WO2023128048A1 - Cancer therapeutic combination comprising cancer-targeting trans-splicing ribozyme and immune checkpoint inhibitor - Google Patents

Abstract

The present invention relates to a cancer therapeutic combination comprising a cancer-targeting trans-splicing ribozyme and an immune checkpoint inhibitor. The ribozyme of the present invention exhibits cancer cell-specific expression, is superb in terms of expression efficiency at a posttranscriptional level, anticancer activity against heterogeneous cancers, and stability in normal liver tissues, has a synergistic therapeutic effect on cancer when administered in combination with an immune checkpoint inhibitor, and can be used as a therapeutic modality for cancer resistant to an immune checkpoint inhibitor.

Description

Cancer therapeutic combination comprising a cancer-specific trans-splicing ribozyme and an immune checkpoint inhibitor

The present invention relates to a cancer therapeutic combination comprising a cancer-specific trans-splicing ribozyme and an immune checkpoint inhibitor, and the like.

Telomerase is a ribonucleoprotein that repeatedly adds TTAGGG sequences to the ends of telomeres present at the 3' end of chromosomes to restore shortened telomere regions during DNA replication, resulting in cell perpetuity. enable proliferation. Telomerase is one of the most important enzymes that regulate the immortality and proliferative capacity of cancer cells. Hematopoietic cells and 80-90% of cancer cells have telomerase activity, while normal cells near cancer cells do not have the activity. Moreover, telomerase reactivation has a major effect on the permanent growth of advanced metastatic cancer.

Human telomerase consists of two components: human telomerase RNA (hTR) acting as substrate RNA and human telomerase reverse transcriptase (hTERT) acting as a catalyst. The human telomerase reverse transcriptase (hTERT) gene is expressed in proportion to telomerase activity, and there is a strong correlation between the intracellular hTERT level and cellular telomerase activity. In particular, TERT activity is observed in more than 90% of cancer patients.

Recently, as a trans-splicing ribozyme targeting hTERT has become known, attempts to develop cancer therapeutics using the trans-splicing ribozyme are being actively pursued. However, the combination of a trans-splicing ribozyme and a tissue-specific promoter exhibits high tissue specificity but very low expression efficiency, resulting in a problem in terms of therapeutic efficiency. In addition, since telomerase activity is also shown in normal cells such as stem cells, hematopoietic stem cells, germ cells, and regenerating normal liver cells, treatment targeting hTERT is unlikely to cause toxicity to these cells. can Although hepatocytes are weak, 5% of normal hepatocytes have telomerase activity, and regenerating liver is known to increase telomerase activity. In particular, hepatocellular carcinoma (HCC) is mostly accompanied by liver cirrhosis, and TERT is expressed at a low level in non-tumorous hepatocytes constituting regenerative nodules at the site of liver cirrhosis.

Korean Patent Publication No. 10-2016-0038674 discloses a ribozyme containing a tissue-specific promoter, a trans-splicing ribozyme targeting a cancer-specific gene, and a target gene linked to the 3' exon of the ribozyme- Disclosed are a recombinant vector in which a nucleic acid sequence recognizing microRNA-122 (microRNA-122, miR-122) is additionally linked to a target gene expression cassette, and a use of a ribozyme expressed therefrom for preventing or treating liver cancer. The miR-122 is a microRNA known to be highly overexpressed in normal liver, and its expression decreases when liver cancer progresses. Based on this phenomenon, the prior art introduces the miR-122 target site into the 3' UTR region of the ribozyme expression vector, so that the ribozyme delivered to the liver is not expressed by the overexpressed miR-122 in the normal liver. In liver cancer where the level of miR-122 is lowered, it is expressed and able to act.

Immune checkpoint inhibitors for maintaining the immune function of T cells are the fastest-growing field in the cancer treatment market. there is. In addition, there is a problem in that resistant cancer cells having resistance to the drug are generated due to repeated drug exposure even in patients who show a response. As an alternative to this, a combined treatment method with gene therapy to overcome the limitations of immunotherapeutic agents is attracting attention.

The present invention is to solve the above problems, and to overcome the limitations of immunotherapy, a cancer-specific trans-strain with excellent safety and expression efficiency and excellent anticancer activity against heterogeneous cancer as a gene therapy used in combination with an immune checkpoint inhibitor in order to overcome the limitations of immunotherapy. Its purpose is to provide a plicing ribozyme.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

As a result of intensive research on a gene therapy method for resolving the side effects and limitations of cancer immunotherapy, the inventors of the present invention found that a trans-strain targeting a cancer-specific gene sequence in cancer cells in combination with the immunotherapy The present invention was completed by confirming that the side effects of the immunotherapy method can be reduced and the cancer treatment effect can be improved when the plicing ribozyme is expressed or administered.

That is, the present invention was completed after the present inventors confirmed a synergistic effect that exceeded the sum of the anticancer effects of each when the present inventors administered the gene therapy and existing immunotherapies together, and the present invention is a cancer-specific gene sequence in a therapeutically effective amount A trans-splicing ribozyme expression vector targeting, a gene delivery system comprising the vector, or a ribozyme expressed from the vector; and a therapeutically effective amount of an immune checkpoint inhibitor; a treatment for use in a cancer treatment method comprising administering the therapeutic combination to a mammal in a combined formulation or alternatingly; Provides enemy combinations.

In the present invention, a therapeutically effective amount means to reduce the number of cancer cells and/or; reduce tumor size and/or; inhibit (ie, slow to some extent and preferably stop) cancer cell invasion into peripheral organs; inhibit (ie, slow to some extent and preferably stop) tumor metastasis; inhibit to some extent tumor growth; It refers to a dose that can relieve one or more symptoms associated with cancer to some extent.

In one embodiment of the present invention, the expression vector may include a cytomegalovirus (CMV) promoter operably linked to the ribozyme gene, and splicing donor / at the 5' end of the ribozyme gene It may include a splicing donor/splicing acceptor sequence (SD/SA sequence), and may include a Woodchuck hepatitis virus Posttranscriptional Regulatory Element (WPRE) at the 3' end.

As another embodiment of the present invention, the expression vector may include a target gene linked to the 3' exon of the ribozyme gene.

As another embodiment of the present invention, the expression vector contains a gene encoding a sequence complementary to part or all of microRNA-122 (microRNA-122, miR-122) at the 3'-UTR end of the ribozyme gene. can do.

As another embodiment of the present invention, the cancer-specific gene sequence is essential for the growth, proliferation, and/or metastasis of cancer cells and is not limited as long as it is a gene that is overexpressed in cancer cells than in normal cells, but is preferably TERT (Telomerase Reverse Transcriptase) mRNA, alphafetoprotein (AFP) mRNA, carcinoembryonic antigen (CEA) mRNA, prostate-specific antigen (PSA) mRNA, cytoskeleton-associated protein 2 (CKAP2) mRNA, and mutant rat sarcoma (RAS) mRNA. There can be more than one gene.

As another embodiment of the present invention, the TERT mRNA sequence may consist of or include the nucleotide sequence of SEQ ID NO: 2.

As another embodiment of the present invention, the trans-splicing ribozyme may consist of or include the nucleotide sequence of SEQ ID NO: 3.

As another embodiment of the present invention, the target gene may be a cancer treatment gene or a reporter gene, and the cancer treatment gene may be a drug susceptibility gene, an apoptosis gene, a cell proliferation inhibitory gene, a cytotoxic gene, or a tumor suppressor gene. It may be at least one gene selected from the group consisting of a gene, an antigenic gene, a cytokine gene, and an angiogenesis inhibitor gene, and the drug susceptibility gene may be a Herpes Simplex Virus thymidine kinase (HSVtk) gene.

As another embodiment of the present invention, the HSVtk gene may consist of or include the nucleotide sequence of SEQ ID NO: 4.

As another embodiment of the present invention, the reporter gene is luciferase, green fluorescent protein (GFP), modified green fluorescent protein (mGFP), enhanced green fluorescent protein (enhanced green fluorescent protein) EGFP), red fluorescent protein (RFP), modified red fluorescent protein (mRFP), enhanced red fluorescent protein (ERFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), yellow fluorescent protein (YFP) ), enhanced yellow fluorescent protein (EYFP), indigo fluorescent protein (CFP) or enhanced indigo fluorescent protein (ECFP).

As another embodiment of the present invention, the gene encoding a sequence complementary to miR-122 may express a sequence complementary to part or all of miR-122, preferably a sequence complementary to all of miR-122. The sequence may be expressed, but is not limited to substitution, deletion or insertion of some bases as long as it binds to miR-122 in the cell and induces degradation of the ribozyme of the present invention. In the present invention, the gene encoding a sequence complementary to miR-122 may consist of or include the nucleotide sequence of SEQ ID NO: 5, and may repeatedly include the nucleotide sequence of SEQ ID NO: 5.

As another embodiment of the present invention, the gene delivery system may be a viral vector including the expression vector, and the viral vector may be an adenoviral vector.

