KR100875198B1 - Lava cell comprising a viral vector and anticancer composition comprising the same - Google Patents

Abstract

The present invention relates to a lava cell comprising a viral vector and an anticancer composition comprising the same. In particular, the present invention provides a lava cell having an anticancer effect specifically located in tumor cells, an anticancer composition comprising the same, and a method for killing tumor cells using the same.

Description

Lava cell comprising a viral vector and anticancer composition comprising the same {CELL TRANSDUCED BY VIRUS VECTOR AND ANTI-CANCER COMPOSITION}

1 is a gene structure diagram of an Ad / E- vector,

FIG. 2 shows intravenous injection of 293adv in rats 30 weeks after NNM injection and changes in HCC nodules every two weeks.

Figure 3 is a picture of histology after 2 weeks of injection of 293adv in HCC mice,

Figure 4 is a 293adv injection through the tail vein and the liver was extracted from 2 weeks old rats and X-Gal stained pictures,

5 is a photograph confirming the histological characteristics of tumor cells after injection of 293adv or PBS + adv into tumor nodules of immunodeficient mice.

Figure 6 is a cell photo stained after injecting 10 ⁶ 293adv in the left lobe of HCC-produced mice, PBS in the right lobe into the tumor.

[TECHNICAL FIELD OF THE INVENTION]                         

The present invention relates to a lava cell comprising a viral vector and an anticancer composition comprising the same, and more particularly, to produce E1 through genetic manipulation of an adenovirus that has lost its self-replicability by deleting E1. The present invention relates to a lava cell prepared by infecting immortalized cells and an anticancer composition capable of treating the lava cells with tumor cells to increase an immune response or specifically remove tumor cells.

[Private Technology]

Gene therapy refers to the treatment of intractable diseases such as cancer and AIDS by introducing missing or completely new genes into cells. Common cancer treatment methods include surgery, chemotherapy, radiotherapy, and immunotherapy, but these procedures not only have side effects, but also have anti-cancer effects, which require the development of new chemotherapy.

Since gene therapy was first performed in ADA (adenosine deaminase) deficient patients in 1990, numerous clinical trials have been conducted in patients with incurable diseases with cancer or AIDS.

Gene therapy used in cancer treatment is largely classified as follows.

A direct attack strategy is to increase the function by suppressing the oncogene or by introducing a tumor suppressor gene. In addition, a method of removing cancer cells through a suicide gene that induces suicide of cancer cells also belongs to this method.

Indirect attack strategy is implemented by inducing immune strengthening. Passive immunotherapy is a method of injecting an immune enhancing gene into cells in vitro and then injecting the cells, and active immunotherapy is a method of directly injecting an immune enhancing gene to enhance immunity.

However, the effect of cancer treatment by these specific genes does not produce satisfactory results at the clinical level. Instead, the most effective anticancer gene therapy at the clinical level is the result of phase II, III by topical administration of adenovirus (ONYX-015) with cancer specific self-replicability. However, ONYX-015 is effective when used in combination with chemotherapy, and the clinical effect when administered alone is relatively small. The clinical effect of topical administration is excellent, but the conclusions about the clinical effect of systemic administration are unknown. In addition, expensive treatment is expected because an overdose of ONYX-015 is used. Structures for the treatment of various solid tumors with structures similar to those of ONYX-015 have been developed and are undergoing preclinical experiments.

There are two methods of transferring genes to a living body: physicochemical methods and biological methods. Physicochemical method It is a method that uses chemicals that can impose an arbitrary impact on the cell membrane of a target cell or bind to the cell membrane. The representative ones are CaPO ₄ , microinjection, electroporation, and liposome. There is this. A biological method is a method of inserting a therapeutic gene into an organism such as a microorganism or a virus and then delivering the therapeutic gene to a target cell. Currently, viruses such as adenoviruses (including ONYX-015), retroviruses, and adeno-associated viruses (AAV) are being studied. This method relies on the local administration of the tumor site, and the cancer cell specificity in the systemic administration is very insignificant and has a limitation of paying high side effects compared to the efficacy.

