Cancer cell-specific gene expression system

Abstract

The present invention relates to recombinant nucleic acid constructs useful in cancer therapy and gene expression systems containing the same. [Solution] The recombinant nucleic acid construct of the present invention is characterized in that it comprises, from the 5' to 3' positions, the first exon, the first intron, the second intron, and the second exon of the granzyme B (GZMB) gene, and contains nucleotides encoding at least one interfering RNA (RNAi) between the first intron and the second intron.

Description

[Technical Field]

[0001] The present invention relates to recombinant nucleic acid constructs useful in cancer therapy, and gene expression systems containing the same. [Background technology]

[0002] Various treatments have been developed to treat cancer, which has the highest mortality rate among various diseases both in Korea and overseas. However, traditional treatments using small molecule anticancer drugs are unable to distinguish between cancer cells and normal cells, which results in non-specific drug effects, the emergence of cancer cells that are resistant to anticancer treatment, or serious side effects due to excessive cytotoxicity.

[0003] However, advances in anticancer drug development have significantly increased tumor specificity and therapeutic efficacy compared to previous studies. Despite this, these improved anticancer therapeutics still have limitations. For example, bevacizumab, a single antibody that binds to VEGF-A, and cetuximab, a single antibody that binds to the EGF receptor, have been used to treat colon cancer, lung cancer, and brain tumors. However, frequent mutations in the target protein reduce the specificity of the antibody. Furthermore, immune-mediated anticancer therapies using immune barrier inhibitors, such as pembrolizumab, ipilimumab, and nivolumab, which have attracted attention in recent years, have limited efficacy in cold tumor patients with extremely low T cell activity. Therefore, there is a need in the medical field for new therapeutic concepts that address the limitations of these therapeutics. In recent years, gene-level targeted therapy has been developed to overcome problems associated with mutations in target proteins found in tumors, and the correlation between miRNAs and diseases has been attracting particular attention.

[0004] MicroRNAs (miRNAs) are short RNAs of approximately 19-25 nucleotides that do not carry genetic information themselves but are known to regulate the expression of various genes. miRNAs can regulate the ultimate function of specific genes by participating in various biological processes such as cell cycle, differentiation, proliferation, death, stress resistance, metabolism, and immune response. In particular, the expression of genetic information induced by miRNA regulates final gene expression by the miRNA-induced silencing complex (miRISC) binding to the 3'-untranslated regions (UTR) of target mRNAs and inhibiting mRNA cleavage or translation. In this way, miRNAs have been found to regulate the expression of approximately 30% of genetic information in human genes, and about half of these are involved in the induction of tumors, and reduced expression of miRNAs in tumor cells is thought to contribute to the development of tumors.

[0005] On the other hand, several pathogenic microorganisms, including Salmonella and Listeria, cause disease when they infect laboratory rats. However, numerous studies have shown that when these attenuated microorganisms are administered to laboratory rats, they attack tumors, inhibit their growth, and extend the survival time of laboratory rats that develop tumors. In particular, since preclinical experiments suggested the possibility of using Salmonella to treat tumors, active research has been conducted into the use of Salmonella in tumor therapy. It has been found that Salmonella either forms clusters in tumor tissue and directly kills tumor cells, or changes the immune microenvironment around the tumor, allowing the patient's immune cells to recognize the tumor and lead to its death. While there has been controversy regarding anti-cancer therapy using Salmonella, clinical trials have progressed from treating tumors with Salmonella alone to methods and approaches that involve delivering therapeutic drugs. [Prior art documents] [Non-patent literature]

[0006]

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[0007] It is an object of the present invention to provide recombinant nucleic acid constructs that can be used in cancer therapy through post-transcriptional modification.

[0008] Another object of the present invention is to provide a recombinant nucleic acid construct comprising a polynucleotide encoding multiple miRNAs that target cancer cells.

[0009] A further object of the present invention is to provide an expression vector containing the above recombinant nucleic acid construct.

