive studies on the above-mentioned problems, and as a result, it was found that the combined use of a cytokine gene-introduced effector cell and a tumor vaccine in which a cytokine gene was introduced into a tumor cell resulted in efficient systemic immunity. Were found to enhance the antitumor effect and the metastasis suppressing effect, and completed the present invention.

That is, the present invention provides an agent for cancer gene therapy comprising an effector cell into which a cytokine gene has been introduced and a tumor pectin having a cytokinin gene introduced into a tumor cell. "

The present invention also relates to the simultaneous or sequential administration of a preparation containing a cytokine gene into which an effector cell has been introduced and a preparation containing a cytokine vaccine into which a cytokine gene has been introduced into a fistula cell to a cancer patient. To provide a gene therapy method for cancer, characterized in that

Further, the present invention relates to a pharmaceutical composition used for gene therapy of cancer, wherein the composition is a combination of an effector cell into which a cytokine gene has been introduced and a tumor vaccine having a cytokine gene introduced into a tumor cell. And a pharmaceutically acceptable carrier A pharmaceutical composition comprising:

The term “gene therapy for cancer” as used in the present invention intends to treat cancer from both the antitumor effect and the metastasis inhibitory effect of cytokine genes. Hereinafter, the present invention will be described in detail.

The cytokine gene used in the present invention includes granulocyte macrophage colony stimulating factor (GM-CSF), interleukin (1-2, IL-3, IL_4, IL-6, IL-7, IL) -10, IL-12, IL-13, IL-15, interleukin-la (IL-1), interleukin receptor 1 antagonist (1 ぃ 1RA), tumor necrosis factor (TNF) Lymphotoxin (LT) -15, granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), interferon (IFN) -α, macrophage migration inhibitory factor (MIF), leukemia inhibitory factor ( LIF), a gene encoding various cytokines such as T cell activation co-stimulator B7 (CD80) and B7-2 (CD86), kit 'ligand, oncostatin M, etc. Already various cytokines cDNA has been cloned [for human GM-CSF cDNA, see Wong et al., Science, 2 28, 810-815 (1985), Taniguchi et al., Nature, 302, 305-310 (1983) for human IL-2 cDNA, and Gray et al., Nature, 298, 859 for human IFN-r cDNA. -863 (1982)] The cytokine gene used in the present invention may be a cDNA isolated from cells using a known technique, or may be disclosed in the above-mentioned literature and the like. Although it may be chemically synthesized according to methods such as the polymerase chain reaction (PCR) based on information, humans may be used to minimize immune rejection and to increase the therapeutic effect. The origin is desirable.

In the present invention, the effector cell refers to a cell population that is in charge of the final stage of destruction of a cancer cell and is directly involved in the destruction, and specifically, a tumor infiltrating lymphocyte (TIL), a lymphokine-activated killer cell ( LAK), cytotoxic T cells (CTL) and the like.

The introduction of the cytokine gene into these effector cells can be performed very efficiently by using an adenovirus vector. As the adenovirus vector used here, any vector containing an insertion site for the cytokine gene and capable of expressing the cytokine in the introduced effector cell can be used. Although not particularly limited, Adexl derived from human type 5 adenovirus [Sai to, 1. et al., J. Viol., 54, 711-719 (1985)] is preferably used.

On the other hand, the term "tumor vaccine" as used in the present invention refers to the above-mentioned cytokine gene, which is isolated (cultured) by a retrovirus vector, introduced into tumor cells, and irradiated with X-rays. Production was stopped without inhibiting production. By administering this tumor pectin to the host, tumor-specific immune cells of the host can be induced.

