阅读说明：本技术 安全性和抗癌作用得到改善的溶瘤病毒 (Oncolytic virus with improved safety and anti-cancer effect ) 是由 黄泰皓 赵懞 于 2018-12-28 设计创作，主要内容包括：本发明涉及安全性和抗癌作用有所改善的溶瘤病毒及其用途。通过将编码HSV-TK片段的基因插入TK基因区域以缺失牛痘病毒的TK,可以获得本发明的安全性和抗癌作用得到改善的溶瘤病毒。另外,本发明的溶瘤病毒表达HSV-TK片段以使GCV磷酸化,从而可以杀死被溶瘤病毒感染的癌细胞,甚至杀死它们附近的癌细胞。另外,GCV还参与病毒增殖的抑制,因此即使施用高剂量的病毒也可以控制由病毒引起的副作用。此外,即使由于GCV对抗病毒增殖的抑制而减少了病毒颗粒的数量,还是提高了抗癌作用。因此,本发明的安全性和抗癌作用得到改善的溶瘤病毒可以有效地用于癌症的治疗。(The present invention relates to an oncolytic virus having improved safety and anticancer effects and use thereof. The oncolytic virus of the present invention having improved safety and anticancer effects can be obtained by inserting a gene encoding an HSV-TK fragment into a TK gene region to delete TK of vaccinia virus. In addition, the oncolytic virus of the present invention expresses an HSV-TK fragment to phosphorylate GCV, thereby killing cancer cells infected by the oncolytic virus, and even cancer cells in their vicinity. In addition, GCV is also involved in the inhibition of virus proliferation, and thus side effects caused by viruses can be controlled even by administering a high dose of viruses. Furthermore, even if the number of virus particles is reduced due to the inhibition of the anti-viral proliferation by GCV, the anti-cancer effect is improved. Therefore, the oncolytic virus of the present invention, which has improved safety and anticancer effects, can be effectively used for the treatment of cancer.)

1. A recombinant viral vector comprising a nucleotide sequence encoding an HSV-TK (herpes simplex virus thymidine kinase) fragment or variant thereof.

2. The recombinant viral vector according to claim 1, wherein the HSV-TK fragment is an HSV1-TK (herpes simplex virus type 1 thymidine kinase) fragment.

3. The recombinant viral vector according to claim 2, wherein the HSV1-TK fragment is SEQ ID NO: 1, wherein 1 to 195 amino acid residues from the C-terminus of the amino acid sequence shown in 1 are continuously deleted.

4. The recombinant viral vector according to claim 2, wherein the HSV1-TK fragment is SEQ ID NO: 1, wherein the C-terminal amino acid sequence of the fragment has a continuous deletion of 24 to 149 amino acid residues.

5. The recombinant viral vector according to claim 2, wherein the HSV1-TK fragment is SEQ ID NO: 1, wherein the C-terminal amino acid sequence of the fragment has a consecutive deletion of 30 to 46 amino acid residues.

6. The recombinant viral vector according to claim 2, wherein the HSV1-TK fragment is SEQ ID NO: 1, wherein 46 amino acid residues at the C-terminal end of the amino acid sequence shown in 1 are continuously deleted.

7. The recombinant viral vector according to claim 2, wherein the HSV1-TK fragment has any one of the amino acid sequences selected from the group consisting of: SEQ ID NO: 2-6.

8. The recombinant viral vector according to claim 1, wherein the variant of the HSV-TK fragment is a variant of the HSV1-TK fragment.

9. The recombinant viral vector according to claim 8, wherein the variant of the HSV1-TK fragment has the sequence as shown in seq id NO: 7 or 8.

10. The recombinant viral vector according to claim 1, wherein the viral vector is derived from an adenovirus, a herpes simplex virus, a lentivirus, a retrovirus, an adeno-associated virus, a vaccinia virus, or a poxvirus.

11. An oncolytic virus comprising a nucleotide sequence encoding an HSV-TK fragment or variant thereof.

12. An oncolytic virus according to claim 11, wherein the HSV-TK fragment is an HSV1-TK fragment.

13. The oncolytic virus of claim 12, wherein the HSV1-TK fragment is SEQ ID NO: 1, wherein 1 to 195 amino acid residues from the C-terminus of the amino acid sequence shown in 1 are continuously deleted.

14. The oncolytic virus of claim 12, wherein the HSV1-TK fragment is SEQ ID NO: 1, wherein the C-terminal amino acid sequence of the fragment has a continuous deletion of 24 to 149 amino acid residues.

15. The oncolytic virus of claim 12, wherein the HSV1-TK fragment is SEQ ID NO: 1, wherein the C-terminal amino acid sequence of the fragment has a consecutive deletion of 30 to 46 amino acid residues.

16. The oncolytic virus of claim 12, wherein the HSV1-TK fragment is SEQ ID NO: 1, wherein 46 amino acid residues at the C-terminal end of the amino acid sequence shown in 1 are continuously deleted.

17. An oncolytic virus according to claim 12, wherein said HSV1-TK fragment has any one of the amino acid sequences selected from the group consisting of: SEQ ID NO: 2-6.

18. An oncolytic virus according to claim 11, wherein the variant of the HSV-TK fragment is a variant of the HSV1-TK fragment.

19. The oncolytic virus of claim 18, wherein the variant of HSV1-TK fragment has the sequence as shown in seq id NO: 7 or 8.

20. The oncolytic virus of claim 11, wherein the oncolytic virus is derived from adenovirus, measles virus, herpes simplex virus, lentivirus, retrovirus, cytomegalovirus, baculovirus, reovirus, adeno-associated virus, myxoma virus, vesicular stomatitis virus, poliovirus, newcastle disease virus, parvovirus, coxsackie virus, seneca virus, vaccinia virus, or poxvirus.

21. The oncolytic virus of claim 11, derived from a vaccinia virus.

22. A pharmaceutical composition for preventing or treating cancer, comprising the oncolytic virus of any one of claims 11 to 21 as an active ingredient.

23. The pharmaceutical composition for preventing or treating cancer according to claim 22, wherein the pharmaceutical composition is for preventing or treating cancerThus, the oncolytic virus is 1 × 10³pfu to 1 × 10¹⁰Doses of pfu were administered.

24. The pharmaceutical composition for preventing or treating cancer according to claim 22, wherein the cancer is selected from the group consisting of: lung cancer, large intestine cancer, prostate cancer, thyroid cancer, breast cancer, brain cancer, head and neck cancer, esophageal cancer, skin cancer, thymus cancer, stomach cancer, colon cancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer, rectal cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, non-small cell lung cancer, bone cancer, intraocular melanoma, perianal cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulval cancer, hodgkin's disease, small intestine cancer, endocrine adenocarcinoma, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic leukemia, acute leukemia, lymphocytic lymphomas, kidney cancer, ureter cancer, renal cell cancer, renal pelvis cancer, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brain stem glioma, pituitary adenoma, or a combination thereof.