Accordingly, an object of the present invention is to provide a method capable of specifically killing tumor cells through local or systemic administration of tumor cells.

It is another object of the present invention to provide an anticancer composition capable of specifically killing tumor cells.

It is also an object of the present invention to provide a method for delivering a foreign gene of the present invention in vivo.

In order to achieve the above object, the present invention provides an anticancer composition comprising a lava cell infected with an immortalized cell comprising a viral vector lacking a self-replicating gene.

In another aspect, the present invention is to prepare an anti-cancer composition comprising lava cells by infecting a viral vector lacking a self-replicating gene, the immortalized cells comprising the self-replicating gene and to treat the lava cells to tumor cells It provides a method for killing tumor cells comprising.

The present invention also includes a viral vector lacking a self-replicating gene including a promoter, a foreign gene, a transcriptional regulator, and an immortalized cell comprising a self-replicating gene, wherein the viral vector is infected with an immortal cell. Provide foreign gene carriers.

Hereinafter, the present invention will be described in detail.

The inventors have found that adenosine lacks a self-replicating gene. The lava cells infected with the immortalized cells containing the self-replicating genes were prepared by viral vectors.

The oncolytic cell of the present invention refers to a cell that is infected with a virus and causes anticancer activity after systemic administration. Since the virus is a non-replicating virus that lacks a gene capable of self-replicating, in normal cells, Although proliferation is not possible, proliferation is possible in the immortalized cells used in this experiment. However, as non-transfected replicas of non-fected immunized cells administered in vivo replicated, the immortalized cells are killed or immunologically removed along with cancer cells.

Non-viral replicating viral vectors of the present invention are viral vectors used in conventional gene therapy, including promoters and transcriptional regulators, and further include a number of cloning sites, selectors, and the like, included in conventional viral vectors. In addition, non-viral replicating viral vectors further comprise an expression system capable of expressing further including foreign genes.

The non-viral replicating viral vector of the present invention is preferably selected from the group consisting of adenovirus vectors, retroviral vectors and Adeno-associated viruses, more preferably Ad / E-vectors.                     

The self-replicating gene of the present invention is a gene encoding a protein required for the viral vector to replicate. More specifically, it is a general transcription factor, a factor involved in viral particle formation, and the like, and examples thereof include E1, E2, C, NS2, NS3, NS4A, NS4B, NS5A, NS5B, and the like (Graham, FL (1984) EMBO). J. 3, 2917-2922; McGroy WJ, Bautista, DS, and Graham FL. (1988) Virology 163, 614-617; Hanke, T., Graham, FL, Lulitanond, V., and Johnson, DC (1990) Virology 177, 437-444; Bett, AJ, Prevec, L., and Graham, FL, (1993) J. Virol 67, 5911-5921; Graham, FL, Smiley, J., Russel, WC, and Nairn, R (1977) J. Gen. Virol. 36, 59-72).

Immortalized cells of the invention are for propagation against adenovirus vectors, retroviral vectors or adeno-associated viruses As a host cell, it is an animal cell having semi-permanent proliferative activity under cell culture conditions. Preferably, the visa is an animal cell that enables replication of the viral vector, including a self-replicating gene that is lacking in the replicative viral vector. More preferably human cells or human derived cells, most preferably 293 cells (human embryonic kidney). 293 cells impart self proliferation of E1 deficient viral vectors, including E1. When viral vectors proliferate, 293 cells undergo a lytic pathway and die in the body.

The lava cells of the present invention enable self-replication of viruses by genes related to viral replication contained within the cells, so that cells are lysed 1 to 3 days after infection of the virus vector in vitro, and a plurality of replicated viral vectors Is generated.                     