The retroviral vector used here is not particularly limited as long as it contains an insertion site for a cytokine gene and can express cytokines in the introduced tumor cells. MFG, α-SCG, PLJ, pEm and the like disclosed in JP-A-6-503968. The retrovirus vector inserted with the site force gene transfer was mixed with a plasmid having a neomycin resistance gene as a marker to select that the desired gene was introduced, and Ψ2, Ψ- Am, CRIP, WCRE [Danos et al., PNAS, 85, 6460-6464 (1988)], etc., are introduced by a calcium coprecipitation method (cotransfection). Further, this is cultured in the presence of the drug G418, and by collecting cells that survive and form a colony, only cells into which the target gene has been introduced can be collected. Next, the culture supernatant of these cells was used to infect various types of fistula cells, such as N1H3T3 mouse fibroblasts and B16 mouse melanoma cells. It can be confirmed by hybridization. The amount of cytokines secreted from infected cells can be measured by immunological assays such as ELISA.

Next, the tumor cells into which the cytokine II gene has been introduced are usually irradiated with X-rays at a concentration of 0.0000 to 150,000 rad, and then used as a tumor vaccine.

The tumor cells here 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, etc. Can be used.

The cytokine cytokine-introduced effector cells prepared as described above and the tumor vaccine can be used as they are, but they must be combined with a pharmaceutically acceptable carrier. In addition, the composition can be administered as a pharmaceutical composition in the form of a solid such as tablets, powders, granules, and pills, or in the form of a solution, suspension, gel, or the like.

Examples of the carrier include excipients or diluents such as a filler, a bulking agent, a binder, a disintegrant, and a surfactant, which are commonly used for preparing a preparation according to a use form. The administration forms of the gene therapy agent include the usual intravenous, intraarterial, subcutaneous, etc. systemic administration, local injection into the tumor lesion, or into the expected metastatic site corresponding to the cancer type. Local administration such as oral administration can be performed. Further, the administration of the gene therapy agent of the present invention may be in the form of administration in combination with catheter technology, gene transfer technology, surgical operation, or the like.

In addition, as an administration method, the cytokine hepatocyte guide effector one cell and the tumor pectin prepared as described above may be simultaneously administered, or the tumor vaccine may be administered after the effector one cell is first administered. The tumor vaccine may be administered first and then sequentially, or the effector cells may be administered sequentially and subsequently.

The dosage of the gene therapy agent of the present invention varies depending on the age, sex, symptom, administration route, number of administrations, and dosage form. In general, in adults, about 0.1 to 100% by weight of cytokine gene per day is used. A range of 100 mg is appropriate. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the construction of an expression cassette to be used for the introduction of the gene.

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

Figure 3 shows the effector cell (TIL) derived from mouse melanoma (B16F10) transfected with the cytokine gene (I2) and the murine melanoma transfected with the cytokine gene (I2 + GM-CSF). The results of the cancer metastasis inhibitory effect of Kuma (B16F10) Pectin are shown.

Figure 4 shows mouse melanoma (B16F10) -derived effector cells (TIL) transfected with the cytokine gene (IFN-a) and mouse melanoma cells transfected with the cytokine gene (GM-CSF). (B16F10) shows the results of the cancer metastasis inhibitory effect of Pectin. Fig. 5 shows the effector cells (TIL) derived from mouse colon cancer (Colon26) transfected with the site force gene (I-2 or IFN-a) and the site force gene (I-2). The results of mouse metastatic cancer (Colon 26) inhibitory effect on cancer metastasis by pectin are shown. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described specifically with reference to examples. The scope of the present invention is not limited by these examples.

(Reference Example 1)

The cDNA of mouse 1-2, mouse GM-CSF, and mouse IFN-α were obtained by RT-PCR (Reverse transcripi ton-pol ymera.se chain react ion) using mRNA of mouse spleen lymphocyte. Prepared. In performing PCR, primers # 479 and # 480 shown below for mouse 1L-2, primers # 477 and # 478 shown below for mouse GM-CSF, and mouse IFN- Used primers # 485 and # 486 shown below, respectively. Primer (# 479): CCGAATTCTAGACACC ATG TAC AGC ATG CAG CTC GCA TCC TG T G