25. A pharmaceutical composition for preventing or treating cancer, comprising the oncolytic virus of any one of claims 11 to 21, and GCV (ganciclovir) or ACV (acyclovir) as active ingredients.

26. The pharmaceutical composition for preventing or treating cancer according to claim 25, wherein the oncolytic virus and GCV or ACV are administered simultaneously or sequentially.

27. The pharmaceutical composition for preventing or treating cancer according to claim 25, wherein the oncolytic virus is 1 × 10³pfu to 1 × 10¹⁰Doses of pfu were administered.

28. The pharmaceutical composition for preventing or treating cancer according to claim 25, wherein GCV or ACV is administered at a dose of 0.1 μ g/kg/day to 50 mg/kg/day.

29. The pharmaceutical composition for preventing or treating cancer according to claim 25, wherein the cancer is selected from the group consisting of: lung cancer, large intestine cancer, prostate cancer, thyroid cancer, breast cancer, brain cancer, head and neck cancer, esophageal cancer, skin cancer, thymus cancer, stomach cancer, colon cancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer, rectal cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, non-small cell lung cancer, bone cancer, intraocular melanoma, perianal cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulval cancer, hodgkin's disease, small intestine cancer, endocrine adenocarcinoma, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic leukemia, acute leukemia, lymphocytic lymphomas, kidney cancer, ureter cancer, renal cell cancer, renal pelvis cancer, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brain stem glioma, pituitary adenoma, or a combination thereof.

30. A method for treating cancer comprising administering an oncolytic virus according to any one of claims 11 to 21 and GCV or ACV.

31. The method of claim 30, wherein the oncolytic virus is 1 × 10³pfu to 1 × 10¹⁰Doses of pfu were administered.

32. The method for treating cancer of claim 30, wherein the GCV or ACV is administered at a dose of 0.1 μ g/kg/day to 50 mg/kg/day.

33. The method of claim 30, wherein the GCV or ACV is administered at least once during or after the administration of the oncolytic virus.

34. The method of treating cancer according to claim 30, wherein GCV or ACV is administered for about two weeks from 24 hours after administration of the oncolytic virus.

35. The method of treating cancer according to claim 30, wherein GCV or ACV is administered twice weekly for 5 to 18 days.

36. The method for treating cancer according to claim 30, wherein said cancer is selected from the group consisting of: lung cancer, large intestine cancer, prostate cancer, thyroid cancer, breast cancer, brain cancer, head and neck cancer, esophageal cancer, skin cancer, thymus cancer, stomach cancer, colon cancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer, rectal cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, non-small cell lung cancer, bone cancer, intraocular melanoma, perianal cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulval cancer, hodgkin's disease, small intestine cancer, endocrine adenocarcinoma, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic leukemia, acute leukemia, lymphocytic lymphomas, kidney cancer, ureter cancer, renal cell cancer, renal pelvis cancer, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brain stem glioma, pituitary adenoma, or a combination thereof.

37. The method for treating cancer according to claim 30, wherein the oncolytic virus is administered intratumorally, intraperitoneally, or intravenously.

38. A method of producing a recombinant vaccinia virus expressing HSV-TK fragment or variant thereof comprising the steps of: i) transfecting a shuttle plasmid comprising a nucleotide sequence encoding an HSV-TK fragment or variant thereof into a host cell and treating the host cell with a wild-type vaccinia virus; ii) culturing the resulting host cell; and iii) obtaining the recombinant vaccinia virus from the resulting culture.

39. The method of making a recombinant vaccinia virus expressing an HSV-TK fragment or variant thereof according to claim 38, wherein the HSV-TK fragment has any one of the amino acid sequences selected from the group consisting of: SEQ ID NO: 2-6.

40. The method of making a recombinant vaccinia virus expressing an HSV-TK fragment or variant thereof according to claim 38, wherein the nucleotide sequence encoding the HSV-TK fragment is as set forth in SEQ ID NO: 9, or a nucleotide sequence shown in the specification.

41. The method of making a recombinant vaccinia virus expressing an HSV-TK fragment or variant thereof according to claim 38, wherein the nucleotide sequence encoding the HSV-TK fragment variant is as set forth in SEQ ID NO: 10 or 11.

42. Use of an oncolytic virus according to any one of claims 11 to 21 for the treatment of cancer.

43. Use of a pharmaceutical composition according to any one of claims 25 to 29 in the treatment of cancer.

44. Use of an oncolytic virus according to any one of claims 11 to 21 in the manufacture of a medicament for the prevention or treatment of cancer.

45. Use of a pharmaceutical composition of any one of claims 25 to 29 in the manufacture of a medicament for the prevention or treatment of cancer.

Technical Field

The present invention relates to an oncolytic virus having improved safety and anticancer effects and uses thereof.

Background

With the comprehensive application of gene recombination technology, clinical studies using oncolytic viruses having improved tumor selectivity and anticancer efficacy have been initiated. The first recombinant oncolytic virus reported in the literature was herpes simplex virus. Since then, studies have been actively undertaken to use other viruses for oncolytic applications (Martuza et al, 1991; Hwang et al, 2011; Kaufaman et al, 2015; Khuri et al, 2000; Park et al, 2008).

The use of oncolytic viruses has received much attention due to the recent successful commercialization of herpes virus based T-vecs (oncolytic viruses) for the treatment of advanced melanoma in the united states and europe. Meanwhile, vaccinia virus lacking the Thymidine Kinase (TK) gene has great clinical utility, but has limitations in maximizing its clinical effects due to its narrow therapeutic window. Vaccinia viruses lacking TK have a narrow therapeutic window, meaning that high viral doses have great clinical efficacy, but are at clinical risk due to viral toxicity.

In fact, in Pexa-Vec (JX)-594; SillaJen corporation) in a phase II clinical trial (the trial was performed in thirty (30) primary liver cancer patients), the results indicated that the treatment was comparable to the low dose group (10)⁸pfu) high dose group (10)⁹pfu) increased survival (Heo et al, 2013). However, in phase I clinical trials with intratumoral dosing, at 3x10⁹Dose-limiting toxicity (DLT) was observed at pfu, so that the Maximum Tolerated Dose (MTD) was limited to 1x10⁹pfu. This is reported to be drug independent. However, many cases of death soon after oncolytic virus therapy have been reported, suggesting that unwanted viral replication may lead to unpredictable results. These dose-dependent increases in efficacy and dose-limiting toxicity indicate the need to develop safer, more effective vaccinia viruses.