In another aspect, the present invention provides an anticancer composition comprising the lava cells. The anticancer composition preferably comprises 10 ⁴ to 10 ¹³ lava cells.

The lava cells of the present invention specifically survive around tumor cells when used in an animal body. This means that tumor cells do not induce a response by the immune system Because of the mechanism of causing immune evasion, the environment around the tumor cells is strongly suppressed the immune response, and provides an environment in which non-self cells can survive.

The anticancer composition of the present invention is specifically located in tumor cells to induce the death of tumor cells. In addition, the anticancer composition may further include foreign genes to increase immune responses through expression of foreign genes in or outside tumor cells, or induce death of tumor cells. This can be achieved by inserting a foreign gene into the viral vector during lava cell production. Therefore, the viruses constituting the lava cells include adenoviruses that have lost their self-replicability or adenoviruses containing tumor suppressors, and the like are 293 cells that produce E1.

The anticancer composition of the present invention further comprises a pharmacologically acceptable solvent. The solvent is an excipient or diluent, and is at least one selected from the group consisting of saline, buffered saline, dextrose, water, glycerol, and ethanol. However, without limitation, suitable formulations known in the art are described in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA.

The composition can only be used for injection and can be used for topical administration, preferably for vascular administration, to the tumor site. The maximum possible number of cells for the production of lava cells identified to date during vascular administration is 10 ⁸ and may be higher depending on future studies. The maximum possible number of viruses is from 10 to 100 times the number of cells used. The capacity is not limited to this. The exact dosage may vary depending on the condition of the patient, the type of disease, and the drug being used.

The lava cells of the present invention were injected intravenously into rats that developed hepatocellular carcinoma (hereinafter referred to as "HCC"), resulting in a significant decrease in tumor size, extensive cell death and restoration of normal liver tissue. The aspect was confirmed (FIG. 2). Rat HCC was induced by drinking N-nitrosomorpholine (NNM), a cancer-inducing substance.

Hemoglobin was observed in tumor nodules two weeks after injection of lava cells into HCC mice, and tumor nodules were almost detached from liver due to hemolytic apoptosis of tumor cells. Was confirmed to be attached to normal liver tissue (FIG. 3).

Lava cells were found to be involved in anticancer activity by being specifically located at the contact surface between tumor cells and normal cells or surrounding cells at the apoptosis site of tumor nodules. This was confirmed by X-gal staining after preparing lava cells using a virus containing LacZ and intravenous injection (Fig. 4).                     

   Immunodeficiency mice were treated with the anticancer composition of the present invention to exclude the occurrence of immune responses except lava cells, and then anticancer activity was observed. As a result, a wide range of tumor cell death was confirmed by the lava cells, but progressed slowly (FIG. 5). This is evidence that the intrinsic immune mechanism is involved in the death of tumor cells by the lava cells. In addition, when the lava cells were used, the expression of the inserted foreign gene (LacZ) was higher than that of the virus-only treatment, which indicates that the lava cells of the present invention are specifically located around the tumor cells, and then the lytic response of the lava cells. This is evidence that the virus has replicated by occurrence.

Therefore, the anticancer composition of the present invention is not only useful for the treatment of tumor cells, but the lava cells are specifically located around the tumor cells, and then the cells are killed by the lytic phenomenon. Because it is not infected with normal cells, it is a safe sample because it is removed by an in vivo immune response.

The present invention also includes the steps of (a) infecting the viral vector to an immortalized host cell capable of replicating a replicative viral vector to produce lava cells, and (b) treating the lava cells to tumor cells. It provides a method for killing tumor cells.

 Lava cells of the present invention is prepared by introducing a non-viral virus vector of 10 to 100 MOI into immortalized cells, the introduction method uses intracellular delivery by conventional viral infection.

The method for killing tumor cells may include a method of directly injecting or intravenously injecting tumor cells, but is not limited thereto.                     