Primer (# 480): C TGT CAA AGC ATC ATC TCA ACA AGC CCT CAA TAA GGATC C CC

Primer (# 477): CCGAATTCTAGACACC ATG TGG CTG CAG AAT TTA CTT TTC CT G GGC

Primer (# 478): C CCC TTT GAA TGC AAA AAA CCA AGC CAA AAA TGA GGATC C GG

Primer (# 485): CC GAA TTC TAGA CACC ATG AAC GCT ACA CAC TGC ATC TT G GC

Primer (# 486): C AGG AAG CGG AAA AGG AGT CGC TGC TGA GGATCCGG

[Example 1] Preparation of site-transduced effector cells (TI L / I2) (1) Preparation of mouse melanoma B16F10-derived T1L

The preparation of T1L is described in Alexander, RB et al., J. Immunol., 145, 1615-1620 (199 0), Mat is, LA et al., Methods Enzymol., 150, 342-351 (1987), Livingst one, A. et al., Methods Enzymol., 150, 325-333 (1987) Was performed as follows. Fresh B16F10 (obtained from Whitehead Institute Dr. Glenn Dranoff), a highly metastatic strain of mouse melanoma B16 (ATCC CRL6322) transplanted into female C57BL / 6 mice (purchased from Charles River Japan) of 6 to 10 weeks of age 5 x 10 ⁷ cells / ml were suspended in complete culture medium (CM) at 4 ° C. CM was heat-inactivated 10¾ fetal serum, 2 mM L-glutamine, 5 × 10 " ⁵ M 2-mercaptoethanol, 100 U / ml penicillin, 100 g / ml streptomycin, 0.5 ^ g / ml RPMI 1640 with addition of mlhotericin B, lOmM 3- (N-morpholino) propanesulfonic acid, and 70 U / ml recombinant human IL-2 (obtained from Shionogi & Co., Ltd.) TIL, etc. Volume of anti-CD-8-conjugated immunoadsorbent beads at 1 × 10 ⁸ / ml and incubated for 2 hours at 4 ° C. Beads with T1L were pelleted and washed three times with cold CM and, suspended 1 xi0 ⁷ beads / ml in CM, were seeded in tissue culture play Bok 24 Uweru, 37 ° C, and Peretz collected by reduction Inkyubeto was. after one day incubation, the beads separated from TIL under 5¾C0 _2, removed Separated TILs were irradiated at 2 x 10 ⁵ cells per well (10,000 rad) tumor cells, and 1 x 10 ⁶ irradiations per well (3,000 rad) Stimulated with normal splenocytes In vitro stimulation was repeated every 14 days from 7 days When confluence, TIL was collected and added to a new CM in 2 × 10 ⁵ cells. The cells / ml were resuspended.

(2) Adenovirus transduction of mouse IL-2 gene into TIL

The method for preparing the recombinant adenovirus was carried out by a modification of Saito. I. et al., J. Viol., 54, 711-719 (1985). That is, an expression unit consisting of a cytomegalovirus enhancer, a chicken / S-actin promoter, a mouse 1L-2 cDNA sequence prepared in Reference Example 1, and a rabbit-1 yS-globin poly (A) signal sequence. [Niwa, H. et al., J. Gene 108, 193-200 (1991)] were converted to pAdexlvv, a 42 kb cosmid containing a 31 kb adenovirus type 5 lacking the E1A, E1B, and E3 遗 genes. Hiromi Kae, Shizuko Harada, Izumi Saito, Experimental Medicine Biomanual 4, 189-204, 1994; Yodosha) constructed a cosmid cassette for expression (Fig. 1). The cosmid cassette for expression and adenovirus DNA-terminal protein complex (DNA-TPC) were co-precipitated into 293 cells (ATCC CRL1573). Co-transfected by The recombinant virus containing the expression cassette was confirmed by digestion with an appropriate restriction enzyme. The recombinant virus was subsequently propagated on 293 cells, and the virus solution was stored at -80 ° C. Virus stock titers were determined by plaque assay on 293 cells. For in vitro infection of the adenovirus to the TIL prepared in (1), the culture was removed from TIL cells seeded on a 12-well culture plate, and 150 μl of the virus stock was added to each mouse. After incubation at 37 ° C for 1 hour, a growth medium was added, and TIL cells were cultured for 2 to 3 days, to obtain mouse-transduced T1L cells (TIL / -2).