[0010] Another object of the present invention is to provide a host cell transformed with the above expression vector.

[0011] It is yet another object of the present invention to provide a composition for preventing, ameliorating, or treating cancer, which comprises the host cells.

[0012] However, the technical problems of the present invention are not limited to those mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description. [Means for solving the problem]

[0013] The recombinant nucleic acid construct of the present invention is characterized in that it comprises, from the 5' to 3' positions, the first exon, the first intron, the second intron, and the second exon of the granzyme B (GZMB) gene, and contains nucleotides encoding at least one interfering RNA (RNAi) between the first intron and the second intron.

[0014] In the present invention, the "nucleic acid construct" refers to any polynucleotide designed to transcribe RNA.

[0015] In the present invention, "granzyme B (GZMB)" is one of the serine proteases most abundantly found in the granules of natural killer cells (NK cells) and cytotoxic T cells. It is secreted by these cells along with the pore-forming protein perforin to mediate target cell death. The human GrB gene (GZMB) has been mapped to the "chymase locus" on chromosome 14q11.2. This locus contains three functional genes: granzyme H gene (GZMH), cathepsin G gene (CTSG), and mast cell chymase gene (CMA1). The GZMB gene is located at the 5' end of the cluster and is followed by the GZMH, CTSG, and CMA1 genes. The GZMB gene is approximately 3.2 kb in length and consists of five exons and four introns. The signal sequence of the GrB preproprotein is encoded and conserved in exon 1, and the amino acid residues that form the catalytic triad, namely, His57, Asp102, and Ser195, are encoded and conserved in exons 2, 3, and 5, respectively.

[0016] As used herein, the term "exon" refers to a nucleic acid sequence (or the like) that encodes one or more amino acids of a transcript (e.g., a protein of interest) expressed from a nucleic acid, e.g., within an open reading frame. Exon sequences can be transcribed from DNA to RNA (i.e., can exist in the pre-mRNA state) or can exist in mature mRNA (i.e., the processed form of RNA after splicing, for example) that is translated into a polypeptide.

[0017] As used herein, the term "intron" refers to a nucleic acid sequence (or the like) that does not encode one or more amino acids of a polypeptide transcript (e.g., a protein of interest) expressed from a nucleic acid, e.g., within an open reading frame. Intron sequences can be transcribed from DNA to RNA (i.e., can be present in pre-mRNA), but can be removed from the mature mRNA before the protein is expressed, e.g., via splicing.

[0018] In the present invention, the granzyme B gene (GZMB) may be derived from humans and may, for example, contain the base sequence shown in SEQ ID NO: 1, preferably consist of the base sequence shown in SEQ ID NO: 1, but is not limited thereto.

[0019] In the present invention, the granzyme B gene (GZMB) may be derived from humans and may encode, for example, the amino acid sequence shown in SEQ ID NO: 2, but is not limited thereto.

[0020] In the present invention, exon 1 of the granzyme B gene (GZMB) may include the coding sequence shown in SEQ ID NO: 3, but is not limited thereto.

[0021] In the present invention, exon 1 of the granzyme B gene (GZMB) may contain the base sequence shown in SEQ ID NO: 4, preferably consisting of the base sequence shown in SEQ ID NO: 4, but is not limited thereto.

[0022] In the present invention, exon 1 of the granzyme B gene (GZMB) may encode the amino acid sequence shown in SEQ ID NO: 12, but is not limited thereto.

[0023] In the present invention, exon 2 of the granzyme B gene (GZMB) may contain the base sequence shown in SEQ ID NO: 5, preferably consisting of the base sequence shown in SEQ ID NO: 5, but is not limited thereto.

[0024] In the present invention, exon 2 of the granzyme B gene (GZMB) may encode the amino acid sequence shown in SEQ ID NO: 13, but is not limited thereto.

[0025] In the present invention, exon 3 of the granzyme B gene (GZMB) may contain the base sequence shown in SEQ ID NO: 6, preferably consisting of the base sequence shown in SEQ ID NO: 6, but is not limited thereto.