Ganciclovir (GCV), on the other hand, is an antiviral drug against herpes simplex virus, cytomegalovirus and varicella zoster virus. When bound to the TK of herpes simplex virus, the 5' end of GCV is phosphorylated and then converted to ganciclovir triphosphate (GCV-TP). GCV-TP inhibits the activity of viral DNA polymerase and attaches to the 3' end of viral DNA, thereby terminating DNA extension. GCV-TP is a highly toxic substance that can exhibit cytotoxicity by preventing DNA synthesis in cells.

Currently, there has been a study to use the HSV-TK/GCV system in oncolytic virus therapy by inserting HSV-TK into oncolytic virus to induce apoptosis of cancer cells by co-administration of GCV. In this therapy, the oncolytic virus first infects tumor cells to induce a direct anticancer effect, and GCV phosphorylated by HSV1-TK (suicide gene) inhibits proliferation of tumor cells, thereby exhibiting an additional anticancer effect (Oliver W et al, human gene therapy, vol 10, stage 16, 1999). The HSV1-TK/GCV system is mainly used for oncolytic virus therapy taking adenovirus as a vector. However, other cytotoxic effects expected from co-administration of GCV remain controversial.

In particular, it was observed that the cytotoxic effect on glioma cells was significantly enhanced when a replication competent adenovirus with HSV-TK was administered in combination with GCV. However, not all studies have shown consistent results (Lambirght ES et al, Gene therapy, p.8: 946-53). The reason for this is that the HSV-TK/GCV system is not only involved in viral replication, but also in tumor cell proliferation, the overall effect of which is counteracted by conflicting effects (Widner O, Morris JC, 2000).

Therefore, there is a need to study a specific method for applying the HSV-TK/GCV system to oncolytic virus therapy while ensuring safety and enhancing anticancer effects.

Disclosure of Invention

Technical problem

Accordingly, the present inventors have conducted studies to develop oncolytic viruses having improved safety and anticancer effects, and completed the present invention by developing recombinant vaccinia virus comprising a gene encoding HSV1-TK fragment (HSV1-TKmut) in which the C-terminal part of HSV1-TK is truncated or a variant thereof and confirming that the recombinant vaccinia virus has good safety and anticancer effects when administered in combination with GCV.

Solution scheme

To achieve the above objects, one aspect of the present invention provides a recombinant viral vector comprising a nucleotide sequence encoding an HSV-TK (herpes simplex virus thymidine kinase) fragment or variant thereof.

In another aspect of the invention there is provided an oncolytic virus comprising a nucleotide sequence encoding an HSV-TK fragment, or a variant thereof.

Another aspect of the present invention provides a pharmaceutical composition for treating cancer, comprising the oncolytic virus.

Another aspect of the present invention provides a pharmaceutical composition for treating cancer, comprising the oncolytic virus, and GCV (ganciclovir) or ACV (acyclovir) as active ingredients.

Another aspect of the invention provides a method of treating cancer comprising administering the oncolytic virus, and GCV or ACV.

In another aspect of the present invention, there is provided a method for preparing a recombinant vaccinia virus comprising the steps of: i) transfecting a shuttle plasmid comprising a nucleotide sequence encoding an HSV-TK fragment or variant thereof into a host cell and treating the host cell with a wild-type vaccinia virus; ii) culturing said host cell; and iii) obtaining the recombinant vaccinia virus from the culture.

Another aspect of the invention provides the use of the oncolytic virus in the treatment of cancer.

Another aspect of the invention provides the use of the pharmaceutical composition in the treatment of cancer.

Another aspect of the invention provides the use of the oncolytic virus for the manufacture of a medicament for the treatment of cancer.

Another aspect of the invention provides the use of the pharmaceutical composition in the manufacture of a medicament for the treatment of cancer.

Advantageous effects of the invention

The oncolytic virus of the present invention having improved safety and anticancer effects is due to its coding gene encoding HSV-TK fragment or a variant thereof, which is inserted into a deleted region of the native TK gene of vaccinia virus. Furthermore, the oncolytic virus of the invention may express an HSV-TK fragment or variant thereof to phosphorylate GCV and thereby kill cancer cells infected by the oncolytic virus, even surrounding cancer cells. In addition, GCV can also inhibit viral replication, so it can control adverse side effects caused by the virus in the case of administration of high doses of virus. Furthermore, although the number of virus particles is reduced due to GCV inhibition of virus replication, its anticancer effect is enhanced. Therefore, the oncolytic virus of the present invention having improved safety and anticancer effects can be used for treating cancer.

Brief description of the drawings

FIG. 1 is a schematic representation of a shuttle plasmid vector carrying the HSV1-TK gene for use in the preparation of recombinant vaccinia virus.

FIG. 2 is a schematic diagram showing an example of recombinant vaccinia virus (OTS-412) in which HSV1-TKmut gene and firefly luciferase gene were inserted in its TK gene region.

FIG. 3 shows the procedure for preparing recombinant vaccinia virus (OTS-412) expressing HSV1-TK fragment.

FIG. 4 depicts the results of a Western blot used to confirm the expression of the HSV1-TK fragment in OTS-412.

FIG. 5 shows a schematic diagram for identifying HSV1-TK_mutThe result of restriction enzyme mapping of the insertion of the gene in OTS-412 DNA.

FIG. 6 shows a graph of replication of OTS-412 24 and 48 hours after infection in the HCT-116 cancer cell line.

FIG. 7 shows a graph of the inhibition of viral replication by GCV in the OTS-412 treated NCI-H460 and NCI-H23 cancer cell lines.

FIG. 8 shows a graph of the enhancement of cytotoxicity of GCV in the OTS-412 treated HCT-116 cancer cell line.

FIG. 9 shows a graph of cell viability of A549, NCI-H460, HT-29, and HCT-116 cancer cell lines following administration of OTS-412 alone or in combination with GCV, respectively.

FIG. 10 shows the apoptosis and necrosis patterns of A549 and NCI-H460 cancer cell lines following administration of GCV, OTS-412, respectively, or OTS-412 and GCV in combination.

FIG. 11 shows a graph of cell viability of A549 and NCI-H460 cancer cell lines following administration of GCV, OTS-412, respectively, or OTS-412 and GCV in combination.

FIG. 12 shows a graph of tumor size change 12 days after administration of saline, OTS-412 or combined OTS-412 and GCV to mice transplanted with HCT-116 cancer cell line.

FIG. 13 is a bioluminescence image showing the distribution of OTS-412 in mice transplanted with HCT-116 cancer cell line after administering PBS, OTS-412, or a combination of OTS-412 and GCV to the mice.

FIG. 14 shows graphs of viral DNA copy number counted in tumor tissues obtained from mice transplanted with HCT-116 cancer cell line after OTS-412 was administered alone or in combination with GCV to the mice.

FIG. 15 shows a graph of body weight change 12 days after administration of saline, OTS-412, or a combination of OTS-412 and GCV to HCT-116 tumor-bearing mice.