The present inventors confirmed the anticancer effect of the combination in various carcinomas through specific experiments, and verified the synergistic effect in a liver cancer animal model and a brain tumor animal model (orthotopic tumor model), which is the target of prevention or treatment of the present invention The cancer is one or more cancers selected from the group consisting of liver cancer, glioblastoma, bile duct cancer, lung cancer, pancreatic cancer, melanoma, bone cancer, breast cancer, colon cancer, stomach cancer, prostate cancer, leukemia, uterine cancer, ovarian cancer, lymphoma, and brain cancer However, preferably, it may be a cancer in which miR-122 is not substantially expressed in cancer tissue, and specifically, the copy number of miR-122 in cancer tissue is the copy number of the ribozyme expressed in cancer tissue by the pharmaceutical composition. It may be cancer less than 100 times.

In particular, it can be seen from the microenvironment mimic characteristics (BBB, cerebral blood vessel specific immune activity, etc.) of the orthotopic tumor model that the combination of the present invention can be provided as an effective anticancer agent for brain cancer that is difficult to penetrate. As another embodiment of the present invention, the liver cancer is not limited to the etiology, and non-limiting examples of the etiology include hepatitis B virus, hepatitis C in which the expression of miR-122 is reduced in liver cancer tissue. Virus, alcohol, chronic hepatitis, cirrhosis, non-alcoholic fatty liver, aflatoxin, and family history.

In another embodiment of the present invention, the therapeutically effective amount of the expression vector, gene delivery system, or ribozyme and the therapeutically effective amount of the immune checkpoint inhibitor may be administered as a combined formulation.

In another embodiment of the present invention, the therapeutically effective amount of the expression vector, gene delivery system, or ribozyme and the therapeutically effective amount of the immune checkpoint inhibitor may be administered alternately.

As another embodiment of the present invention, the immune checkpoint inhibitor is CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, TIGIT, CD47, VISTA and /or an inhibitor of A2aR.

As another embodiment of the present invention, the therapeutically effective amount of the expression vector, gene delivery system, or ribozyme may be administered by a route selected from the group consisting of intravenous, intraarterial, intramuscular and subcutaneous administration, orally or It may be administered in the form of an injection, but is preferably administered directly into a tumor.

In addition, the present invention provides a pharmaceutical composition for the treatment of immune checkpoint inhibitor-resistant cancer comprising the above expression vector, gene transfer system, or ribozyme as an active ingredient, wherein the immune checkpoint inhibitor is CTLA-4, PD-1 , PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, TIGIT, CD47, VISTA and/or A2aR inhibitors, but non-limiting examples thereof include nivolumab , pembrolizumab, atezolizumab, avelumab, durvalumab, and ipilimumab.

In addition, the present invention provides a cancer treatment method comprising the step of simultaneously or sequentially administering the above-described expression vector, gene delivery system, or ribozyme and an immune checkpoint inhibitor to a subject.

As an embodiment of the present invention, the cancer treatment method may further include administering GCV to the subject, and GCV may be administered to the subject by intraperitoneal injection.

More specifically, the immune checkpoint inhibitor may be administered after the administration of the above-described expression vector, gene delivery system, or ribozyme is completed.

Administration of the expression vector, gene delivery system, or ribozyme described above in the present invention may be performed by intratumoral injection, and may be administered twice or more at intervals of 24 to 48 hours, and may be administered once. The dose at the time of administration may be 1 x 10 ⁹ VP to 2.5 x 10 ¹² VP, but the present inventors confirm that the synergistic anti-cancer effect that occurs when administered in combination with an immune checkpoint inhibitor increases in a concentration-dependent manner of RZ-001 As such, the administration dose is preferably administered at a high concentration within a range that does not induce liver toxicity in the subject in consideration of the type of the gene therapy delivery system.

In addition, in the present invention, the immune checkpoint inhibitor may be administered by intravenous injection, may be administered after completion of administration of the above-described gene therapy, may be administered twice or more at intervals of 24 to 48 hours, and may be administered 1 The dosage for each administration may be 1 to 30 mg/kg, preferably 3 to 20 mg/kg, but the dosage may be applied variably depending on the type of immune checkpoint inhibitor to be administered and the condition of the subject. For example, 20 mg/kg for atezolizumab and 3 mg/kg for nivolumab can be administered, for example, for human administration, typically 1200 mg for atezolizumab and 240 mg for nivolumab in a single administration. can be administered.

In addition, the present invention provides a method for treating immune checkpoint inhibitor-resistant cancer comprising administering the expression vector, gene delivery system, or ribozyme described above to a subject.

As another embodiment of the present invention, the subject is not limited to any mammal in need of cancer treatment, but may be preferably a human.

In addition, the present invention provides a trans-splicing ribozyme expression vector targeting the cancer-specific gene sequence for the preparation of a drug for the treatment of immune checkpoint inhibitor-resistant cancer, a gene delivery system including the vector, or a vector from the vector. Uses of the expressed ribozymes are provided.

The trans-splicing ribozyme according to the present invention does not act on normal tissues, is specifically expressed in cancer tissues, has high safety, and has excellent anticancer activity because it induces the expression of anticancer genes at the same time as the removal of target RNA. can be useful for In addition, since the ribozyme according to the present invention exhibits an increased cancer treatment effect in combination with an immune checkpoint inhibitor, it can be used as a drug for the treatment of carcinoma resistant to immunotherapeutic agents.

1 is a schematic diagram showing the construction of a CMV promoter-based hTERT-targeting trans-splicing ribozyme and a target gene expression cassette of the present invention, and a composition related to the combination including an immune checkpoint inhibitor.

FIG. 2 is a graph showing tumor size (FIG. 2a) and tumor weight (FIG. 2b) after tumor formation was induced by injecting glioblastoma cell line LN229 into nude mice and ECRT-122T adenovirus was administered.

3 is a graph showing tumor size by inducing tumor formation by injecting U87MG cell line, which is a glioblastoma cell, into nude mice and then administering ECRT-122T adenovirus.

4 is a graph showing the results of measuring AST and ALT levels after injecting ECRT-122T adenovirus into normal ICR mice.

5 is a graph showing the results of measuring body weight (A), food consumption (B), and liver weight (C) of mice after injecting ECRT-122T adenovirus into normal ICR mice.

Figure 6 shows the results of histopathological examination of the liver performed after injecting different doses of ECRT-122T adenovirus into normal ICR mice.

7 is a graph showing the results of measuring AST and ALT levels after injecting ECRT-122T adenovirus into normal ICR mice and treating them with GCV.

Figure 8 is a graph showing the results of measuring body weight (Figure 8a), food consumption (Figure 8b) and liver weight (Figure 8c) of normal ICR mice injected with ECRT-122T adenovirus and treated with GCV. .

9 shows the results of histopathological examination of the liver performed after injecting different doses of ECRT-122T adenovirus into normal ICR mice and treating them with GCV.

10a to 10e show results of comparing anticancer efficacy by inducing tumor formation by injecting SNU398 cells into a mouse xenograft subcutaneous model and then administering CRT-122T or ECRT-122T adenovirus.

11 is a schematic diagram of an experiment to confirm the effect of the combined administration of adenovirus and an immune checkpoint inhibitor.

Figure 12 is a diagram comparing body weight changes in mouse xenograft subcutaneous model mice administered with GCV and adenovirus and/or IgG after administration of PBMC and FACS analysis of the degree of humanization of mice after PBMC injection targeting CD45+. .

Figure 13 shows the results of comparing changes in tumor tissue size according to administration of an immune checkpoint inhibitor (nivolumab (Fig. 13a) or atezolizumab (Fig. 13b)) and/or adenovirus in a PBMC-humanized liver cancer model.

Figure 14 shows the results of comparison of blood cytokine levels following administration of an immune checkpoint inhibitor (nivolumab or atezolizumab) and/or adenovirus in a PBMC-humanized liver cancer model.

Figure 15 shows the results of confirming the degree of T cell infiltration into tumor tissue at the RNA level following administration of an immune checkpoint inhibitor (nivolumab or atezolizumab) and/or adenovirus in a PBMC-humanized liver cancer model.

Figure 16 shows the results of confirming the degree of T cell infiltration into tumor tissue following the administration of an immune checkpoint inhibitor (nivolumab or atezolizumab) and/or adenovirus in a PBMC-humanized liver cancer model using immunohistochemical staining.

17 shows liver toxicity evaluation results of immune checkpoint inhibitors (nivolumab or atezolizumab) and/or adenovirus administration in a PBMC-humanized liver cancer model.

18a and 18b show the results of comparing changes in tumor tissue size according to the administration concentration of an immune checkpoint inhibitor (atezolizumab) and/or adenovirus in a PBMC-humanized liver cancer model.

Figure 19 shows the results of confirming the degree of T cell infiltration into tumor tissue according to the administration of an immune checkpoint inhibitor (atezolizumab) and/or low concentration adenovirus in a PBMC-humanized liver cancer model using immunohistochemical staining.