[0026] In the present invention, exon 3 of the granzyme B gene (GZMB) may encode the amino acid sequence shown in SEQ ID NO: 14, but is not limited thereto.

[0027] In the present invention, exon 4 of the granzyme B gene (GZMB) may contain the base sequence shown in SEQ ID NO: 7, preferably consisting of the base sequence shown in SEQ ID NO: 7, but is not limited thereto.

[0028] In the present invention, exon 4 of the granzyme B gene (GZMB) may encode the amino acid sequence shown in SEQ ID NO: 15, but is not limited thereto.

[0029] In the present invention, exon 5 of the granzyme B gene (GZMB) may include the coding sequence shown in SEQ ID NO: 8, but is not limited thereto.

[0030] In the present invention, exon 5 of the granzyme B gene (GZMB) may contain the base sequence shown in SEQ ID NO: 9, preferably consisting of the base sequence shown in SEQ ID NO: 9, but is not limited thereto.

[0031] In the present invention, exon 5 of the granzyme B gene (GZMB) may encode the amino acid sequence shown in SEQ ID NO: 16, but is not limited thereto.

[0032] In the present invention, the first exon of the granzyme B gene may be at least one selected from the five exons contained in the granzyme B gene or fragments thereof, i.e., exon 1, exon 2, exon 3, exon 4, exon 5, or at least one selected from fragments thereof.

[0033] In the present invention, the second exon of the granzyme B gene may be at least one selected from the five exons contained in the granzyme B gene or fragments thereof, i.e., at least one selected from exon 1, exon 2, exon 3, exon 4, exon 5, or fragments thereof.

[0034] In the present invention, the first exon and the second exon may be the same or different from each other. Preferably, the first exon and the second exon may be different from each other.

[0035] In one example of the present invention, the first exon may be at least one selected from exon 1, exon 2, exon 3, exon 4, exon 5, or fragments thereof contained in the granzyme B gene, and the second exon may be at least one selected from the remaining exons or fragments thereof.

[0036] In one embodiment of the present invention, the first exon may comprise exon 1 of the granzyme B gene (GZMB) or a fragment thereof, and the second exon may comprise exon 5 of the granzyme B gene (GZMB) or a fragment thereof, but is not limited thereto.

[0037] In one embodiment of the present invention, the first exon may include exon 1 of the granzyme B gene (GZMB) or a fragment thereof, and the second exon may include exons 2, 3, and 4 of the granzyme B gene (GZMB) and exon 5 or a fragment thereof, but is not limited thereto.

[0038] In one embodiment of the present invention, the first exon may include exon 1 or a fragment thereof and exon 2 of the granzyme B gene (GZMB), and the second exon may include exon 3 and exon 4 of the granzyme B gene (GZMB), and exon 5 or a fragment thereof, but is not limited thereto.

[0039] In another embodiment of the present invention, the first exon may include exon 1 or a fragment thereof and exons 2 and 3 of the granzyme B gene (GZMB), and the second exon may include exon 4 and exon 5 or a fragment thereof of the granzyme B gene (GZMB), but is not limited thereto.

[0040] In one embodiment of the present invention, the first exon may include exon 1 or a fragment thereof, and exon 2, exon 3, and exon 4 of the granzyme B gene (GZMB), and the second exon may include, but is not limited to, exon 5 or a fragment thereof of the granzyme B gene (GZMB).

[0041] In one example of the present invention, the first exon may include the nucleotide sequence shown in SEQ ID NO: 3, but is not limited thereto.

[0042] In one example of the present invention, the first exon may include the nucleotide sequence shown in SEQ ID NO: 4, but is not limited thereto.

[0043] As one example of the present invention, the second exon may include the base sequence shown in SEQ ID NO: 10 (the sequences of SEQ ID NOs: 5 to 8 are linked sequentially from the 5' position to the 3' position), but is not limited thereto.