FIG. 16 shows a graph of the change in tumor size 24 days after administration of saline, OTS-412, or a combination of OTS-412 and GCV to Renca tumor-bearing mice.

FIG. 17 shows a graph of the total number of viral particles in tumor tissue obtained from Renca tumor-bearing mice following administration of saline, OTS-412, or a combination of OTS-412 and GCV.

FIG. 18 shows TUNEL staining of tumor tissue obtained from mice transplanted with Renca cancer cell line following administration of saline, OTS-412, or a combination of OTS-412 and GCV to the mice.

FIG. 19 shows a graph of the quantification of apoptotic moieties observed in TUNEL staining of tumor tissue obtained from mice transplanted with Renca cancer cell line following administration of saline, OTS-412, or a combination of OTS-412 and GCV to the mice.

FIG. 20 shows a graph of tumor size change 21 days after administration of saline, OTS-412, or a combination of OTS-412 and GCV to HCT-116 tumor-bearing mice.

FIG. 21 shows Immunohistochemical (IHC) results of tumor tissues obtained from mice transplanted with HCT-116 cancer cell line following administration of saline, OTS-412, or combined administration of OTS-412 and GCV.

FIG. 22 shows a graph of the quantification of the stained sections observed in Immunohistochemistry (IHC) results for tumor tissues obtained from mice transplanted with the HCT-116 cancer cell line following administration of saline, OTS-412, or combined administration of OTS-412 and GCV.

FIG. 23 shows a graph of the quantification of necrotic parts observed in H & E staining of tumor tissues obtained from mice transplanted with the HCT-116 cancer cell line after administration of saline, OTS-412, or combined administration of OTS-412 and GCV.

FIG. 24 shows a graph of post-tumor survival zones resulting from H & E staining of tumor tissue obtained from HCT-116 tumor-bearing mice following administration of saline, OTS-412, or a combination of OTS-412 and GCV to the mice.

FIG. 25 shows a graph of body weight of mice of each group treated with OTS-412 and GCV, which were transplanted with HCT-116 cancer cell line, administered saline, OTS-412 or a combination thereof for 21 days.

FIG. 26 shows a graph of cell viability assessment for thirteen (13) different cancer cell lines after treatment with OTS-412.

FIG. 27 shows a schematic representation of HCT-116. IC of OTS-412 administered in SK-MEL-28 and DU145 cancer cell lines₅₀The figure (a).

FIG. 28 is a bioluminescence image showing the viral distribution of intraperitoneal injected OTS-412 in HCT-116 tumor-bearing mice on days 3 and 7.

Best mode for carrying out the invention

Hereinafter, the present invention will be described in detail.

The present invention provides a recombinant viral vector comprising a nucleotide sequence encoding an HSV-TK (herpes simplex virus thymidine kinase) fragment, or a variant thereof.

As used herein, the term "TK (thymidine kinase)" refers to thymidine kinase, an enzyme involved in the biosynthesis of nucleotides. TK is an enzyme used for biosynthesis of nucleotides in cells and viruses. TK may be derived from herpes simplex virus. Here, the herpes simplex virus may be herpes simplex virus type 1 (HSV1) or herpes simplex virus type 2 (HSV 2).

As used herein, the term "HSV-TK (herpes simplex virus thymidine kinase)" refers to the thymidine kinase of herpes simplex virus. Specifically, the HSV-TK may be HSV1-TK (herpes simplex virus type 1 thymidine kinase). HSV1-TK is an enzyme which participates in the initial phosphorylation reaction during DNA synthesis of herpes simplex virus. HSV-TK is also involved in the phosphorylation of the antiviral agents GCV (ganciclovir) or ACV (acyclovir). In particular, HSV1-TK is about ten times more sensitive to GCV or ACV than any other TK present in other types of viruses.

As used herein, the term "HSV-TK fragment" refers to a fragment of HSV-TK following deletion of some of the amino acid residues. Specifically, the HSV-TK fragment may be a fragment in which a part of the N-terminus or C-terminus of HSV-TK is deleted. Preferably, the HSV-TK fragment may be a fragment in which a part of the C-terminus of HSV-TK is deleted. In one embodiment, the HSV-TK fragment may be a fragment in which 1 to 195 amino acid residues of the C-terminus of HSV-TK are continuously deleted. The HSV-TK fragment may be a fragment in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 amino acid residues are continuously deleted from the C-terminus of the HSV-TK.

The HSV1-TK fragment of the invention or variants thereof may be derived from a virus mutated by induction of adaptive evolution. To develop vaccinia virus expressing the optimized HSV1-TK fragment or variant thereof, the inventors prepared recombinant vaccinia virus containing the HSV1-TK gene and induced adaptive evolution in the absence of TK. The resulting recombinant vaccinia virus was subjected to experiments for confirming luciferase activity, genomic analysis, and experiments for confirming sensitivity to GCV, and screened for vaccinia virus OTS-412 expressing HSV1-TK fragment or variant thereof. Despite the important truncation at the C-terminus of HSV1-TK, the HSV1-TK fragment or variant thereof remains sensitive to GCV. Furthermore, it has been demonstrated that anti-cancer effects are enhanced when vaccinia virus expressing the HSV1-TK fragment or variant thereof is administered in combination with GCV.

The HSV-TK fragment may be an HSV1-TK fragment. An embodiment of an HSV1-TK fragment may be any one of the following fragments: SEQ ID NO: 1, wherein the C-terminal of the amino acid sequence is a fragment obtained by continuous deletion of 1 to 195, 24 to 149, or 30 to 46 amino acid residues. In particular, the HSV-TK fragment may be one of the following fragments: SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 195, 180, 190 of the amino acid residues of the C-terminal of the amino acid sequence shown in 1. Preferably, the HSV1-TK fragment may be one of the following fragments: SEQ ID NO: 1, wherein 24, 30, 70, 99, 149 or 195 amino acid residues are continuously deleted from the C-terminal end of the amino acid sequence shown in the specification. The HSV1-TK fragment may have reduced sensitivity to GCV due to deletion of certain amino acid residues at the C-terminus.

An example of the HSV1-TK fragment may be one of the following fragments: SEQ ID NO: 1, wherein 195, 149, 99, 70, 46, 30 or 24 amino acid residues are continuously deleted from the C-terminal end of the amino acid sequence shown in the figure. Specifically, the HSV1-TK fragment may have any one of the amino acid sequences selected from the group consisting of: SEQ ID NO: 2-6.

The nucleotide sequence encoding the HSV1-TK fragment may be a nucleotide sequence encoding any of the following HSV1-TK fragments: SEQ ID NO: 1, 1 to 195, 24 to 149, or 30 to 46 amino acid residues from the C-terminus of the amino acid sequence shown in 1. Specifically, the nucleotide sequence encoding the HSV1-TK fragment may be a nucleotide sequence encoding any one of the following amino acid sequences: SEQ ID NO: 2-6. Preferably, the nucleotide sequence encoding the HSV1-TK fragment may be SEQ ID NO: 9, or a nucleotide sequence shown in the specification.