[Example 2] Preparation of tumor vaccine (B16F10 / IL-2 + GM-CSF vaccine)

(1) Preparation of recombinant retrovirus-producing clone of mouse IL-2 and mouse GM-CSF

Retroviral vector MF G (Dranoff, G. et al., Proc. Natl. Acad. Sci. USA, 90, 3539-3543, 1993) Eagl / B amHI fragment containing two LTRs (Long Terminal Repeat) (5200bp :), Eagl / Xbal fragment (10OObp :), and Reference Examples

Three of the Xbal / BamHI fragments of each of the mouse I and mouse GM-CSF cDNAs prepared in 1 were ligated with T4 DNA ligase and incorporated into plasmid (Fig. 2). The obtained plasmid and pPGKneo [H. Takeshima et al., Nature, 369, 556-559 (1994;)] were co-precipitated with WCRIPCDanos et al., Proc. Natl. Acad. Sci. USA, 85 , 6460 (1988)] in a medium containing G-418 (GIBC0, lmg / ml).

After culturing for one week, cells forming colonies were collected. Next, culture supernatants of these cells were infected to NIH3T3 mouse fibroblasts (ATCC CRL 1658) in the presence of polybrene (Sigma, 8 g / ml). Genomic DNA from infected cells was isolated and virus titer was determined by Southern blotting and evaluated as the copy number of the integrated provirus. The transfection efficiency into N1H3T3 was usually 1-3 copies per cell with provirus integration. Cytokines secreted from infected cells were identified by ELISA (Endogen) 48 hours after seeding 1 × 10 ⁶ cells in a 10-cm dish containing 10 ml of medium (Table 1). 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) Preparation of tumor vaccine (B16F10 / IL-2 + GM-SCF vaccine)

The high titer recombinant retrovirus-producing clones of mouse 1 and mouse GM-CSF selected in (1) were cloned into B16F10 (Whitehead Institute Dr. Glenn Dranof), a highly transgenic strain of mouse melanoma B16 (ATCC CRL 6322). obtained from f). The transfected cells were maintained in Dulbecco's Eagle's medium supplemented with 10% fetal serum and 2 mM glutamine, treated with trypsin / EDTA, and treated with 10,000 rad using a HITACHI MBR-1505R X-ray generator. X-rays were irradiated. Irradiated cells were washed twice with HBSS (Hank's Balanced Salt Solution), resuspended in HBSS at a concentration of 5 xiO ⁶ cells / ml, and immunized with tumor vaccine (B16F10 / IL-2 + GM-CSF vaccine). Obtained.

[Example 3] Preparation of cytokine II gene transfer effector cells (TIL / IFN-a)

(1) Preparation of TIL derived from mouse melanoma B16F10

Prepared in the same manner as in Example 1 (1).

(2) Introduction of mouse IFN-7 gene into T1L by adenovirus

The method for preparing the recombinant adenovirus was performed according to a modification of Saito. I. et al., J. Viol., 54, 711-719 (1985). That is, an expression unit consisting of a cytomegalovirus enhancer, a chicken yS-actin promoter, a mouse IFN-a cDNA sequence prepared in Reference Example 1, and a rabbit-β-globin poly (A) signal sequence [ Niwa, H. et a, J. Gene 108, 193-200 (1991)], and pAdexlcw, a 42 kb cosmid containing 31 kb adenovirus type 5 lacking the E1A, BIB, and E3 遗 genes (Yumi Kanegae, Shizuko Harada, Izumi Saito, Biomedical Manual for Experimental Medicine 4, 189-204, 19 94, Yodosha) to construct a cosmid cassette for expression by inserting it into the Clal restriction enzyme site (Fig. 1). This cosmid cassette for expression and adenovirus DNA-terminal protein complex (DNA-TPC) were co-transfected into 293 cells (ATCC CRL1573) by a calcium coprecipitation method. The recombinant virus containing the expression cassette was confirmed by digestion with an appropriate restriction enzyme. The recombinant virus was subsequently grown on 293 cells and the virus solution was stored at -80 ° C. The titer of the virus stock was determined by plaque assay on 293 cells. For in vitro infection of the adenovirus with the TIL prepared in (1), the culture solution was removed from T1L cells seeded on a 12-well culture plate, and 150 μl of the virus stock was added to each well. After incubating at 37 ° C for 1 hour, a growth medium was added, and TIL cells were cultured for 2 to 3 days to obtain mouse 1FN-7 transgenic T1L cells (T1 / IFN-7).