[0044] As one example of the present invention, the second exon may include the base sequence shown in SEQ ID NO: 11 (the sequences of SEQ ID NOs: 5 to 7 and 9 linked sequentially from the 5' position to the 3' position), but is not limited thereto.

[0045] In the present invention, the first intron and the second intron may contain a splice site.

[0046] In the present invention, the term "splice site" refers to a specific nucleic acid sequence that can be recognized by the splicing machinery of a eukaryotic cell and ligated to a corresponding splice site. Splice sites allow the excision of introns present in a pre-mRNA transcript. Typically, the 5' portion of the splice site is referred to as the splice donor site, and the 3' portion of the splice site is referred to as the splice acceptor site. The term splice site includes, for example, naturally occurring splice sites, engineered splice sites, e.g., synthetic splice sites, canonical or consensus splice sites, and / or non-canonical splice sites, e.g., cryptic splice sites.

[0047] In the present invention, the first intron or a fragment thereof may contain a splice donor site.

[0048] In the present invention, the second intron or a fragment thereof may contain a splice acceptor site.

[0049] Introns that can be used in the present invention may be introns isolated from genes derived from animals, for example, mammals (e.g., humans), and there are no particular limitations on the type of intron, as long as it can be removed by splicing after transcription of the region.

[0050] In the present invention, the intron may contain splice donor and acceptor sites, a triple guanine motif, and / or a branch point sequence.

[0051] As one example of the present invention, the first intron may include a splice donor site of the base sequence shown in SEQ ID NO: 17, preferably consisting of the base sequence shown in SEQ ID NO: 17, but is not limited thereto.

[0052] As one example of the present invention, the second intron may include a splice acceptor site of the base sequence shown in SEQ ID NO: 18, preferably consisting of the base sequence shown in SEQ ID NO: 18, but is not limited thereto.

[0053] The recombinant nucleic acid construct of the present invention may contain a polynucleotide encoding a tumor antigen, e.g., an interfering RNA (miRNA) having immune checkpoint-specific inhibitory activity, between the first intron and the second intron.

[0054] In the present invention, the "immune checkpoint" is a protein in the immune system that suppresses excessive immune responses, and is a surface protein of immune cells that cancer cells use to avoid attacks by the immune system. In the present invention, examples of the immune barrier protein include, but are not limited to, PD-1, PD-L1, PD-L2, IDO, 2B4 (CD244), 4-1BB, A2Ar, B7.1, B7.2, B7-H2, B7-H3, B7-H4, B7-H6, BTLA, butyrophilin, CD160, CD48, CTLA4, GITR, gp49B, HHLA2, HVEM, ICOS, ILT-2, ILT-4, KIR family receptors, LAG-3, OX-40, PIR-B, SIRPalpha (CD47), TFM-4, TIGIT, TIM-1, TIM-3, TIM-4, and VISTA.

[0055] In the present invention, the "interfering RNA (miRNA)" may be an shRNA, siRNA, or microRNA having a sequence homologous to a target gene, and preferably may be a microRNA, but is not limited thereto.

[0056] In the present invention, the term "miRNA" or "microRNA" refers to a small, non-coding RNA consisting of 18-25 nucleotides (nt) that regulates gene expression by binding to the 3'-untranslated region (UTR) of target genes (Non-Patent Documents 1 and 2). It is assembled through a series of processes from introns, exons, or intergenic regions (Non-Patent Document 3). First, miRNAs are transcribed by RNA polymerase into nascent miRNA (pri-miRNA) molecules containing several thousand nucleotides. The pri-miRNA is then assembled through successive processes by the microprocessor Drosha RNase endonuclease and DiGeorge syndrome region gene 8 protein (DGCR8) to form an approximately 70-nt stem-loop intermediate known as a miRNA precursor (Non-Patent Documents 4 and 5). The pre-miRNA is then transported from the nucleus to the cytoplasm via exportin-5 (EXP5) and the cofactor Ran-GTP, where it is processed by the RNase endonuclease Dicer to form an 18-25 nt mature miRNA duplex (Non-Patent Documents 6 and 7). The mature miRNA duplex is integrated as a single stranded RNA with Argonaute protein into the RNA-induced silencing complex (Non-Patent Document 7), which induces either cleavage or translational repression of the target mRNA (Non-Patent Documents 8, 9, and 10).