20 shows the results of confirming the expression levels of AKT, S6, and PDL1 in the cancer signaling pathway according to administration of an immune checkpoint inhibitor (atezolizumab) and/or low concentration adenovirus in a PBMC-humanized liver cancer model.

21 shows liver toxicity evaluation results of administration of an immune checkpoint inhibitor (atezolizumab) and/or low concentration adenovirus in a PBMC-humanized liver cancer model.

22 shows the results of comparison of changes in tumor tissue size according to administration of an immune checkpoint inhibitor and/or mCRT-122T in a liver cancer subcutaneous transplantation model.

23 shows liver toxicity evaluation results of administration of an immune checkpoint inhibitor and/or mCRT-122T in a liver cancer subcutaneous transplant model.

The present invention can apply various transformations and can have various embodiments. Hereinafter, specific embodiments will be illustrated in the drawings and described in detail in the detailed description. However, it should be understood that this is not intended to limit the present invention to specific embodiments, and includes all transformations, equivalents, and substitutes included in the spirit and scope of the present invention. In describing the present invention, if it is determined that a detailed description of related known technologies may obscure the gist of the present invention, the detailed description will be omitted.

[Example]

Hereinafter, the present invention will be described in more detail.

In the present invention, when a part "includes" a certain component, it means that it may further include other components, not excluding other components unless otherwise stated.

[recombinant vector]

One aspect of the present invention is

(i) a cytomegalovirus promoter; and

(ii) a ribozyme-target gene expression cassette comprising a trans-splicing ribozyme targeting a cancer-specific gene sequence and a target gene linked to the 3' exon of the ribozyme;

The expression cassette has a splicing donor/splicing donor sequence (SD/SA sequence) linked to the 5' end of the ribozyme-target gene expression cassette and WPRE linked to the 3' end,

상기 WPRE의 3' 말단에는 마이크로 RNA-122를 인식하는 핵산 서열이 추가로 연결된 것을 특징으로 하는 재조합 벡터이다.

It is a recombinant vector characterized in that a nucleic acid sequence recognizing microRNA-122 is additionally linked to the 3' end of the WPRE.

The cytomegalovirus (CMV) promoter included in the recombinant vector according to the present invention can increase ribozyme expression in Hep3B, SNU398, and SNU449 cell lines more than the PEPCK promoter. In addition, when the SD/SA sequence and WPRE are included as components at both ends of the ribozyme and the target gene along with the CMV promoter, the expression efficiency of the ribozyme in vivo of the recombinant vector is higher. Based on this, the recombinant vector of the present invention simultaneously uses the CMV promoter, SD/SA sequence, and WPRE, and additionally includes a nucleic acid sequence (miR-122T) that recognizes miR-122, thereby enabling treatment of various cancer cells as well as liver cancer cells. It is characterized by what made it possible.

As used herein, the term "vector" is an expression vector capable of expressing a target gene in a suitable host cell, and refers to a gene construct containing essential regulatory elements operably linked to express a gene insert contained in the vector.

As used herein, the term “operably linked” refers to functional linkage between a nucleic acid expression control sequence that performs a general function and a nucleic acid sequence encoding a gene of interest.

For example, operably linking a ribozyme coding sequence to a promoter brings expression of the ribozyme coding sequence under the influence or control of the promoter. Two nucleic acid sequences (a ribozyme coding sequence and a promoter region sequence at the 5' end of the sequence) are operably linked when the action of a promoter is induced and the ribozyme coding sequence is transcribed, and the linkage between the two sequences changes the frame. If a frameshift mutation is not induced and the expression control sequence does not inhibit the expression of the ribozyme, it can be considered to be operably linked. Operable linkage with the recombinant vector can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cutting and linking can use enzymes generally known in the art.

The vector according to the present invention includes a signal sequence or leader sequence for membrane targeting or secretion in addition to expression control elements such as a promoter, operator, initiation codon, stop codon, polyadenylation signal, and enhancer, and can be prepared in various ways according to the purpose. there is. The vector's promoter may be constitutive or inducible. In addition, the expression vector may include a selectable marker for selecting host cells containing the vector, and may include an origin of replication in the case of a replicable expression vector. Vectors can replicate autonomously or integrate into host DNA.

The vector according to the present invention may preferably be a plasmid vector, a cosmid vector or a viral vector, and most preferably a viral vector. The viral vector is preferably a retrovirus, for example, human immunodeficiency virus (HIV), mouse leukemia virus (MLV), avian sarcoma / leukemia virus (Avian sarcoma / leucosis virus (ASLV), spleen necrosis virus (SNV), Rous sarcoma virus (RSV), mouse mammary tumor virus (MMTV), adenovirus, adeno-related virus (Adeno-associated virus, AAV), or a vector derived from a herpes simplex virus (Herpes simplex virus, HSV), etc., but is not limited thereto. The recombinant vector according to the present invention may most preferably be a recombinant adenoviral vector.

The term "expression cassette" used in the present invention includes a CMV promoter and a trans-splicing ribozyme-target gene, and at each end of the trans-splicing ribozyme-target gene, an SD/SA sequence and a WPRE sequence is present, and a nucleic acid sequence recognizing miR-122 is additionally linked to the 3' end of the WPRE, thereby trans-splicing ribozyme - refers to a unit cassette capable of expressing a target gene.

The trans-splicing ribozyme-target gene expression cassette according to the present invention may further include a sequence regulating the transcription level, that is, a regulatory derivative, to the sequence linked to the trans-splicing ribozyme and the target gene. . Although not limited thereto, in the present invention, in particular, a splicing donor/splicing acceptor sequence (SD/SA sequence) and/or a Woodchuck hepatitis virus Posttranscriptional Regulatory Element (WPRE) are linked, A sequence recognizing miR-122 may be additionally linked to the 3' end of WPRE. As a result, the expression level of the ribozyme-target gene can be controlled, and the effect on normal hepatocytes can be minimized by allowing the ribozyme to be expressed only when miR-122 is below a certain level.

The ribozyme-target gene expression cassette according to the present invention preferably has a splicing donor/splicing donor sequence (SD/SA) linked to the 5' end of the ribozyme, and WPRE at the 3' end of the target gene. and a sequence recognizing miR-122 may be linked to the 3' end of the WPRE.

SD/SA according to the present invention increases transcription initiation, processing of RNA polymerase II and nucleocytoplasmic export of mRNA from the nucleus to the cytoplasm, and the WPRE according to the present invention It can increase the level of pre-mRNA by increasing mRNA processing and transport from the nucleus to the cytoplasm, respectively. Through the configuration as described above, the RNA level of the ribozyme can be significantly increased in the cell to increase the death of cancer cells in vivo while allowing the specific expression of the ribozyme to be expressed in a cancer cell, thereby reducing toxicity to normal cells.

The SD/SA sequence according to the present invention is a sequence corresponding to the beginning and end of an intron cut out in a splicing reaction to remove an intron of an RNA transcript. In general, the SD sequence is a GU sequence at the 5' end of the intron , and the SA sequence may be an AG sequence at the 3' end of the intron.

WPRE (Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE)) according to the present invention refers to a sequence that induces a tertiary structure that promotes transcription in DNA and increases gene expression.

In the present invention, the SD / SA sequence and the WPRE sequence may include the sequences of SEQ ID NO: 6 and SEQ ID NO: 7, respectively, but are not limited thereto as long as they are present in the target gene expression cassette and can promote the expression of the target gene.

The nucleic acid sequence recognizing miR-122 according to the present invention is referred to as miR-122T (microRNA-122 target site) within the specification. The miR-122T may include the sequence of SEQ ID NO: 5 one or more times, for example, 1 to 10 times, preferably 1 to 5 times, more preferably 1 to 3 times. can miR-122 is normally expressed in normal hepatocytes, but its expression level is reduced in hepatoma cells. Using this, it is possible to develop a therapeutic agent with increased sensitivity and specificity for liver cancer cells. In the present invention, by linking a nucleic acid sequence recognizing miR-122 to a ribozyme to which a target gene is linked, expression of a liver cancer cell-specific ribozyme can be achieved. made it possible.

In one embodiment of the present invention, when SD/SA and WPRE are additionally linked, the expression of ribozymes is increased to further increase the effect of inducing cell death. In addition, by linking miR-122T targeting miR-122, cell death is not induced in normal hepatocytes with normal expression of miR-122, but only in liver cancer cells in which miR-122 expression is reduced, thereby inducing cell death in liver cancer cells. It was confirmed that a specific treatment was possible.