[Example 4] Preparation of tumor vaccine (B16F10 / GM-CSF vaccine)

(1) Preparation of mouse GM-CSF high titer recombinant retrovirus-producing clone Prepared in the same manner 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 introduced into B16F10 (available from Whitehead Institute Dr. Glenn Dranoff), which is a highly metastatic strain of mouse melanoma B16 (ATCC CRL 6322). The transfected cells were maintained in Dulbecco's Eagle's medium supplemented with 10X fetal serum and 2 mM glutamine, treated with tribsine / EDTA, and treated with X-ray of 10, OOOrad using a HITACHI MBR-1505R X-ray generator. The line was irradiated. Irradiated cells were washed twice with HBSS and resuspended in HBSS at a concentration of 5 xiO ⁶ cells / ml to obtain a tumor vaccine (B16F10 / GM-CSF vaccine).

[Example 5] Preparation of cytokine gene transfer effector cell (TIL / I-2, TIL / IFN-7)

(1) Preparation of TIL from mouse colorectal cancer Colon 26

TIL was prepared according to Alexander, RB et al., J. Immunol., 145, 1615-1620 (1990), Matis, A. et al., Methods Bnzymol., 150, 342-351 (1987), Livingst lo One, A. et al., Methods Enzymol., 150, 325-333 (1987) was modified as described below. A 6-10 week old female BALB / C mouse (purchased from Charles River Japan) was transplanted with a fresh tumor mass of colon cancer Colon 26 at 4 ° C in a complete culture medium (CM) at 5 × 10 ⁷ cells / cell. ml was suspended. 10¾ © Shi calf serum CM is obtained by heat-inactivated, 2mM have glutamicum emissions, ⁵ X10- 5 M 2- mercaptoethanol, lOOU / ml Bae Nishiri down, 100 g / ml scan Torre but-mycin, 0.5 g / ml Anhoteri Shin B, RPMI 1640 supplemented with 10 mM 3- (N-morpholino) propanesulfonic acid and 70 U / ml recombinant human I2 (obtained from Shionogi & Co., Ltd.). TIL was mixed with an equal volume of anti-CD-8-conjugated immunosorbent beads at 1 × 10 ⁸ / ml and incubated at 4 ° C. for 2 hours.

Beads TIL is attached is Perez toy spoon, washed 3 times with cold CM, suspended 1 xl0 ⁷ b eads / ml in CM, were seeded in tissue culture plates in 24 Uweru, 37 ° C, 5¾C0 ₂ below Incubated. One day after culture: The beads separated from TIL were removed by pelleting. The isolated TILs were stimulated with 2 x 10 ⁵ irradiated (10,000 rad) tumor cells per well and 1 x 10 ⁶ irradiated (3, OOOrad) normal spleen cells per well. In vitro stimulation was repeated every 7 to 14 days. Sample was collected TIL when it is Konfuruen bets and resuspended 2 xi0 ⁵ cell / ml in fresh CM.

(2) Adenovirus-mediated transduction of mouse I2 gene into TIL

In the same manner as in Example 1 (2), the mouse I-2 cDNA prepared in Reference Example 1 was introduced into the TIL prepared in (1) above, and the mouse IL-2 gene-introduced TIL cells (TIL / I-2) I got

(3) Introduction of mouse IFN-A gene into TIL by adenovirus

Example 3 The mouse IFN-7 CDNA prepared in Reference Example 1 was introduced into T1L prepared in (1) by the same method as in (2), and the mouse IFN-7 gene-transfected TIL cells (TIL / 1FN-α ) Got.