[0057] In the present invention, the polynucleotide encoding the miRNA may be not only a polynucleotide encoding the mature form of the miRNA, but also a polynucleotide encoding an miRNA analog, which may include a miRNA precursor, a primary transcript miRNA (pri-miRNA), or a plasmid-form miRNA precursor.

[0058] In the present invention, the miRNA may include, without limitation, any miRNA having cancer prevention, amelioration, or treatment activity, for example, an miRNA having an inhibitory activity on a gene encoding human PD-L1 (Programmed Cell Death-Ligand-1) or human IDO (indoleamine dioxygenase), among immune barrier proteins (immune checkpoints), but is not limited thereto.

[0059] In one embodiment of the present invention, the miRNA may be, but is not limited to, miR-138.

[0060] In one example of the present invention, the miRNA may be, but is not limited to, miR-153.

[0061] In one example of the present invention, the miRNA may be, but is not limited to, miR-138 and miR-153.

[0062] In the present invention, the mature sequence of miR-138 may be human hsa-miR-138, which is hsa-miR-138-5p consisting of the base sequence shown in SEQ ID NO: 19, but is not limited thereto.

[0063] In the present invention, the sequence encoding the mature sequence of miR-138 may include, but is not limited to, the nucleotide sequence represented by SEQ ID NO: 20, and preferably consists of the nucleotide sequence represented by SEQ ID NO: 20.

[0064] In the present invention, the sequence encoding the precursor (pre-miRNA) of miR-138 may include, but is not limited to, the base sequence represented by SEQ ID NO: 21, and preferably consists of, the base sequence represented by SEQ ID NO: 21.

[0065] In the present invention, the sequence encoding the initial transcript (pri-miRNA) of miR-138 may include the base sequence shown in SEQ ID NO: 22, and preferably consists of the base sequence shown in SEQ ID NO: 22, but is not limited thereto.

[0066] In the present invention, the mature sequence of miR-153 may be hsa-miR-153 derived from humans (homo sapiens), which may be hsa-miR-153-5p consisting of the base sequence shown in SEQ ID NO: 23, but is not limited thereto.

[0067] In the present invention, the sequence encoding the mature RNA sequence of miR-153 may include the base sequence shown in SEQ ID NO: 24, and preferably consists of the base sequence shown in SEQ ID NO: 24, but is not limited thereto.

[0068] In the present invention, the sequence encoding the precursor (pre-miRNA) of miR-153 may include the base sequence shown in SEQ ID NO: 25, and preferably consists of the base sequence shown in SEQ ID NO: 25, but is not limited thereto.

[0069] In the present invention, the sequence encoding the initial transcript (pri-miRNA) of miR-153 may include the base sequence shown in SEQ ID NO: 26, and preferably consists of the base sequence shown in SEQ ID NO: 26, but is not limited thereto.

[0070] In the present invention, when two or more interfering RNAs are contained between the first intron and the second intron, the order in which they are bound is not particularly limited.

[0071] In one embodiment of the present invention, when both a polynucleotide encoding miR-138 and a polynucleotide encoding miR-153 are contained between the first and second introns, the order of their binding is not particularly limited, and they may be linked in the order of the sequence encoding miR-138 (or an analog thereof) followed by the sequence encoding miR-153 (or an analog thereof) from the 5' to 3' position, or the sequence encoding miR-153 (or a mimetic thereof) followed by the sequence encoding miR-138 (or an analog thereof).

[0072] As an example of the present invention, the polynucleotide may include, but is not limited to, a polynucleotide comprising the base sequence shown in SEQ ID NO: 20 and a polynucleotide comprising the base sequence shown in SEQ ID NO: 24 between the first intron and the second intron.