The term "cancer-specific gene" used in the present invention refers to a gene that is specifically expressed or markedly overexpressed only in cancer cells. The cancer-specific gene may add a feature that allows the ribozyme according to the present invention to act in a cancer-specific manner. These cancer-specific genes are preferably TERT (Telomerase reverse transcriptase) mRNA, AFP (alphafetoprotein) mRNA, CEA (carcinoembryonic antigen) mRNA, PSA (Prostate-specific antigen) mRNA, CKAP2 (Cytoskeleton-associated protein 2) mRNA, or It may be a mutant rat sarcoma (RAS) mRNA, more preferably a telomerase reverse transcriptase (TERT) mRNA, and most preferably a human telomerase reverse transcriptase (hTERT) mRNA sequence. The ribozyme of the present invention exhibits an anticancer effect even in cancer cell lines of tissues that do not substantially express miR-122, including the cancer-specific gene, and the present inventors are particularly interested in glioblastoma cell lines, colorectal cancer cell lines, melanoma cell lines, uterine When cervical cancer cell lines, lung cancer cell lines, osteosarcoma cell lines, breast cancer cell lines, and cholangiocarcinoma cell lines were treated with adenovirus expressing ECRT-122T, it was confirmed that cell death increased.

The term used in the present invention, “TERT (Telomerase reverse transcriptase)” is one of the most important enzymes that regulate the immortality and proliferation ability of cancer cells, and forms telomere structures on chromosomes to form chromosome ends. It means an enzyme that inhibits cell aging through its role in protecting cells. In normal cells, each time the cell divides, the length of the telomeres decreases little by little, and eventually the genetic material is lost and the cell dies. However, in cancer cells, this enzyme continuously elongates telomeres, so the cells do not die, and it is known as a serious obstacle to cancer treatment by directly contributing to the immortality of cancer cells. In the present invention, hTERT mRNA including the sequence of SEQ ID NO: 2 may be used as a cancer-specific gene, but is not limited thereto.

As used herein, the term "promoter" is involved in the binding of RNA polymerase to initiate transcription as a portion of DNA. In general, it is located adjacent to and upstream of the target gene, and is a binding site for RNA polymerase or a transcription factor, a protein that induces RNA polymerase, and can induce the enzyme or protein to be located at the correct transcription start site. can That is, it is located at the 5' site of the gene to be transcribed in the sense strand and induces RNA polymerase to bind to the corresponding position directly or through a transcription factor to initiate mRNA synthesis for the target gene. A specific gene sequence have

The promoter according to the present invention is preferably a cytomegalovirus (CMV) promoter comprising the sequence of SEQ ID NO: 1 from the viewpoint of increasing gene expression.

In one embodiment of the present invention, when the CMV promoter is introduced into the recombinant vector according to the present invention, the ribozyme expression efficiency is excellent, and miR-122T possessed by the recombinant vector is regulated by miR-122, but high ribozyme By expression, it was confirmed that cell death was induced and anticancer effects were exhibited despite a certain degree of miR-122 expression.

The term "ribozyme" used in the present invention is a molecule composed of an RNA molecule that acts like an enzyme or a protein containing the RNA molecule, and is also called an RNA enzyme or catalytic RNA. An RNA molecule with a clear tertiary structure that performs chemical reactions and has catalytic or autocatalytic properties. Some ribozymes inhibit their activity by cleaving their own or other RNA molecules, and other ribozymes are known to catalyze the activity of ribosome aminotransferases. Such ribozymes may include hammerhead ribozymes, VS ribozymes, and hairpin ribozymes.

The ribozyme according to the present invention inhibits the activity of cancer-specific genes through the trans-splicing reaction of Group I introns, thereby exhibiting a selective anti-cancer effect, and is expressed in a conjugated form with a cancer therapeutic gene to treat cancer. genes can be activated. Therefore, any type of material may be used as long as it exhibits characteristics capable of inactivating cancer-specific genes and activating cancer therapeutic genes.

The ribozyme according to the present invention may preferably be a ribozyme targeting hTERT mRNA as described above, targeting cancer cells in which hTERT is overexpressed, specifically cleaving hTERT mRNA to inhibit its expression, and generating a therapeutic gene. It can play a role in specific expression.

As used herein, the term “trans-splicing” means linking RNAs from different genes to each other. Preferably, an hTERT target trans-splicing group I ribozyme that has been verified to have the ability to trans-splice by recognizing cancer-specific hTERT mRNA may be used.

The term "target gene" used in the present invention refers to a gene whose expression is induced by being linked to mRNA of a cancer-specific gene by the ribozyme.

The target gene according to the present invention may preferably be a gene for cancer treatment or a reporter gene, and most preferably a gene for cancer treatment.

As used herein, the term "anti-cancer therapeutic gene" refers to a polynucleotide sequence encoding a polypeptide that exhibits a therapeutic effect when expressed in cancer cells. The cancer therapeutic gene may be expressed in a conjugated form with the ribozyme or expressed independently to exhibit anticancer activity. These cancer therapeutic genes are preferably selected from the group consisting of drug susceptibility genes, apoptosis genes, cytostatic genes, cytotoxic genes, tumor suppressor genes, antigenic genes, cytokine genes, and anti-angiogenic genes. It may be one or more, and most preferably, it may be a drug susceptibility gene.

In the present invention, the cancer treatment gene may be used alone or two or more genes may be used in combination.

The drug-sensitizing gene according to the present invention is a gene encoding an enzyme that converts a non-toxic prodrug into a toxic substance, and is also called a suicide gene because cells into which the gene is introduced die. . That is, when a precursor that is not toxic to normal cells is systemically administered, the precursor is converted into a toxic metabolite only in cancer cells, thereby changing the sensitivity to the drug, thereby destroying cancer cells. These drug susceptibility genes are preferably HSVtk (Herpes simplex virus-thymidine kinase) genes using ganciclovir as a precursor, or E. coli using 5-fluorocytosine (5-FC) as a precursor. It may be a cytosine deaminase (CD) gene, and most preferably, it may be an HSVtk gene including the sequence of SEQ ID NO: 4.

A proapoptotic gene according to the present invention refers to a nucleotide sequence that induces programmed cell death when expressed. Apoptosis genes known to those skilled in the art, encoding p53, adenovirus E3-11.6K (derived from Ad2 and Ad5) or adenovirus E3-10.5K (derived from Ad), adenovirus E4 gene, p53 pathway gene and caspase genes may be included.

A cytostatic gene according to the present invention refers to a nucleotide sequence that is expressed in cells and stops the cell cycle during the cell cycle. Examples include p21, retinoblastoma gene, E2F-Rb fusion protein gene, genes encoding cyclin-dependent kinase inhibitors (e.g., p16, p15, p18 and p19), growth arrest specific homeobox , GAX) gene, etc., but is not limited thereto.

A cytotoxic gene according to the present invention refers to a nucleotide sequence that is expressed in cells and exhibits a toxic effect. Examples include, but are not limited to, nucleotide sequences encoding Pseudomonas exotoxin, ricin toxin, diphtheria toxin, and the like.

A tumor suppressor gene according to the present invention refers to a nucleotide sequence capable of suppressing a tumor phenotype or inducing apoptosis by being expressed in a target cell. Representatively, tumor necrosis factor-α (TNF-α), p53 gene, APC gene, DPC-4/Smad4 gene, BRCA-1 gene, BRCA-2 gene, WT-1 gene, retinoblastoma gene, MMAC- 1 gene, adenomatous polyposis coil protein, missing colon tumor (DCC) gene, MMSC-2 gene, NF-1 gene, nasopharyngeal tumor suppressor gene located on chromosome 3p21.3, MTS1 gene , CDK4 gene, NF-1 gene, NF-2 gene, VHL gene, or programmed death-1 (sPD-1).

An antigenic gene according to the present invention refers to a nucleotide sequence that is expressed in a target cell and produces a cell surface antigenic protein that can be recognized by the immune system. Examples of antigenic genes known to those skilled in the art may include carcinoembryonic antigen (CEA) and p53.

A cytokine gene according to the present invention refers to a nucleotide sequence that is expressed in a cell to produce a cytokine. Representatively, GMCSF, interleukins (IL-1, IL-2, IL-4, IL-12, IL-10, IL-19, IL-20), interferon α, β, γ (interferon α-2b) and interferon α Fusions such as -2α-1 may be included.

An anti-angiogenic gene according to the present invention refers to a nucleotide sequence that is expressed and releases the anti-angiogenic factor out of the cell. Examples include angiostatin, vascular endothelial growth factor (VEGF) inhibitor, endostatin, and the like.

As used herein, the term “HSV-tk (Herpes simplex virus-thymidine kinase)” refers to a thymidine kinase derived from herpes simplex virus. This enzyme is a representative example of a drug susceptibility gene that converts a non-toxic prodrug into a toxic substance, causing the transfected cell to die. In the present invention, the HSVtk gene is expressed in a form conjugated to the ribozyme according to the present invention and can be used as a cancer therapeutic gene exhibiting anticancer activity. These HSVtk genes are preferably genbank accession numbers AAP13943, P03176, AAA45811, P04407, Q9QNF7, KIBET3, P17402, P06478, P06479, AAB30917, P08333, BAB84107, AAP13885, AAL73990, AAG4 0842, BAB11942, NP_044624, NP_044492, It may be described in CAB06747 or the like.