The variant of the HSV-TK fragment may be a variant of the HSV1-TK fragment. In particular, the variant of the HSV1-TK fragment may be a variant in which at least one amino acid residue constituting the HSV1-TK fragment is substituted. In particular, the variant of the HSV1-TK fragment may comprise the amino acid sequence as set forth in SEQ ID NO: 1 to 145 amino acid residues of the amino acid sequence shown in 1. In one embodiment, the variant of the HSV1-TK fragment may have an amino acid sequence as set forth in SEQ ID NO: 7 or 8. The nucleotide sequence encoding the variant of the HSV1-TK fragment may be SEQ ID NO: 10 or 11.

Viral vectors are vectors used for introducing genes into cells or producing viruses. The viral vector may be derived from an adenovirus, a herpes simplex virus, a lentivirus, a retrovirus, an adeno-associated virus, a vaccinia virus or a poxvirus.

In another aspect of the invention there is provided an oncolytic virus comprising a nucleotide sequence encoding an HSV-TK fragment, or a variant thereof.

As used herein, the term "oncolytic virus" refers to a recombinant virus that destroys cancer cells, the genes of which are manipulated to replicate specifically in cancer cells. The oncolytic virus may be derived from adenovirus, herpes simplex virus, measles virus, lentivirus, retrovirus, cytomegalovirus, baculovirus, reovirus, adeno-associated virus, myxoma virus, vesicular stomatitis virus, poliovirus, newcastle disease virus, parvovirus, coxsackie virus, seneca virus, vaccinia virus or poxvirus. Preferably, the oncolytic virus may be derived from a vaccinia virus.

The vaccinia virus may be, but is not limited to, Western Reserve (WR), NYVAC (New York vaccinia virus), Wyeth (New York City health office; NYCBOH), LC16m8, Listeria (Lister), Copenhagen (Copenhagen), Temple (TianTan), USSR, Tash stem (TashKent), Evens (Evans), IHD-J (International sanitation-J), or IHD-W (International sanitation-white) strains.

The HSV-TK fragment may be an HSV1-TK fragment. An embodiment of an HSV1-TK fragment may be any one of the following fragments: SEQ ID NO: 1, 1 to 195, 24 to 149, or 30 to 46 amino acid residues from the C-terminus of the amino acid sequence shown in 1. In particular, the HSV-TK fragment may be one of the following fragments: SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 195, 180, 190 of the amino acid residues of the C-terminal of the amino acid sequence shown in 1. Preferably, the HSV1-TK fragment may be one of the following fragments: SEQ ID NO: 1, wherein 24, 30, 70, 99, 149 or 195 amino acid residues are continuously deleted from the C-terminal end of the amino acid sequence shown in the specification. The HSV1-TK fragment may have reduced sensitivity to GCV due to deletion of certain amino acid residues at the C-terminus.

An example of the HSV1-TK fragment may be one of the following fragments: SEQ ID NO: 1, wherein 195, 149, 99, 70, 46, 30 or 24 amino acid residues are continuously deleted from the C-terminal end of the amino acid sequence shown in the figure. Specifically, the HSV1-TK fragment may have any one of the amino acid sequences selected from the group consisting of: SEQ ID NO: 2-6.

The nucleotide sequence encoding the HSV1-TK fragment may be a nucleotide sequence encoding any of the following HSV1-TK fragments: SEQ ID NO: 1, 1 to 195, 24 to 149, or 30 to 46 amino acid residues from the C-terminus of the amino acid sequence shown in 1. Specifically, the nucleotide sequence encoding the HSV1-TK fragment may be a nucleotide sequence encoding any one of the following amino acid sequences: SEQ ID NO: 2-6. Preferably, the nucleotide sequence encoding the HSV1-TK fragment may be SEQ ID NO: 9, or a nucleotide sequence shown in the specification.

The variant of the HSV-TK fragment may be a variant of the HSV1-TK fragment. In particular, the variant of the HSV1-TK fragment may be a variant in which at least one amino acid residue constituting the HSV1-TK fragment is substituted. In particular, the variant of the HSV1-TK fragment may comprise the amino acid sequence as set forth in SEQ ID NO: 1 to 145 amino acid residues of the amino acid sequence shown in 1. In one embodiment, the variant of the HSV1-TK fragment may have an amino acid sequence as set forth in SEQ ID NO: 7 or 8. The nucleotide sequence encoding the variant of the HSV1-TK fragment may be SEQ ID NO: 10 or 11.

The oncolytic virus may lack the native TK gene. In particular, the oncolytic virus may be a virus lacking the native TK gene by inserting or replacing the nucleotide sequence encoding the HSV1-TK fragment in the native TK gene region. In one embodiment of the invention, the natural TK gene of vaccinia virus is deleted by inserting the nucleotide sequence encoding the HSV1-TK fragment into the natural TK gene region of vaccinia virus.

As used herein, the term "gene deletion" refers to the absence of expression of a gene due to partial or complete deletion of the gene or insertion of a foreign gene into the gene. The partial gene deletion may be a partial deletion at the 5 'end or the 3' end. That is, the natural TK gene of the oncolytic virus may be partially or fully deleted.

In one example of the present invention, a recombinant vaccinia virus OTS-412 comprising a gene encoding the HSV-TK fragment was infected into the HCT-116 cancer cell line, and infection and replication efficiencies were confirmed (FIG. 6). In addition, OTS-412 was administered in thirteen (13) different cancer cell lines, and after 72 hours of culture, cell viability was measured to show cytotoxicity of OTS-412 (fig. 26). Thus, the oncolytic virus of the present invention comprising a nucleotide sequence encoding the HSV-TK fragment is effective for the treatment of cancer.

Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising an oncolytic virus comprising a nucleotide sequence encoding the HSV-TK fragment or variant thereof as an active ingredient.

The oncolytic virus included in the pharmaceutical composition as an active ingredient is as described above.

The dose of the oncolytic virus varies depending on the condition and weight of an individual, severity of disease, type of drug, route and time of administration, and may be appropriately selected by those skilled in the art³To 1 × 10¹⁸Individual virus particles, infectious virus units (TCID)₅₀) Or plaque forming units (pfu) in particular, the dose may be 1 × 10³、2×10³、5×10³、1×10⁴、2×10⁴、5×10⁴、1×10⁵、2×10⁵、5×10⁵、1×10⁶、2×10⁶、5×10⁶、1×10⁷、2×10⁷、5×10⁷、1×10⁸、2×10⁸、5×10⁸、1×10⁹、2×10⁹、5×10⁹、1×10¹⁰、5×10¹⁰、1×10¹¹、5×10¹¹、1×10¹²、1×10¹³、1×10¹⁴、1×10¹⁵、1×10¹⁶、1×10¹⁷、1×10¹⁸Or more virus particles, infectious virus units (TCID)₅₀) Or plaque forming units (pfu), which may include various values and ranges therebetween, preferably, the oncolytic virus may be at 1 × 10³To 1 × 10¹⁰In one example of the invention, the oncolytic virus is 1 × 10⁶、1×10⁷And 1 × 10⁸Doses of pfu were administered.