[0073] As an example of the present invention, the polynucleotide may include, but is not limited to, a polynucleotide comprising the base sequence represented by SEQ ID NO: 21 and a polynucleotide comprising the base sequence represented by SEQ ID NO: 25 between the first intron and the second intron.

[0074] As an example of the present invention, the polynucleotide may include, but is not limited to, a polynucleotide comprising the base sequence shown in SEQ ID NO: 22 and a polynucleotide comprising the base sequence shown in SEQ ID NO: 26 between the first intron and the second intron.

[0075] The mRNA obtained after transcription of the recombinant nucleic acid construct provided in the present invention undergoes a splicing process in which the first and second intron regions are removed, and the first and second exon mRNAs are combined with each other. The polypeptide obtained through translation then expresses cancer cell killing activity.

[0076] Furthermore, the miRNA or its analog (e.g., pre-miRNA or pre-miRNA) separated during the splicing process can undergo additional processes as needed and exert therapeutic activity by targeting cancer cells and suppressing their growth or inducing their death.

[0077] Another embodiment of the present invention relates to a recombinant nucleic acid construct comprising a polynucleotide encoding each of miR-138 and miR-153.

[0078] In the present invention, the polynucleotide encoding miR-138 or miR-153 may be a polynucleotide encoding not only the mature form of these miRNAs but also a polynucleotide encoding the miRNA analogue, where the miRNA mimic may include a miRNA precursor, a primary transcript miRNA (pri-miRNA), or a plasmid-form miRNA precursor.

[0079] In the present invention, the mature sequence of miR-138 may be hsa-miR-138 derived from humans (homo sapiens), which may be hsa-miR-138-5p consisting of the base sequence shown in SEQ ID NO: 19, but is not limited thereto.

[0080] In the present invention, the sequence encoding the mature sequence of miR-138 may include the nucleotide sequence shown in SEQ ID NO: 20, and preferably consists of the nucleotide sequence shown in SEQ ID NO: 20, but is not limited thereto.

[0081] In the present invention, the sequence encoding the precursor (pre-miRNA) of miR-138 may include the base sequence shown in SEQ ID NO: 21, and preferably consists of the base sequence shown in SEQ ID NO: 21, but is not limited thereto.

[0082] In the present invention, the sequence encoding the initial transcript (pri-miRNA) of miR-138 may include the base sequence shown in SEQ ID NO: 22, and preferably may consist of the base sequence shown in SEQ ID NO: 22, but is not limited thereto.

[0083] In the present invention, the mature sequence of miR-153 may be hsa-miR-153-5p, which is human-derived hsa-miR-153 and consists of the base sequence shown in SEQ ID NO: 23, but is not limited thereto.

[0084] In the present invention, the sequence encoding the mature sequence of miR-153 may include the nucleotide sequence shown in SEQ ID NO: 24, and preferably consists of the nucleotide sequence shown in SEQ ID NO: 24, but is not limited thereto.

[0085] In the present invention, the sequence encoding the precursor (pre-miRNA) of miR-153 may include the base sequence shown in SEQ ID NO: 25, and preferably consists of the base sequence shown in SEQ ID NO: 25, but is not limited thereto.

[0086] In the present invention, the sequence encoding the initial transcript (pri-miRNA) of miR-153 may include the base sequence shown in SEQ ID NO: 26, and preferably consists of the base sequence shown in SEQ ID NO: 26, but is not limited thereto.

[0087] The recombinant nucleic acid construct of the present invention may contain both a sequence encoding miR-138 or a mimic thereof and a sequence encoding miR-153 or an analog thereof in a single construct. However, the scope of the present invention also includes constructs that separately contain a polynucleotide containing a sequence encoding miR-138 or a mimic thereof and a polynucleotide containing a sequence encoding miR-153 or an analog thereof.