As used herein, the term "reporter gene" is a gene used to monitor the introduction of a recombinant vector or the expression efficiency of a ribozyme according to an embodiment of the present invention, which can be monitored without damaging infected cells or tissues. Genes may be used without limitation. Preferably, luciferase, green fluorescent protein (GFP), modified green fluorescent protein (mGFP), enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), Modified red fluorescent protein (mRFP), enhanced red fluorescent protein (ERFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), yellow fluorescent protein (YFP), enhanced yellow fluorescence protein (EYFP), indigo fluorescent protein (CFP) or enhanced indigo fluorescent protein (ECFP).

By inserting a reporter gene into the target gene, the expression level of the cancer cell-specific ribozyme can be observed. In particular, the ribozyme of the present invention contains a promoter and a microRNA target site, so it is not expressed in normal cells and is specific to cancer cells. can manifest. It is obvious to those skilled in the art that it can be applied to diagnose whether cancer has occurred in a specific tissue using this.

In the present specification, "ECRT" means a vector driven by the CMV promoter and containing the trans-splicing ribozyme of the hTERT target, the target gene, SD/SA sequence, and WPRE sequence, and "ECRT-122T" refers to the above It means a vector that additionally contains the miR-122T sequence at the 3' end of the gene expression cassette, and “CRT” means a vector that does not contain the SD/SA sequence and WPRE sequence among the gene expression cassettes. On the other hand, vectors using mouse TERT sequences are marked with “m” for use in specific experiments below.

[Gene Delivery System]

Another aspect of the present invention is a gene delivery system comprising a recombinant vector according to the present invention.

As used herein, the term “gene delivery system” refers to a system capable of increasing expression efficiency by increasing the transfer efficiency of a desired gene and/or nucleic acid sequence into a cell, and is a virus-mediated system. and non-viral systems.

Virus-mediated systems use viral vectors such as retroviral vectors and adenovirus vectors, and have relatively higher efficiency of intracellular gene transfer than non-viral systems because they use the unique intracellular penetration mechanism of viruses that infect human cells. It is known. In addition, after entering the cell, the non-viral vector has a problem in that the endosome fuses with the lysosome and then the genes are degraded in the endolysosome, but the viral vector does not pass through the lysosome and transfers the gene into the nucleus by a mechanism It has the advantage of high gene delivery efficiency because the loss of is small.

Viral vectors that can be used in the present invention may be vectors derived from retroviruses, adenoviruses, adeno-associated viruses, and the like, as described above for recombinant vectors. These viral vectors can be assembled into viral particles and then introduced into cells by a transduction method such as infection.

In one embodiment of the present invention, a recombinant adenovirus containing the above-described recombinant vector as an example of a gene transfer vehicle was designed. That is, the recombinant adenovirus performs a function of delivering a recombinant vector expressing a trans-splicing ribozyme specific to a cancer-specific gene to a target cell (eg, cancer cell), and the recombinant delivered into the cell Vectors are expressed by the intracellular transcription system. The expressed trans-splicing ribozyme can insert a target gene linked to the ribozyme into the transcript of a cancer-specific gene present in large numbers in cancer cells. In the present specification, RZ-001 refers to adenovirus containing ECRT-122T described above, and mRZ-001 refers to adenovirus containing mCRT-122T.

The non-viral system is a method using a cationic lipid delivery system or a cationic polymer carrier or the like as a delivery medium for nucleic acids and/or genes, or using an electroporation method.

Cationic lipid carriers use the positive charge of nanometer-sized liposomes or lipid nanoparticles mainly composed of cationic lipids to form complexes with negatively charged genes, expression vectors or nucleic acids containing genes, and then form the complexes by phagocytosis. method of delivery into cells. Complexes delivered into cells are first delivered from endosomes to lysosomes and then released into the cytosol to be expressed. Cationic polymer carriers deliver genes in a similar way to cationic lipid carriers, except that polymers are used instead of lipids. Representative cationic polymers include polyethyleneimine, poly-L-lysine, and chitosan. (chitosan), etc.

Therefore, a complex formed by combining the recombinant vector of the present invention with a cationic lipid carrier or a cationic polymer carrier can be used as a gene carrier.

In the present invention, the gene delivery system includes the recombinant vector described above, and both virus-mediated systems and non-viral systems may be used, but it is preferable to use a virus-mediated system.

[ribozyme]

Another aspect of the present invention is a ribozyme expressed from a recombinant vector according to the present invention.

Details of the recombinant vector or ribozyme according to the present invention are as described above.

[Pharmaceutical composition]

Another aspect of the present invention is a recombinant vector according to the present invention, a gene delivery system comprising the recombinant vector, or a gene therapy comprising a ribozyme and CTLA-4, PD-1, PD-L1, PD-L2, LAG- 3, BTLA, B7H3, B7H4, TIM3, KIR, TIGIT, CD47, VISTA and / or an immunotherapeutic agent exhibiting an inhibitory activity of A2aR as an active ingredient is a pharmaceutical composition for preventing or treating cancer. The pharmaceutical composition of the present invention may be provided as a cancer therapeutic combination in which the gene therapy agent and the immunotherapeutic agent are administered simultaneously or alternately, and each component of the combination may be applied simultaneously or sequentially in a cancer treatment method. there is.

Details of the recombinant vector, gene delivery system, or ribozyme according to the present invention are as described above.

The term "cancer" used in the present invention refers to a problem in the control function of normal division, differentiation, and death of cells, which abnormally proliferates and infiltrates surrounding tissues and organs to form lumps and destroy existing structures. denotes a deformed state.

The cancer according to the present invention may preferably be liver cancer, glioblastoma, biliary tract cancer, lung cancer, pancreatic cancer, melanoma, bone cancer, breast cancer, colon cancer, stomach cancer, prostate cancer, leukemia, uterine cancer, ovarian cancer, lymphoma, or brain cancer, , more preferably liver cancer, glioblastoma, or cholangiocarcinoma, and most preferably liver cancer and/or brain cancer.

In addition, the cancer according to the present invention may preferably have a copy number (expression level) of miR-122 expressed in cancer tissue that is less than 100 times the copy number of the ribozyme expressed in cancer tissue by the pharmaceutical composition. .

On the other hand, the present inventors compared the expression level of hTERT target ribozyme with miR-122T capable of inducing cell death in a previous study and the expression level of miR-122 in cells, and the ratio of miR-122 to ribozyme was It was confirmed that the higher the expression of the ribozyme, the lower the cell death inducing effect. Therefore, the amount of adenovirus expressing the ribozyme can be determined by inferring the amount of ribozyme to exhibit the anticancer effect according to the expression level of miR-122 in the cancer tissue. Specifically, if the minimum copy number of miR-122 is about 100 times or more than the ribozyme copy number, the function (expression) of the ribozyme having a miR-122 target site is weakened. It can be seen that high anticancer efficacy can be obtained when the number of copies of the ribozyme expressed in cancer tissue by the pharmaceutical composition according to is less than 100 times.

Also, the cancer according to the present invention may preferably be a cancer in which miR-122 is not substantially expressed in cancer tissue. The above “cancer in which miR-122 is not substantially expressed in cancer tissue” means that although miR-122 is expressed in cancer tissue, it is expressed in cancer tissue to the extent that it does not substantially affect the function of the ribozyme having a miR-122 target site. It means cancer with a low copy number of miR-122.

Through previous studies, the present inventors have found that the anticancer efficacy of the ribozyme according to the present invention in colorectal cancer, glioblastoma, melanoma, cervical cancer, lung cancer, osteosarcoma, breast cancer and cholangiocarcinoma cell lines in which miR-122 is not substantially expressed in cancer tissues confirmed.

In addition, liver cancer according to the present invention is preferably hepatitis B virus, hepatitis C virus in which the expression of miR-122 is reduced in liver cancer tissues, alcohol, chronic hepatitis, cirrhosis, nonalcoholic fatty liver disease, aflatoxin It may be due to one or more causes selected from the group consisting of, and family history.

The present inventors analyzed the expression level of miR-122 in liver cancer caused by various etiological factors. As a result, among liver cancers caused by HCV, the expression level of miR-122 in normal liver tissues was higher than that in liver cancer tissues. Therefore, it was confirmed that the function of the ribozyme according to the present invention is weakened in normal liver tissue by miR-122 and can act in liver cancer tissue in which the expression of miR-122 is reduced.

In addition, the cancer according to the present invention may be resistant to tyrosine kinase inhibitors (TKI), and more specifically, may be liver cancer resistant to sorafenib. Sorafenib is a TKI, a first-line treatment for advanced liver cancer that inhibits the growth and differentiation of cancer cells by inhibiting protein kinase, which plays an important role in cell signaling pathways. The present inventors confirmed that the ribozyme according to the present invention induces cell death in both cell lines sensitive to sorafenib and cell lines without it through specific experiments.