The cancer may be selected from the group consisting of: lung cancer, large intestine cancer, prostate cancer, thyroid cancer, breast cancer, brain cancer, head and neck cancer, esophageal cancer, skin cancer, thymus cancer, stomach cancer, colon cancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer, rectal cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, non-small cell lung cancer, bone cancer, intraocular melanoma, perianal cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulval cancer, hodgkin's disease, small intestine cancer, endocrine adenocarcinoma, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic leukemia, acute leukemia, lymphocytic lymphomas, kidney cancer, ureter cancer, renal cell cancer, renal pelvis cancer, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brain stem glioma, pituitary adenoma, or a combination thereof.

The pharmaceutical composition of the present invention may further comprise a physiologically acceptable carrier. In addition, the pharmaceutical composition of the present invention may further comprise suitable excipients and diluents conventionally used in the preparation of pharmaceutical compositions. In addition, it can be formulated into an injectable form according to a conventional method.

The pharmaceutical compositions may be formulated as sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, suppositories, and the like for parenteral administration. For non-waterSolutions and suspensions, propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), injectable esters (e.g., ethyl oleate), and the like may be used. As suppository base, Witepsol can be used^TMPolyethylene glycol (macrogol) and Tween^TM61. Cocoa butter, lauryl butter, glycerogelatin, and the like.

The pharmaceutical composition may be administered to a subject in various routes and amounts depending on the condition of the patient and whether there are side effects, and the optimal route, dose and frequency of administration may be selected by those skilled in the art within an appropriate range. In addition, the pharmaceutical composition may be administered in combination with other drugs or physiologically active substances whose therapeutic effects on diseases are known, or may be formulated in combination with other drugs.

The pharmaceutical compositions may be administered parenterally, including intratumoral, intraperitoneal, subcutaneous, intradermal, intranodal, and intravenous administration, and the like. Preferably, it may be intratumoral, intraperitoneal or intravenous administration. In another aspect, the dosage of the pharmaceutical composition may be determined according to the administration regimen, the dosage and the health condition of the patient.

The present inventors administered OTV-412 in combination with GCV in A549, NCI-H460, HT-29 and HCT-116 cancer cell lines, evaluated the viability of the cancer cell lines, and confirmed that anti-cancer effects were enhanced by combination therapy (FIG. 9). In addition, it was confirmed that the virus maintained a certain level of replication ability even when OTS-412 and GCV were administered in combination to cancer cell lines (FIG. 14). Thus, the combination of an oncolytic virus comprising a nucleotide sequence encoding the HSV-TK fragment or variant thereof and GCV can be effectively used for the treatment of cancer.

Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising an oncolytic virus comprising a nucleotide sequence encoding the HSV-TK fragment or variant thereof, and GCV (ganciclovir) or ACV (acyclovir) as an effective ingredient.

The oncolytic virus and GCV or ACV comprised in the pharmaceutical composition may be administered simultaneously, sequentially or in reverse order. In particular, the oncolytic virus and GCV or ACV comprised in the pharmaceutical composition may be administered simultaneously. Alternatively, the oncolytic virus may be administered first, followed by GCV or ACV. In addition, the oncolytic virus is administered first, followed by GCV or ACV, and then again.

The oncolytic virus included in the pharmaceutical composition as an active ingredient is as described above.

The dose of the oncolytic virus varies depending on the condition and weight of an individual, severity of disease, type of drug, route and time of administration, and may be appropriately selected by those skilled in the art³To 1 × 10¹⁸Individual virus particles, infectious virus units (TCID)₅₀) Or plaque forming units (pfu) in particular, the dose may be 1 × 10³、2×10³、5×10³、1×10⁴、2×10⁴、5×10⁴、1×10⁵、2×10⁵、5×10⁵、1×10⁶、2×10⁶、5×10⁶、1×10⁷、2×10⁷、5×10⁷、1×10⁸、2×10⁸、5×10⁸、1×10⁹、2×10⁹、5×10⁹、1×10¹⁰、5×10¹⁰、1×10¹¹、5×10¹¹、1×10¹²、1×10¹³、1×10¹⁴、1×10¹⁵、1×10¹⁶、1×10¹⁷、1×10¹⁸Or more virus particles, infectious virus units (TCID)₅₀) Or plaque forming units (pfu), which may include various values and ranges therebetween, preferably, the oncolytic virus may be at 1 × 10³To 1 × 10¹⁰In one example of the invention, the oncolytic virus is 1 × 10⁶、1×10⁷And 1 × 10⁸Doses of pfu were administered.

As used herein, the term "GCV" refers to an antiviral agent, which is called ganciclovir, and which is effective against herpes simplex virus, cytomegalovirus, and varicella zoster virus. GCV is phosphorylated at the 5' end by the TK of the virus and then converted to ganciclovir triphosphate (GCV-TP). GCV-TP inhibits the activity of viral DNA polymerase and attaches to the 3' end of viral DNA, thereby terminating DNA extension. In addition, phosphorylated GCV can prevent cellular DNA replication, thereby inhibiting cell growth. GCV is represented by the following formula 1.

[ formula 1]

As used herein, the term "ACV" refers to an antiviral agent, which is called acyclovir, and which is effective against herpes simplex virus, varicella zoster virus and epstein-barr virus. ACV is phosphorylated by the viral TK and then converted to acyclovir triphosphate (ACV-TP). ACV-TP inhibits the activity of viral DNA polymerase and attaches to the 3' end of viral DNA, thereby terminating DNA extension. ACV is represented by the following formula 2.

[ formula 2]

Further, the daily dose of GCV or ACV may be 0.1. mu.g/kg to 50 mg/kg. Specifically, the daily dose of GCV or ACV may be 0.1. mu.g/kg to 50mg/kg, 1. mu.g/kg to 40mg/kg, 5. mu.g/kg to 30mg/kg, or 10. mu.g/kg to 20 mg/kg. In one example of the invention, GCV is administered at a daily dose of 50 mg/kg.