[Example 6] Preparation of tumor vaccine (Colon26 / IL-2 vaccine)

(1) Preparation of mouse IL-2 high titer recombinant retrovirus producing clone

It was prepared in the same manner 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 to colon 26 colon cancer. The transfected cells were maintained in RPMI1640 medium supplemented with 10% fetal serum and 2 mM glutamine, and irradiated with 10,000 rad X-rays using a HITACHI MBR-1505R X-ray genera tor. Irradiated cells were washed twice with HBSS and resuspended in HBSS at a concentration of 5 x 10 ⁶ cells / ml to obtain a tumor vaccine (Colon26 / IL-2 vaccine).

[Test Example 1] Effect test in lung metastasis system (1)

Six to ten week old female C57BL / 6 mice (purchased from Charles River Japan) were inoculated with 4 × 10 ⁵ mouse melanoma B16F10 cells into the tail vein to induce lung metastasis. Two days later, TIL alone or TIL / IL-2 prepared in Example 1 was administered through ⁶ veins of 4 xlO [E / T ratio-10: E / T was a single cell (TIL / I 2 ) Number / tumor cell (B16F10) number]. At the same time, 5 × 10 ⁵ B16F10 / IL-2 + GM-CSF vaccines prepared in Example 2 were subcutaneously administered. Dissection was performed 16 days after the lung metastasis was induced, and the number of metastatic nodules in the lung was counted. For comparison, TIL alone or TIL / IL-2 alone and no vaccine were also tested. The results are shown in Table 2 and FIG.

Table 2

In the group treated with TIL / IL-2 and B16F10 / I2 + GM-CSF Pectin in combination, a particularly remarkable decrease in the number of metastatic nodules was observed, confirming that the effect of suppressing lung cancer metastasis was maximized. did it.

[Test Example 2] Effect test in lung metastasis system (2)

Six to 10-week-old female C57BL / 6 mice (purchased from Charles River Japan) were inoculated with 3 xiO ⁵ mouse melanoma B16F10 cells into the tail vein to induce lung metastasis. Two days later, TIL alone or 4.5 × 10 ⁶ TIL / IFN-7 prepared in Example 3 were intravenously administered (E / T ratio = 15) ₀ Simultaneously, B16F10 / GM- prepared in Example 4 ⁵ × 10 ⁵ CSF peptides were subcutaneously administered. Dissection was performed 16 days after the induction of lung metastasis, and the number of metastatic nodules generated in the lung was counted. For comparison, TIL alone, TIL / IFN-α alone, no vaccine, or B16F10 / GM-CSF vaccine alone were also tested. The results are shown in Table 3 and FIG.

Table 3

In the group administered with T1L / IFN-r and B16F10 / GM-CSF Pectin in combination, a particularly remarkable decrease in the number of metastatic nodules was observed, confirming that the effect of suppressing lung cancer metastasis was maximized.

[Test Example 3] Effect test in lung metastasis system (3)

Six to ten-week-old female BALB / C mice (purchased from Charles River Japan) were inoculated into the tail vein with 2 × 10 ⁴ mouse melanoma colon cancer Colon 26 cells to induce lung metastasis. After that, TIL alone or TIL / 1L-2 or TML / IFN-α prepared in Example 5 was intravenously administered at 1 × 10 ⁶ (E / T ratio = 50). At the same time, ⁵ × 10 ⁵ pieces of the Colon 26 / IL-2 vaccine prepared in Example 6 were subcutaneously administered. Dissection was performed 18 days after lung metastasis was induced, and the number of metastatic nodules generated in the lung was counted. For comparison, T1L alone, TIL / IFN-α alone and no vaccine administration, or Colon26 / I or 2 vaccine administration alone were also tested. The results are shown in Table 4 and FIG. Table 4

The group treated with TI L / IFN-α and Colon26 / IL-2 vaccine, or TlL / -2 and Colon26 / IL-2 vaccine, showed a particularly remarkable decrease in the number of metastatic nodules. It was confirmed that the effect of inhibiting cancer metastasis to HT was maximized. Industrial applicability

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to exhibit high antitumor activity on animals such as humans, mice, monkeys, dogs, cats, horses, pigs, etc. Useful cancer gene therapy agents and pharmaceutical compositions, and therapeutic methods are provided.