In addition, the gene therapeutic agent of the present invention can be co-administered with an immune checkpoint inhibitor to enhance the tumor invasiveness of T cells. In addition, since it inhibits PD-1 or PDL-1, which inhibits the activity of infiltrated T-cells, it maximizes the activity of T-cells, thereby increasing the effect of immunotherapy. As an independent gene therapy, it is effective together with immune checkpoint inhibitors. Cancer treatment is possible. Immune checkpoint inhibitors perform the binding inhibitory function of PD-L1 and PD-1 for maintaining the immune function of T cells. As a result of the combination administration of the pharmaceutical composition of the present invention by selecting and atezolizumab, the size of cancer tissue was significantly reduced with a small dose of administration compared to the single administration group, and the tumor penetration ability of T cells during combined administration It was confirmed that this markedly increased.

The combination of the present invention can be co-formulated for concomitant administration of the pharmaceutical composition and the immune checkpoint inhibitor as a pharmaceutical formulation, or they can be administered alternately and separately as a therapeutic combination. In this case, when administered separately, the pharmaceutical composition and the immune checkpoint inhibitor may be administered simultaneously or sequentially.

In the present invention, 'simultaneously' means that the immune checkpoint inhibitor and the gene therapy agent of the present invention are administered within 24 hours, and 'sequentially' means that they are administered over 24 hours.

The term "prevention" used in the present invention refers to any action that suppresses or delays the onset of cancer by administering the combination or pharmaceutical composition according to the present invention.

The term "treatment" used in the present invention refers to all activities that improve cancer or beneficially change its symptoms by administration of the combination or pharmaceutical composition according to the present invention.

The pharmaceutical composition according to the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent. Examples of pharmaceutically acceptable carriers, excipients and diluents that can be used in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, and alginate. , gelatin, calcium phosphate, calcium silicate, calcium carbonate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like.

The pharmaceutical composition of the present invention may be administered orally or parenterally depending on the desired method, but is preferably administered parenterally.

According to one embodiment of the present invention, the pharmaceutical composition according to the present invention may be directly administered intravenously, intraarterially, intramuscularly or subcutaneously, and may be administered orally or by injection. The injection according to the present invention may be in a form dispersed in a sterile medium so that it can be used as it is when administered to a patient, or may be administered after dispersing in an appropriate concentration by adding distilled water for injection. In addition, when prepared as an injection, it may be mixed with buffers, preservatives, analgesics, solubilizers, tonicity agents, stabilizers, etc., and may be prepared in unit dosage ampoules or multiple dosage forms.

The dosage of the pharmaceutical composition of the present invention varies depending on the condition and body weight of the patient, the severity of the disease, the drug type, the administration route and time, but can be appropriately selected by those skilled in the art. Meanwhile, the pharmaceutical composition according to the present invention may be used alone or in combination with auxiliary treatment methods such as surgical treatment.

Hereinafter, examples will be described in detail in order to specifically describe the present specification. However, the embodiments according to the present specification may be modified in many different forms, and the scope of the present specification is not construed as being limited to the embodiments described below. The embodiments herein are provided to more completely explain the present specification to those skilled in the art.

Example 1: Construction of hTERT target trans-splicing ribozyme recombinant vector

In order to prepare an hTERT-targeting trans-splicing ribozyme having miR-122T, a miR-122 target site, and exhibiting a high expression rate, a recombinant vector modified from an existing trans-splicing ribozyme was designed.

Specifically, a conventional trans-splicing ribonucleic acid targeting the +21 site of hTERT mRNA, having an antisense sequence (SEQ ID NO: 8) of 326 nucleotides in length, and having HSV-tk as a therapeutic gene A CMV promoter was introduced into the zyme. Transcription efficiency was increased by inserting an SV40 intron splicing donor/acceptor (SD/SA) sequence between the CMV promoter and the ribozyme. WPRE, a posttranscriptional regulatory element of Woodchuck Hepatitis Virus, was inserted into the 3' region of HSV-tk, a therapeutic gene, to increase protein expression efficiency of the therapeutic gene, and to the 3' end of the construct. 3 copies of miR-122T were inserted so that they could be regulated by miR-122.

The entire vector structure of the designed trans-splicing ribozyme is as shown in Figure 1, and it was named ECRT-122T.

Example 2. Animal experiments to confirm anticancer efficacy of ECRT-122T (in vivo)

Tumor formation was induced by subcutaneous injection of 1x10 ⁷ (100 μl) of LN229 cell line or 5x10 ⁶ (100 μl) of U87MG cell line into 6-week-old male Balb/c-nunu mice. When the tumors grew to a certain size, adenovirus expressing ECRT-122T was injected at a dose of 1.0x10 ⁹ VP (100 µl) once every 2 days for a total of 3 times. GCV was administered at a dose of 50 mg/kg twice a day for 10 days (20 times in total) from 24 hours after the first virus injection.

As a result of the experiment, as shown in FIG. 2, it was found that the tumor size continued to increase in the control group (Ad-EGFP injection), but in the experimental group injected with ECRT-122T, the tumor hardly grew, and the tumor weight also increased with ECRT-122T. In all experimental groups injected, it was less than the control group. In addition, as shown in FIG. 3, the tumor growth inhibitory effect of ECRT-122T was also confirmed in experiments using the U87MG cell line.

From the results of this experimental example, it was confirmed that the ribozyme expressed from ECRT-122T can be effectively applied to various carcinomas that do not express miR-122, especially brain cancer, in addition to liver cancer. In addition, when ECRT-122T is administered systemically or locally as an anticancer agent for other cancers other than liver cancer, it can be introduced into the normal liver. In this case, the action of miR-122T will suppress the induction of toxicity in the normal liver. That is, it can be seen that the ECRT-122T of the present invention can effectively exhibit anticancer effects in various carcinomas including liver cancer, and has low toxicity in normal cells and liver.

Example 3: Confirmation of toxicity of ECRT-122T with and without GCV (Ganciclovir) treatment

3-1. GCV untreated

Normal ICR mice were injected once with ECRT-122T adenovirus, and AST and ALT levels were measured 15 and 29 days after injection.

As a result, as shown in FIG. 4, weak toxicity was shown in the 2.5x10 ¹⁰ VP experimental group, and the remaining doses of the experimental group showed AST and ALT levels similar to those of the PBS injection group, indicating little toxicity.

In addition, as shown in FIGS. 5A to 5C, no abnormalities were found in the body weight, feed consumption, and liver weight of mice during the experimental period.

As a result of histopathological examination of the liver performed 29 days after intravenous administration of adenovirus once, as shown in FIG. 6, hepatocellular necrosis (arrow) and inflammation were observed in the 2.5x10 ¹⁰ VP experimental group (G3), indicating slight liver damage. It was found that this caused In the 1.0x10 ¹⁰ VP experimental group (G2), inflammatory cells were locally infiltrated (arrow), but it was confirmed that liver damage was not induced. In addition, no abnormal findings were observed in the 0.5x10 ¹⁰ VP experimental group (G1) and the PBS administration group (G4). In FIG. 6, cv denotes a central vein and p denotes a portal area.

The histopathological examination results are summarized in Table 1 below.

Histopathology/Groups G1 G2 G3 G4 0.5x10 ¹⁰ 1.0x10 ¹⁰ 2.5x10 ¹⁰ Vehicle (PBS) No. examined 4 5 5 5 No specific lesion 4 (100) 4 (80) 0 (0.00) 5 (100) Inflammatory foci with hepatocyte necrosis 0 (0.00) 1 (20.0) 4 (80.0) 0 (0.00) Grades: Minimal 0 One 3 0 Mild 0 0 One 0 Cell infiltration, mononuclear cells, periductal, focal 0 (0.00) 0 (0.00) 2 (40.0) 0 (0.00) Grades: Minimal 0 0 2 0

3-2. Ganciclovir (GCV) treatment

ECRT-122T adenovirus was injected into normal ICR mice, and GCV was administered twice a day for 10 days, and AST and ALT levels were measured.

As a result, as shown in FIG. 7, in the experimental groups of the remaining doses except for the 2.5x10 ¹⁰ VP experimental group, the levels of AST and ALT were similar to those of the control group (PBS injection group), indicating little toxicity.

In addition, as shown in Figures 8a and 8b, there was no significant change in the body weight and feed consumption of the mouse during the course of the experiment, and as shown in Figure 8c, it was not confirmed that the weight of the liver was also abnormal.

As a result of histopathological analysis of the liver, as shown in FIG. 9 , no abnormal findings were found in the PBS injection group (G1), the PBS+GCV administration group (G2), and the 0.25x10 ¹⁰ VP+GCV administration group (G5). On the other hand, in the 1.0x10 ¹⁰ VP+GCV administration group (G3), local microabscess (thick arrow) formed by neutrophil infiltration was confirmed, but liver damage was not induced. In the 2.5x10 ¹⁰ VP+GCV group (G4), hypertrophic hepatocytes with large nuclei, necrotic hepatocytes (thick arrows), multiple inflammatory cell infiltrates (circles), increased hepatocyte mitosis (thick arrows), and bile ducts (b) Peripheral lymphocytic cell infiltration was observed, suggesting that liver damage was induced. In G6 (1.0x10 ¹⁰ VP injection group) not administered with GCV, the number of mitotic hepatocytes (arrows) slightly increased.