In addition, the tumor cell killing method of the present invention further includes a method of preparing a lava cell by inserting a foreign gene into a viral vector to produce a recombinant vector and then infecting the recombinant vector with immortalized cells. The foreign gene is preferably a conventional gene related to the removal or suppression of tumor cells. Examples are cytokines, genes with immunopotentiating activity, apoptosis genes, and the like.

In another aspect, the present invention (a) a non-replicating viral vector comprising a promoter, a foreign gene, a transcriptional regulator, and (b) an immortalized host cell, wherein the non-replicating viral vector is infected with an immortalized host cell It provides a foreign gene carrier in vivo. The foreign gene can be any conventional gene. The gene carrier of the present invention has a specific position around tumor cells and expresses foreign genes. Therefore, it can exhibit anticancer activity by expression of foreign genes.

Hereinafter, examples of the present invention will be described. The following examples are only for illustrating the present invention and the present invention is not limited to the following examples.

Example 1: Lava Cell Preparation

1. Preparation of Adenovirus Vectors with E1 Deletion

pAvCvSv and pJM17 vectors were co-infected and recombined in 293 cells to prepare adenovirus vectors lacking E1, and beta-galactosidase was prepared using a kit of Microbix Biosystem Inc. Ontario, Canada. Recombinant adenovirus vectors expressing and lacking E1 or recombinant adenovirus vectors that do not express beta-galactosidal were prepared (FIG. 1).                     

 2. Preparation of 293 Cells

293 cells were cultured in ATCC (Manassas, VA) and cultured by known methods.

 3. Lava Cell Preparation

10 ⁶ 293 cells inoculated a day ago were treated with 10 ml PBS and 100 multiplicity of infection (2.8 × 10 ⁷ ) recombinant adenovirus. After one hour, 15 ml of the culture medium was mixed, and after 7 hours, the introduction rate was measured by checking the number of adenoviruses in the culture medium. The amount of viral vector in the medium was quantified by HPLC on a 1 ml Resourse Q (Pharmacia Biotech, Piscataway, NJ) AE column. As a result, the remaining adenovirus in the culture medium was 39.4 ± 7.1% and the adenovirus introduced into 293 cells was 56.7 ± 8.5%.

Infected 293 cells were cultured in a culture medium for 10 hours and then treated with X-gal to confirm the infection rate. Infected 293 cells express the beta galactosidase gene inserted into adenovirus and are stained blue by X-gal. As a result of staining by treating 293 cells with X-Gal for 8 hours, it was confirmed that 100% of 293 cells treated with adenovirus were stained and infected.

Infected 293 cells isolated from adenovirus treated for 8 hours and then trypsin-treated into single cells are lava cells. Thereafter, 10 ⁷ lava cells (hereinafter referred to as "293adv") were mixed with 1 ml of saline solution.

Example 2: Anticancer Effect by Lava Cells

(1) Changes in tumor nodule size following lava cell injection

Treatment of N- nitrosomorpholine (NNM) in rats induces hepatocellular carcinoma (hereinafter referred to as "HCC") (Weber, E. et al. Carcinogenesis 1994: 15, 1227-1234; Weber, E. et. al. Carcinogenesis 1988: 9, 1191-1195) and then treated with 293adv to observe the extent of cancer cell treatment.

Sixty-week-old male rats (Spraue-Dawley) were fed with water containing 200 mg / L of NNM (Sigma, St. Louis, MO) for eight weeks, and only water for ten weeks at the end of the experiment. Mice were removed from the entire abdomen and placed in a sternal mount to facilitate tumor observation using an ultrasound transducer, and the size of the short nodules was observed every two weeks after NNM treatment. Ultrasound images were implemented with an Ultramark 9 HDI ultrasound machine (Advanced Technology Laboratories, Inc., Bothell, WA) and an L10-5 MHz transducer.

Mice were tail vein injected with 30 ml of HCC-generated 1 ml physiological saline or control PBS containing 10 ⁶ 293adv 30 weeks after NNM treatment. After the change of liver nodules were observed by ultrasound.