Claims

The scope of the claims

1. A gene therapy agent for cancer containing an effector cell into which a cytokine gene has been introduced and a tumor actin into which a cytokine gene has been introduced into tumor cells.

2. The gene therapy agent according to claim 1, wherein the effector cell is a lymphocyte involved in cancer cell destruction.

3. The gene therapy 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 method according to claim 1, wherein the tumor cell is a melanoma cell, a cancer cell, a breast cancer cell, a squamous cell carcinoma, an adenocarcinoma, a transitional cell carcinoma, a sarcoma, or a glioma (Darcoma). Gene therapy.

5. The gene therapy agent according to claim 1, wherein the cytokine gene introduced into the effector cell is an interleukin (1L) -2 gene.

6. The cytokine gene to be introduced into a tumor cell is at least one of a granulocyte macrophage colony stimulating factor (GM-CSF) gene and an interleukin (1L) 12 gene. The gene therapy agent according to the above.

7. The gene therapy agent according to claim 1, wherein the cytokine gene has been introduced into the effector cell 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.

9. Simultaneous or sequential administration of a preparation containing an effector cell into which a cytokine gene has been introduced and a preparation containing a tumor vaccine into which a cytokine gene has been introduced into a tumor cell to a cancer patient. A method for gene therapy for cancer, which comprises:

10. The method according to claim 9, wherein the effector cell is a lymphocyte involved in destruction of a cancer cell.

11. The gene therapy according to claim 10, wherein the lymphocyte is a tumor-infiltrating lymphocyte (TI), a lymphokine-activated killer cell (LAK), or a cytotoxic T cell (CT). Method.

12. The tumor cells are melanoma cells, 肾 cancer cells, breast cancer cells, squamous cell carcinoma, adenocarcinoma, metastasis The gene therapy method according to claim 9, wherein the method is epithelial cancer, sarcoma, or glioma (Darcoma).

13. The gene therapy method according to claim 9, wherein the site force gene to be introduced into the effector cell is the interleukin (IL) -2 gene.

14. The method according to claim 9, wherein the site force gene to be introduced into the tumor cell is at least one of a granulocyte macrophage colony stimulating factor (GM-CSF) gene and an interleukin () -2 gene. The method for gene therapy according to the above.

15. The gene therapy method according to claim 9, wherein the cytokine gene is introduced into the effector cells by an adenovirus vector.

16. The gene therapy method according to claim 9, wherein the cytokine gene has been introduced into tumor cells by a retroviral vector.

17. A pharmaceutical composition for use in gene therapy for cancer, wherein the composition comprises an effector cell into which a cytokine gene has been introduced and a tumor vaccine comprising a cytodynamic gene introduced into a tumor cell. And a pharmaceutically acceptable carrier.

18. The pharmaceutical composition according to claim 17, wherein the effector cells are lymphocytes involved in cancer cell destruction.

19. The pharmaceutical composition according to claim 18, wherein the lymphocyte is a tumor-infiltrating lymphocyte (TIL), a lymphokine-activated killer cell (LAK), or a cytotoxic T cell (CTL).

20. The medicament according to claim 6, wherein the tumor cell is a melanoma cell, a renal cancer cell, a breast cancer cell, a squamous cell carcinoma, an adenocarcinoma, a transitional cell carcinoma, a sarcoma, or a glioma (Darcoma). Composition.

21. The pharmaceutical composition according to claim 17, wherein the cytokine gene to be introduced into the effector cell is the interleukin (IL) 12 gene.

22. The pharmaceutical according to claim 2, wherein the cytokine gene to be introduced into the tumor cell is at least one of a granulocyte macrophage colony stimulating factor (GM-CSF) gene and an interleukin () -12 gene. Composition.

23. Cytokine genes are transferred to effector cells by adenovirus vectors 18. The pharmaceutical composition according to claim 17, wherein said pharmaceutical composition is contained.

24. The pharmaceutical composition according to claim 17, wherein the cytokine gene has been introduced into tumor cells by a retroviral vector.