The histopathological examination results are summarized in Table 2 below.

Histopathology/Groups G1 G2 G3 G4 G5 G6 Vehicle (PBS) PBS+GCV 1.0x10 ¹⁰ +GCV 2.5x10 ¹⁰ +GCV 0.25x10 ¹⁰ +GCV 1.0x10 ¹⁰ No. examined 5 5 5 5 5 5 No specific lesion 5 (100) 4 (80.0) 3 (60.0) 0 (0.00) 5 (100) 3 (60.0) Cell infiltration, mononuclear or mixed cells, multifocal 0 (0.00) 1 (20.0) 1 (20.0) 4 (80.0) 0 (0.00) 0 (0.00) Grades: Minimal 0 One One 0 0 0 Mild 0 0 0 4 0 0 Microabcess, focal 0 (0.00) 0 (0.00) 1 (20.0) 0 (0.00) 0 (0.00) 0 (0.00) Grades: Minimal 0 0 One 0 0 0 Hepatocytomegaly, diffuse 0 (0.00) 0 (0.00) 0 (0.00) 5 (100) 0 (0.00) 0 (0.00) Grades: Minimal 0 0 0 One 0 0 Mild 0 0 0 2 0 0 Moderate 0 0 0 2 0 0 Necrotic hepatocytes 0 (0.00) 0 (0.00) 0 (0.00) 3 (60.0) 0 (0.00) 0 (0.00) Grades: Minimal 0 0 0 2 0 0 Mild 0 0 0 One 0 0 Hepatocytic mitosis, increased, diffuse 0 (0.00) 0 (0.00) 0 (0.00) 4 (80.0) 0 (0.00) 1 (20.0) Grades: Minimal 0 0 0 2 0 One Mild 0 0 0 One 0 0 Moderate 0 0 0 One 0 0 Oval cell hyperplasia 0 (0.00) 0 (0.00) 0 (0.00) 3 (60.0) 0 (0.00) 0 (0.00) Grades: Minimal 0 0 0 3 0 0 Pericholangitis, (multi)focal 0 (0.00) 0 (0.00) 0 (0.00) 3 (60.0) 0 (0.00) 1 (20.0) Grades: Minimal 0 0 0 3 0 One

AST and ALT levels, and histopathological examination results: When adenovirus was intravenously administered at a dose of 2.5x10 ¹⁰ VP/head, and GCV was administered twice a day for 10 days, hepatocyte necrosis and inflammation were induced, and liver damage was induced. appeared to be However, when 0.25x10 ¹⁰ VP/head and 1.0 x 10 ¹⁰ VP/head were administered alone or in combination with GCV, AST and ALT levels related to liver damage increased up to 15 days after administration, but histological examination performed on day 29 showed No significant toxicological changes considered to be related to adenovirus administration were observed in .

Example 4: Comparison of anticancer efficacy of CRT-122T and ECRT-122T

Tumor formation is induced by injecting SNU398 cells, a liver cancer cell line, into a mouse xenograft subcutaneous model, and when the tumor grows to a certain size, adenovirus containing CRT-122T or ECRT-122T vector is injected 1x10 ⁹ The VP dose was injected (intratumoral injection, IT injection) into the cancer tissue once every 2 days, a total of 2 times. After adenovirus injection, the tumor size and body weight were measured at 3-day intervals while the mice were bred, and the mice were sacrificed after 22 days, and the final tumor size, liver weight, AST (aspartate transaminase) and ALT (alanine transaminase) levels were measured.

As a result, as shown in FIGS. 10A and 10B , it was confirmed that the anticancer efficacy of ECRT-122T was superior to that of CRT-122T. There was no significant difference in the body weight and liver weight of the mice between the experimental groups, and it was found from the AST and ALT levels that adenovirus did not induce liver toxicity (FIGS. 10C to 10E).

Through the above results, it was confirmed that a sufficient anticancer effect could be obtained by injecting only a small amount of adenovirus, and it was found that the anticancer efficacy of ECRT-122T was significantly superior to that of CRT-122T.

Example 5. Comparison of efficacy according to combination administration with immune checkpoint inhibitors in PBMC-humanized liver cancer model

Immune checkpoint inhibitors are those that inhibit the binding of PD-L1 and PD-1 to maintain the immune function of T cells. There are 　 PD-L1 　 monoclonal antibodies 　 atezolizumab, averumab, durvalumab 　, etc. 　 Additionally, Ipilimumab (a monoclonal antibody targeting CTLA-4) ipilimumab) and others. In this experiment, we tried to confirm the increased anticancer effect when adenovirus and immune checkpoint inhibitor were administered together.

5-1. Confirmation of efficacy of immune checkpoint inhibitors and concomitant administration

PBMC humanized mice were prepared by injecting PBMC 5 x 10 ⁶ cells/head into a mouse xenograft subcutaneous model (6-week-old male PBMC-humice (NOG)), and the body weight and After confirming the condition, subcutaneous injection of 5 x 10 ⁶ cells /50 μl of SNU-398 cells was performed, followed by breeding for 2 weeks to construct a liver cancer tumor model. Subsequently, tumor growth was measured, group separation was performed, and drug administration was performed for each group.

Administration of the drug was performed according to the following dosage and usage (see FIG. 11).

RZ-001 adenovirus is administered directly into the tumor (intratumoral injection) at 1 x 10 ⁹ VP/head twice at 48 hour intervals.

Nivolumab or atezolizumab was used as the antibody, and IgG was administered to the control group. The dose of each antibody is 5 mg/kg, and the average weight per mouse is 20 g. For each administration, 100 μg is injected intravenously three times at 48-hour intervals from the end of RZ-001 administration. Nivolumab and atezolizumab antibodies were purchased from Selleckchem.

The dose of GCV is 50mg/kg, and GCV is injected intraperitoneally 10 times at 24-hour intervals from the end of RZ-001 administration.

The experimental animal groups were divided into immune checkpoint inhibitors alone administration group (nivolumab or atezolizumab), RZ-001 alone administration group, and combination administration group (RZ001/nivolumab or RZ001/atezolizumab), and as a control group, PBS administration group and IgG administration group. separated by

As a result of confirming the size of the tumor by sacrificing the mouse 2 weeks after the start of drug administration, the tumor size of the immune checkpoint inhibitor alone and RZ-001 alone treatment groups was significantly reduced compared to the control group, but the reduction in tumor size in the combination treatment group was significantly greater. I was able to confirm.

In addition, 2 weeks after the start of drug administration, the level of cytokines in the blood was checked (FIG. 14), and the degree of T cell activation and tumor infiltration were confirmed. The degree of T cell activation and tumor infiltration was first confirmed by measuring the expression levels of CD4 and CD8 by measuring RNA expression levels, and then verified by performing immunohistochemical staining. Both primary and secondary antibodies used for immunohistochemical staining were purchased from Roche (CD4: 790-4423, CD8: 790-4460). As a result, high expression levels of CD4 and CD8 were confirmed in the immune checkpoint inhibitor alone administration group, RZ-001 alone administration group, and combination administration group, and in particular, high tumor penetration of T cells was confirmed in the combination administration group.

In the hepatotoxicity test by measuring AST and ALT in the blood, it was an experiment conducted after human PBMC administration in the entire administration group. Graft-versus-host disease (GVHD) occurred in all mice at the time of sacrifice. As a result, hepatotoxicity appears to be more in the nivolumab group than atezolizumab. In addition, it can be seen that the RZ-001/atezolizumab combination administration group exhibited lower hepatotoxicity during the combination administration as the AST and ALT levels were reduced compared to the atezolizumab alone administration group (FIG. 17).

5-2. Confirmation of anticancer effect of combined administration of immune checkpoint inhibitors according to RZ-001 administration concentration

The same experiment as in Example 5-1 was performed by varying the administration concentration of RZ-001.

RZ-001 was set to 1 x 10 ⁹ VP/head (L), 3 x 10 ⁹ VP/head (M), and 10 x 10 ⁹ VP/head (H), and the weight of the tumor after the end of the experiment for each experimental group As a result of the measurement, the size of the tumor was significantly reduced in the combined administration group compared to each single administration group, similar to the result of Example 5-1, and the anticancer effect based on the tumor size was dependent on the RZ-001 administration concentration. It was confirmed that the anti-cancer effect was increased by the combined administration of immuno-anticancer agents (FIGS. 18A and 18B).

In addition, in order to confirm the tumor infiltration ability of T cells when RZ-001 was administered at a low concentration, immunohistochemical staining was performed for CD4, CD8, and CD69 in tumor tissue. CD69 primary antibodies were purchased and used from Abcam (SN: ab233396). During immunohistochemical staining, CD4 and CD8 primary antibodies were treated at a concentration of 250 ng/100 ul for 16 minutes, and CD69 primary antibodies were 1 :500 diluted and treated for 32 minutes. Secondary antibodies were purchased from Roche and used. CD4 and CD8 were treated for 8 minutes, CD69 was treated for 16 minutes, and DAB was stained for 8 minutes. As a result, CD4 and CD8 were hardly detected in the PBS-administered group, which is a control group, but the expression levels of CD4 and CD8 were found to be high in each single-administered group and the combined-administered group. In particular, high tumor penetration of T cells was confirmed in the combination administration group (FIG. 19).