Figure 2 shows the change in HCC nodules every two weeks after intravenous injection of 293adv in mice 30 weeks after NNM injection. a is an ultrasound photograph of one HCC nodule around 1 week after intravenous injection of 293adv, b is a graph showing the change in HCC size after intravenous injection of 293adv or PBS (n = 14, A = 293adv, B = PBS) , c is a hematoxylin-eosin stained picture of liver tissue 10 weeks after 293adv treatment, d is a hematocyllin-eosin stained picture of liver tissue 10 weeks after PBS treatment.

In the case of intravenous injection of 293adv in FIG. 2, the tumor size was significantly decreased, whereas in the PBS treated group, the tumor size was continuously increased (FIG. 2B). In addition, extensive apoptosis was observed in 293adv treated liver tissues by ultrasound (FIG. 2B), and histological staining restored normal liver tissue characteristics (FIG. 2C). HCC was confirmed when the control was treated with PBS (FIG. 2D).

(2) Death pattern of tumor cells following 293adv injection

After injection of 293adv into HCC mice, we observed changes in tumor cells over time. For this purpose, two rats were treated and 2 rats were dissected two weeks later.

Figure 3 is a picture of histology 2 weeks after injection of 293adv in HCC mice. a is a liver photograph, in which hemorrhagic apoptosis was observed in the tumor nodules (FIG. 3A). b, d, f and h are histological images of the tumor nodules and then observed under an optical microscope, and c, e, g and i are identified at high magnification (x40), respectively. In FIG. 3, N represents a site of apoptosis, R represents tumor cells, and H represents normal hepatocytes. As a result of the microscopic observation, it was confirmed that tumor nodules were almost detached from the liver due to hemolytic cell death of tumor cells, and that some tumor cells were attached to normal liver tissue.

Tumor nodules also showed extensive cancer cell death and tumors located in some subcapsular were completely separated from liver tissue due to cell death (FIG. 3B, i and j). However, apoptosis of surrounding normal cells No symptoms or death of non-tumor hepatocytes or hemolysis was observed. On the other hand, bleeding or apoptosis was not observed in the control group injected with PBS.

 (3) tumor cell specific adhesion of 293adv

 293adv containing the beta-galactosidase gene was injected and X-gal staining was performed on the extracted liver tissues to confirm the position of 293adv.

Figure 4 is a 293adv injection through the tail vein and the liver was extracted from 2 weeks old rats and X-Gal stained pictures. a is a hematoxylin-eosin stain for the liver tissue sections, and most tumor nodules showed apoptosis. b is X-gal staining of external cells of tumor nodules surrounding apoptosis, N is a site of apoptosis of tumor nodule, and C is a site expressing beta-galactosidase. In FIG. 4, most of the 293adv was observed in the surrounding cells of the tumor cell-hepatic contact surface or the apoptosis site of the tumor nodule, and was not found in the non-tumorigenic liver tissue or benign cholangiofibromatosis.

Therefore, specific X-Gal staining around tumor cells was inferred that 293adv was specifically attached to tumor cells and 293adv was involved in the death of tumor cells.

Example 3: Confirmation of direct anticancer effect by 293adv

 An experiment was performed to confirm the effect of tumor cell death by 293adv, excluding the anticancer effect by the immune system.

Tumor nodules were induced by extracting small HCC nodules from NNM-treated mice and transplanting liver tumor cells 10 ⁶ into the back of NOD / SCID mice lacking immune function. After 3-4 weeks, when tumor nodules were formed with a diameter of 2-3 cm, 10 ⁶ 293adv or PBS + adv were injected into the tumor nodules, and the cancer volume (0.52 x (width) ² x (length) measured by a clipper at regular intervals) was measured. )) Was calculated.