The cancer may be selected from the group consisting of: lung cancer, large intestine cancer, prostate cancer, thyroid cancer, breast cancer, brain cancer, head and neck cancer, esophageal cancer, skin cancer, thymus cancer, stomach cancer, colon cancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer, rectal cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, non-small cell lung cancer, bone cancer, intraocular melanoma, perianal cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulval cancer, hodgkin's disease, small intestine cancer, endocrine adenocarcinoma, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic leukemia, acute leukemia, lymphocytic lymphomas, kidney cancer, ureter cancer, renal cell cancer, renal pelvis cancer, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brain stem glioma, pituitary adenoma, or a combination thereof.

The pharmaceutical composition of the present invention may further comprise a physiologically acceptable carrier. In addition, the pharmaceutical composition of the present invention may further comprise suitable excipients and diluents conventionally used in the preparation of pharmaceutical compositions. In addition, it can be formulated into an injectable form according to a conventional method.

The pharmaceutical compositions may be formulated as sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, suppositories, and the like for parenteral administration. For non-aqueous solutions and suspensions, propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), injectable esters (e.g., ethyl oleate), and the like may be used. As suppository base, Witepsol can be used^TMPolyethylene glycol (macrogol) and Tween^TM61. Cocoa butter, lauryl butter, glycerogelatin, and the like.

The pharmaceutical composition may be administered to a subject in various routes and amounts depending on the condition of the patient and whether there are side effects, and the optimal route, dose and frequency of administration may be selected by those skilled in the art within an appropriate range. In addition, the pharmaceutical composition may be administered in combination with other drugs or physiologically active substances known for their therapeutic effects on diseases, or may be formulated in the form of a combined preparation with other drugs.

The pharmaceutical compositions may be administered parenterally, including intratumoral, intraperitoneal, subcutaneous, intradermal, intranodal, and intravenous administration, and the like. Preferably, it may be intratumoral, intraperitoneal or intravenous administration. In another aspect, the dosage of the pharmaceutical composition may be determined according to the administration regimen, the dosage and the health condition of the patient.

Another aspect of the invention provides a method of treating cancer comprising administering to a subject in need thereof an oncolytic virus comprising a nucleotide sequence encoding the HSV-TK fragment or variant thereof and GCV or ACV.

The oncolytic viruses, GCV and ACV are as described above.

The dose of the oncolytic virus is determined by the condition and weight of the individual, the severity of the disease, and the drugThe type of substance, the route and time of administration vary and may be suitably selected by those skilled in the art the dosage may be such that the patient receives 1 × 10³To 1 × 10¹⁸Individual virus particles, infectious virus units (TCID)₅₀) Or plaque forming units (pfu) in particular, the dose may be 1 × 10³、2×10³、5×10³、1×10⁴、2×10⁴、5×10⁴、1×10⁵、2×10⁵、5×10⁵、1×10⁶、2×10⁶、5×10⁶、1×10⁷、2×10⁷、5×10⁷、1×10⁸、2×10⁸、5×10⁸、1×10⁹、2×10⁹、5×10⁹、1×10¹⁰、5×10¹⁰、1×10¹¹、5×10¹¹、1×10¹²、1×10¹³、1×10¹⁴、1×10¹⁵、1×10¹⁶、1×10¹⁷、1×10¹⁸Or more virus particles, infectious virus units (TCID)₅₀) Or plaque forming units (pfu), which may include various values and ranges therebetween, preferably, the oncolytic virus may be at 1 × 10³To 1 × 10¹⁰In one example of the invention, the oncolytic virus is 1 × 10⁶、1×10⁷And 1 × 10⁸Doses of pfu were administered.

In addition, GCV or ACV may be administered at a daily dose of 0.1 μ g/kg to 50 mg/kg. Specifically, the daily dose of GCV or ACV may be 0.1. mu.g/kg to 50mg/kg, 1. mu.g/kg to 40mg/kg, 5. mu.g/kg to 30mg/kg, or 10. mu.g/kg to 20 mg/kg. In one example of the invention, GCV is administered at a daily dose of 50 mg/kg.

The GCV or ACV is administered at least once during or after the administration of the oncolytic virus. In particular, GCV or ACV may be administered for about 2 weeks starting 24 hours after administration of the oncolytic virus. After administration of the oncolytic virus, GCV or ACV may be administered twice weekly for 5 to 18 consecutive days.

The cancer may be selected from the group consisting of: lung cancer, large intestine cancer, prostate cancer, thyroid cancer, breast cancer, brain cancer, head and neck cancer, esophageal cancer, skin cancer, thymus cancer, stomach cancer, colon cancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer, rectal cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, non-small cell lung cancer, bone cancer, intraocular melanoma, perianal cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulval cancer, hodgkin's disease, small intestine cancer, endocrine adenocarcinoma, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic leukemia, acute leukemia, lymphocytic lymphomas, kidney cancer, ureter cancer, renal cell cancer, renal pelvis cancer, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brain stem glioma, pituitary adenoma, or a combination thereof.

The pharmaceutical composition of the present invention may further comprise a physiologically acceptable carrier. In addition, the pharmaceutical composition of the present invention may further comprise suitable excipients and diluents conventionally used in the preparation of pharmaceutical compositions. In addition, it can be formulated into an injectable form according to a conventional method.

The pharmaceutical compositions may be formulated as sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, suppositories, and the like for parenteral administration. For non-aqueous solutions and suspensions, propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), injectable esters (e.g., ethyl oleate), and the like may be used. As suppository base, Witepsol can be used^TMPolyethylene glycol (macrogol) and Tween^TM61. Cocoa butter, lauryl butter, glycerogelatin, and the like.

The pharmaceutical composition may be administered to a subject in various routes and amounts depending on the condition of the patient and whether there are side effects, and the optimal route, dose and frequency of administration may be selected by those skilled in the art within an appropriate range. In addition, the pharmaceutical composition may be administered in combination with other drugs or physiologically active substances known for their therapeutic effects on diseases, or may be formulated in combination with other drugs.

The pharmaceutical compositions may be administered parenterally, including intratumoral, intraperitoneal, subcutaneous, intradermal, intranodal, and intravenous administration, and the like. Preferably, it may be intratumoral, intraperitoneal or intravenous administration. In another aspect, the dosage of the pharmaceutical composition may be determined according to the administration regimen, the dosage and the health condition of the patient.

As used herein, the term "individual" refers to a human that can alleviate, inhibit, or treat a disease, or a human suffering from the disease, by administering the cancer cell-targeting composition of the present invention.

In addition, the oncolytic virus and GCV may be administered in combination with other drugs or physiologically active substances known for their therapeutic effects on diseases, or may be formulated in the form of a combined preparation with other drugs.

In another aspect of the invention, there is provided a method of preparing a recombinant vaccinia virus expressing an HSV-TK fragment or variant thereof comprising the steps of: i) transfecting a shuttle plasmid comprising a nucleotide sequence encoding an HSV-TK fragment or variant thereof into a host cell and treating the host cell with a wild-type vaccinia virus; ii) culturing the resulting host cell; and iii) obtaining the recombinant vaccinia virus from the resulting culture.