In addition, the expression levels of AKT, pAKT, S6, pS6, and PDL1 in the tumor tissue of the mouse were checked to confirm the tumorigenic agent inhibitory pathway of the drug. Specifically, the tumor tissue collected at the time of autopsy of the mouse was put in 200 ul RIPA buffer (including 100x protease inhibitor and phosphatase inhibitor), homogenized, ground, and centrifuged at 15,000 rpm for 15 minutes, discarding the pellet at the bottom and discarding the supernatant. only separated. The separated supernatant was placed in a new tube and quantified by BCA assay to make a sample to be 2 ug/ul and reacted at 100°C for 5 minutes to denature the protein. Subsequently, a 10% acrylamide gel was made and the protein was transferred to the membrane through SDS-PAGE electrophoresis and transfer, followed by blocking with 5% Blocking reagent (in PBS-T) at room temperature for 1 hour. The antibody was reacted O/N at 4° C. at a ratio of 1:1,000. After washing three times with PBS-T, a secondary antibody reaction was performed at room temperature for 1 hour at a ratio of 1:5,000. Then, after washing three times with PBS-T, the chemiluminescence reaction was measured using a digital imaging system.

Each antibody was purchased and used.

AKT (cell signaling, 4691s), pAKT (cell signaling, 4060L), S6 (cell signaling, 2271L), pS6 (cell signaling, 4858L), PDL1 (Abcam, ab238697), VEGF-c (Santacruz, sc-374628)

As a result, it was confirmed that the expression levels of pAKT and pS6 were significantly decreased in the RZ-001 single administration group and the combined administration group. That is, it can be seen that the combination of RZ-001 and RZ-001 suppresses resistance by immune checkpoint inhibitors administered to mice, thereby inhibiting AKT and phosphorylation of S6 protein, which is a sub mechanism, thereby blocking cancer signaling pathway (FIG. 20).

In the hepatotoxicity test by measuring AST and ALT in the blood, it was an experiment conducted after human PBMC administration in the entire administration group. Graft-versus-host disease (GVHD) occurred in all mice at the time of sacrifice. It can be seen that the RZ-001/atezolizumab combination administration group showed lower liver toxicity than the combination administration, as AST and ALT levels were reduced compared to the atezolizumab alone administration group (FIG. 21).

Example 6. Confirmation of efficacy of combination administration with immune checkpoint inhibitors in Syngeneic liver cancer subcutaneous transplant model

A syngeneic model was constructed by subcutaneously transplanting Hepa1-6 liver cancer cells (1×10 ⁷ cells/mouse) into 6-week-old male C57BL/6 mice. Subsequently, tumor growth was measured, groups were separated, and drug administration was performed for each group.

Administration of the drug was carried out according to the following dosage and usage.

mRZ-001 is administered directly into the tumor at 3.25 x 10 ⁹ VP/head twice at 48-hour intervals.

Anti-mPDL1 was used as the antibody, and IgG was administered to the control group. The dosage of each antibody is 5mg/kg, and it is intravenously administered 4 times at intervals of 48 hours from the end of mCRT-122T administration. Antibodies were produced by requesting inVivoMab.

The dose of GCV is 50 mg/kg, and GCV is intraperitoneally injected 10 times at 24-hour intervals from the end of mCRT-122T administration.

The experimental animal groups were divided into immune checkpoint inhibitor alone administration group (anti-mPDL1), mCRT-122T alone administration group, and combined administration group (mCRT-122T and anti-mPDL1), and as a control group, PBS administration group and IgG administration group.

As a result of checking the change in tumor size of the mice for 2 weeks after drug administration, the anti-mPDL1 alone group, the mCRT-122T alone group, and the combination group had significantly smaller tumor sizes than the control group. In particular, it was confirmed that the size of the tumor in the combination administration group was significantly reduced compared to each single administration group (FIG. 22). In addition, it was confirmed that the combined administration group had higher anticancer efficacy than the single administration group, and in the liver toxicity test, the problem of liver toxicity was very low, similar to that of the PBS administration group (FIG. 23).

As above, specific parts of the present invention have been described in detail, and for those skilled in the art, it is clear that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

When the trans-splicing ribozyme of the present invention is used in combination with an immune checkpoint inhibitor, liver toxicity is lower than when used alone, and a synergistic effect on anticancer is exhibited. Therefore, it is expected to be used as a drug for the treatment of various cancers, including immune checkpoint inhibitor-resistant cancer.

Claims (18)

A therapeutically effective amount of a trans-splicing ribozyme expression vector targeting a cancer-specific gene sequence, a gene delivery system comprising the vector, or a ribozyme expressed from the vector; and As a cancer therapeutic combination comprising a therapeutically effective amount of an immune checkpoint inhibitor, A therapeutic combination for use in a method of treating cancer, wherein the therapeutic combination is administered to the mammal in a combined dosage form or alternately. According to claim 1, The expression vector includes a cytomegalovirus (CMV) promoter operably linked to the ribozyme gene, A splicing donor/splicing acceptor sequence (SD/SA sequence) is included at the 5' end of the ribozyme gene, A therapeutic combination, characterized in that it comprises a Woodchuck hepatitis virus Posttranscriptional Regulatory Element (WPRE) at the 3' terminal position. According to claim 1, The cancer-specific gene sequences include Telomerase Reverse Transcriptase (TERT) mRNA, alphafetoprotein (AFP) mRNA, carcinoembryonic antigen (CEA) mRNA, prostate-specific antigen (PSA) mRNA, cytoskeleton-associated protein 2 (CKAP2) mRNA, and mutant RAS ( Rat sarcoma) mRNA. According to claim 1, The therapeutic combination, wherein the trans-splicing ribozyme comprises the nucleic acid sequence of SEQ ID NO: 3. According to claim 1, The therapeutic combination, characterized in that the expression vector contains the target gene linked to the 3' exon of the ribozyme gene. According to claim 5, The therapeutic combination, characterized in that the gene of interest is a gene for cancer treatment or a reporter gene. According to claim 6, The gene for cancer treatment is selected from the group consisting of a drug susceptibility gene, an apoptosis gene, a cell proliferation inhibitor gene, a cytotoxic gene, a tumor suppressor gene, an antigenic gene, a cytokine gene, and an anti-angiogenic gene, therapeutic combination. According to claim 7, The therapeutic combination, characterized in that the drug susceptibility gene is a Herpes Simplex Virus thymidine kinase (HSVtk) gene. According to claim 1, The expression vector is characterized in that it contains a gene encoding a sequence complementary to part or all of microRNA-122 (microRNA-122, miR-122) at the 3'-UTR end of the ribozyme gene. water. According to claim 9, The therapeutic combination, characterized in that the gene encoding the sequence complementary to miR-122 contains at least one copy of the nucleotide sequence of SEQ ID NO: 5. According to claim 1, The cancer is at least one selected from the group consisting of liver cancer, glioblastoma, bile duct cancer, lung cancer, pancreatic cancer, melanoma, bone cancer, breast cancer, colon cancer, stomach cancer, prostate cancer, leukemia, uterine cancer, ovarian cancer, lymphoma, and brain cancer A therapeutic combination, characterized in that it is cancer. According to claim 1, Therapeutic combination, characterized in that the cancer is a cancer in which miR-122 is not substantially expressed in cancer tissues. According to claim 12, The cancer is liver cancer, The liver cancer is selected from the group consisting of hepatitis B virus, hepatitis C virus in which the expression of miR-122 is reduced in liver cancer tissue, alcohol, chronic hepatitis, cirrhosis, non-alcoholic fatty liver, aflatoxin, and family history A therapeutic combination, characterized in that any one or more causes. According to claim 1, The therapeutic combination, wherein the therapeutically effective amount of the expression vector, gene delivery system, or ribozyme and the therapeutically effective amount of the immune checkpoint inhibitor are administered in a combined formulation. According to claim 14, The therapeutic combination, wherein the therapeutically effective amount of the expression vector, gene delivery system, or ribozyme and the therapeutically effective amount of the immune checkpoint inhibitor are administered alternately. According to claim 1, Therapeutic combination wherein the immune checkpoint inhibitor is an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, TIGIT, CD47, VISTA or A2aR . According to claim 1, The therapeutic combination, wherein the therapeutically effective amount of the expression vector, gene delivery system, or ribozyme is administered orally or in the form of an injection by a route selected from the group consisting of intravenous, intraarterial, intramuscular and subcutaneous. A trans-splicing ribozyme expression vector targeting a cancer-specific gene sequence, a gene delivery system containing the vector, or an immune checkpoint inhibitor containing the ribozyme expressed from the vector as an active ingredient A pharmaceutical composition for the treatment of resistant cancer.

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