Both control and 293adv injections showed no significant difference in the volume of tumor nodules. However, the control and X-gal staining for each nodule fragment showed a marked difference.

5 is a photograph confirming the histological appearance of tumor cells after injection of 293adv or PBS + adv into tumor nodules of immunodeficient mice. a is 2 weeks of tumor cells after 293adv injection, b is 4 weeks of tumor cells, c is 4 weeks of tumor cells after PBS + adv injection, and d is 8 weeks of tumor cells. e is a graph measuring the portion of the tumor cells stained with X-gal and vaccinated by vaccinating after injection of 293adv or PBS + adv. In the case of 293adv, most tumor cells were stained with X-gal at 8 weeks compared to 4 weeks after injection, and more than about 70% of the cells were killed to confirm the empty space (FIGS. 5A and B). In contrast, the injection of adv vector only reduced the X-gal stained area after 8 weeks. This indicates that the E1 deficiency adv replicates at 293adv, and the empty space means that oncolytic was generated by 293adv.

On the other hand, 8 weeks after 293adv injection, 293adv was observed in some liver tumor nodules in contact with skin cells of rats after extensive liquefied cell death.                     

Thus, 293adv was found to induce apoptosis by directly acting on tumor cells even in the absence of immune responses in vivo.

Example 4: Confirmation of immune enhancing effect of 293adv

When 293adv was administered intravenously in Example 2, it was confirmed whether tumor cell death was caused by immune systemic factors.

HCC-producing mice were opened and 10 ⁶ 293adv was injected into the left lobe and PBS into the right lobe tumor. Twenty-four weeks after the injection, the tumor cells of natural dead mice were observed. Figure 6 is a cell photo stained after injecting 10 ⁶ 293adv in the left lobe of HCC-produced mice, PBS in the right lobe into the tumor. a is a photograph showing the left and right lobes of the rat abdomen, and the overall difference was clearly seen. The right lobe injected with 293adv was very similar to the normal liver, but the left lobe was similar to the typical HCC. Hematoclin & eosin staining results were the same (Fig. 6b and c).

As mentioned above, the lava cells of the present invention are specifically located in tumor cells to induce apoptosis of tumor cells as well as express exogenous genes in vivo and exhibit excellent effects in gene therapy.

Claims (11)

A viral vector lacking an autologous gene comprises lava cells infected with an immortalized cell comprising said autologous gene, The self-replicating gene is any one selected from the group consisting of E1, E2, C, NS2, NS3, NS4A, NS4B, NS5A and NS5B, The viral vector is any one selected from the group consisting of adenovirus vectors, retrovirus vectors and adeno-associated viruses, The immortalized cells are host cells of any one of the viral vectors selected from the group consisting of adenovirus vectors, retroviral vectors and adeno-associated viruses and are non-self cells. Anticancer composition. delete delete delete The anticancer composition according to claim 1, wherein the immortalized cells are animal-derived cells. The anticancer composition according to claim 1, wherein the immortalized cells are human derived cells. The anticancer composition of claim 1, wherein the immortalized cells are 293 cells. The anticancer composition of claim 1, wherein the viral vector further comprises a foreign gene. The anticancer composition of claim 1, wherein the anticancer composition comprises 10 ⁴ to 10 ¹³ lava cells. delete (a) a viral vector lacking a self-replicating gene comprising a promoter, a foreign gene, and a transcriptional regulator, and (b) an immortalized cell comprising the self-replicating gene, wherein the viral vector is directed to an immortalized cell. Infected, The self-replicating gene is any one selected from the group consisting of E1, E2, C, NS2, NS3, NS4A, NS4B, NS5A and NS5B, The viral vector is any one selected from the group consisting of adenovirus vectors, retrovirus vectors and adeno-associated viruses, The immortalized cells are host cells of any one of the viral vectors selected from the group consisting of adenovirus vectors, retroviral vectors and adeno-associated viruses and are non-self cells. Foreign gene carriers.

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