The HSV-TK fragment may be an HSV1-TK fragment. An embodiment of an HSV1-TK fragment may be any one of the following fragments: SEQ ID NO: 1, 1 to 195, 24 to 149, or 30 to 46 amino acid residues from the C-terminus of the amino acid sequence shown in 1. In particular, the HSV-TK fragment may be one of the following fragments: SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 195, 180, 190 of the amino acid residues of the C-terminal of the amino acid sequence shown in 1. Preferably, the HSV1-TK fragment may be one of the following fragments: SEQ ID NO: 1, wherein 24, 30, 70, 99, 149 or 195 amino acid residues are continuously deleted from the C-terminal end of the amino acid sequence shown in the specification. The HSV1-TK fragment may have reduced sensitivity to GCV due to deletion of certain amino acid residues at the C-terminus.

An example of the HSV1-TK fragment may be one of the following fragments: SEQ ID NO: 1, wherein 195, 149, 99, 70, 46, 30 or 24 amino acid residues are continuously deleted from the C-terminal end of the amino acid sequence shown in the figure. Specifically, the HSV1-TK fragment may have any one of the amino acid sequences selected from the group consisting of: SEQ ID NO: 2-6.

The nucleotide sequence encoding the HSV1-TK fragment may be a nucleotide sequence encoding any of the following HSV1-TK fragments: SEQ ID NO: 1, 1 to 195, 24 to 149, or 30 to 46 amino acid residues from the C-terminus of the amino acid sequence shown in 1. Specifically, the nucleotide sequence encoding the HSV1-TK fragment may be a nucleotide sequence encoding any one of the following amino acid sequences: SEQ ID NO: 2-6. Preferably, the nucleotide sequence encoding the HSV1-TK fragment may be SEQ ID NO: 9, or a nucleotide sequence shown in the specification.

The variant of the HSV-TK fragment may be a variant of the HSV1-TK fragment. In particular, the variant of the HSV1-TK fragment may be a variant in which at least one amino acid residue constituting the HSV1-TK fragment is substituted. In particular, the variant of the HSV1-TK fragment may comprise the amino acid sequence as set forth in SEQ ID NO: 1 to 145 amino acid residues of the amino acid sequence shown in 1. In one embodiment, the variant of the HSV1-TK fragment may have an amino acid sequence as set forth in SEQ ID NO: 7 or 8. The nucleotide sequence encoding the variant of the HSV1-TK fragment may be SEQ ID NO: 10 or 11.

Transfection may be performed by various methods. In particular, transfection methods such as CaCl may be applied₂Precipitation, Hanahan method (by in CaCl)₂DMSO (dimethyl sulfoxide) used in precipitation to increase efficiency), electroporation, calcium phosphate precipitation, protoplast fusion, agitation with silicon carbide fibers, agrobacterium-mediated transfection, transfection using PEG, dextran sulfate, lipofectamine, and desiccation/suppression-mediated transfection.

In addition, the host cell can be a yeast cell (e.g., Saccharomyces cerevisiae and Schizosaccharomyces pombe, etc.); fungal cells (e.g., pichia pastoris); insect cells (e.g., drosophila and spodoptera Sf9 cells); animal cells (e.g., CHO cells, HeLa cells, COS cells, NSO cells, 293 cells, and bowmelanoma cells); or a plant cell. In one embodiment, the host cell may be a HeLa cell.

The wild-type vaccinia virus may be, but is not limited to, Western Reserve (WR), NYVAC (New York vaccinia virus), Wyeth (New York City health office; NYCBOH), LC16m8, Listeria (Lister), Copenhagen (Copenhagen), Temple (Tian Tan), USSR, Tash Stem (TashKent), Evens (Evans), IHD-J (International sanitation-J), or IHD-W (International sanitation-white) strains.

The shuttle plasmid and wild-type vaccinia virus may comprise homologous regions of the same vaccinia virus gene. The shuttle plasmid and the wild-type vaccinia virus may preferably comprise different origins of replication and/or markers so that each element can be screened.

Host cells can be cultured using methods known in the art. Specifically, the culture method is not particularly limited as long as it can produce a recombinant vaccinia virus expressing the HSV1-TK fragment of the present invention. Specifically, the culture may be continuously performed in a fed-batch or a repeated fed-batch process.

The medium used for the culture may be a conventional medium containing an appropriate carbon source, nitrogen source, amino acid, vitamin, etc., wherein the requirements of a specific strain can be satisfied in an appropriate manner by adjusting temperature, pH, etc. under aerobic conditions. As the carbon source, a mixed sugar of glucose and xylose may be used as a main carbon source. Other carbon sources may include sugars and carbohydrates, such as sucrose, lactose, fructose, maltose, starch, and cellulose; fats and oils (e.g., soybean oil, sunflower oil, castor oil, and coconut oil); fatty acids (e.g., palmitic acid, stearic acid, and linoleic acid); alcohols (e.g., glycerol and ethanol); an organic acid (e.g., acetic acid). In addition, the carbon source may be used alone or in combination. In one embodiment, the medium may be an EMEM medium containing fetal bovine serum.

In addition, suitable precursors may be used in the culture medium. The raw materials may be added to the culture in a batch, fed-batch or continuous manner by a suitable method during the culture, but are not particularly limited thereto. The pH of the culture can be adjusted in a suitable manner by using basic compounds, such as sodium hydroxide, potassium hydroxide, ammonia, or acidic compounds, such as phosphoric acid or sulfuric acid.

In addition, the formation of bubbles can be suppressed by using an antifoaming agent (e.g., fatty acid polyglycol ester). To maintain aerobic conditions, oxygen or an oxygen-containing gas (e.g., air) is injected into the culture. The temperature of the culture is usually in the range of 27 ℃ to 37 ℃ and preferably in the range of 30 ℃ to 35 ℃. The incubation time may be 2 hours to 80 hours. Preferably, the culture time may be 4 to 76 hours.

The method for obtaining virus from culture comprises: harvesting the host cells, subjecting the cells to repeated freeze/thaw cycles to obtain a cell lysate, performing repeated freeze/thaw cycles on the cell lysate to obtain crude viruses, and repeating plaque isolation on the crude viruses to obtain pure recombinant vaccinia virus. However, the method is not limited thereto.

Another aspect of the invention provides the use of the oncolytic virus in the treatment of cancer.

Another aspect of the invention provides the use of the pharmaceutical composition in the treatment of cancer.

Another aspect of the invention provides the use of the oncolytic virus for the manufacture of a medicament for the treatment of cancer.

Another aspect of the invention provides the use of the pharmaceutical composition in the manufacture of a medicament for the treatment of cancer.

MODE OF THE INVENTION

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.