[0088] In the present invention, when a single construct simultaneously contains a sequence encoding miR-138 or an analog thereof and a sequence encoding miR-153 or an analog thereof, the order in which the two genes are linked is not particularly limited. The sequences may be linked from the 5' to 3' positions in the order of miR-138 (or mimic) coding sequence-miR-153 (or mimic) coding sequence, or alternatively, miR-153 (or mimic) coding sequence-miR-138 (or mimic) coding sequence.

[0089] Yet another embodiment of the present invention relates to an expression vector comprising the recombinant nucleic acid construct of the present invention.

[0090] In the present invention, in order to introduce the recombinant nucleic acid construct into an appropriate host cell, the above-described construct can be inserted into a vector so that it can be cloned.

[0091] In the present invention, the term "vector" refers to a recombinant vector that can be introduced into a suitable host cell to express a target protein, and is a gene expression system that includes essential regulatory elements operably linked to express a gene insert.

[0092] In the present invention, various expression systems, such as chromosomes, episomes, and derived viruses, can be used as vectors for inserting the recombinant nucleic acid construct. More specifically, recombinant expression vectors that can be used include, but are not limited to, those derived from bacterial plasmids, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses such as baculovirus, papilloma viruses such as SV40, vaccinia viruses, adenoviruses, adeno-associated viruses, retroviruses, fowlpox viruses, and pseudorabies viruses. These recombinant expression vectors may also be cosmid or phagemid derivatives.

[0093] In an exemplary aspect, one type of vector may be a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments may be ligated. Yet another type of vector may be a phage vector. Yet another type of vector may be a viral vector, in which additional DNA segments may be ligated into the viral genome.

[0094] Certain vectors may be capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) may be integrated into the genome of a host cell upon introduction into the host cell, and thereby be replicated along with the host genome. Certain vectors may also be capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "recombinant vectors").

[0095] In the present invention, the term "operatively linked" refers to a functional connection between an expression regulatory site (e.g., promoter, signal sequence, ribosome binding site, transcription termination sequence, transcription regulatory factor binding site, etc.) of a nucleic acid molecule of interest and another nucleic acid sequence, whereby the regulatory sequence can regulate the transcription and / or decoding of the other nucleic acid sequence.

[0096] The vector system of the present invention can be constructed by various methods known to those skilled in the art, and specific methods for this are disclosed in Non-Patent Document 11, which is incorporated herein by reference.

[0097] As an example of the present invention, vectors that can be used in the present invention can be produced by manipulating plasmids (e.g., Psc101, ColE1, Pbr322, Puc8 / 9, Phc79, Puc19, Pet, etc.), phages (e.g., λgt4Λb, λ-Charon, λΔz1, Λgem.TM.-11, and M13, etc.) or viruses (e.g., SV40, etc.) known to those skilled in the art.

[0098] In one embodiment of the present invention, the recombinant nucleic acid construct may be inserted into a host cell using a viral expression system (vaccinia or other poxvirus, retrovirus or adenovirus). For example, viral vectors may include, but are not limited to, retroviral vectors derived from HIV, SIV, murine retroviruses, gibbon ape leukemia virus, AAVs (adeno-associate viruses), and adenoviruses (Non-Patent Documents 12, 13, 14). Additionally, retroviral vectors derived from murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), ecotropic retroviruses, SIV (simian immunodeficiency virus), and HIV (human immunodeficiency virus) are widely used.

[0099] In one embodiment of the present invention, when the recombinant nucleic acid construct is DNA, the so-called "naked DNA" method can be utilized, or a plasmid vector or a viral vector can be utilized, and the nucleic acid can be contained in an expressible form as a vector targeted to prokaryotic or eukaryotic cells.

[0100] The expression vector of the present invention may contain functional sequences for expressing the nucleic acid sequence of the recombinant nucleic acid construct as an expression target sequence. In the present invention, the functional sequences may include one or more promoters capable of driving transcription, one or more introns, one or more transcription termination regions, one or more start codons, and one or more termination or stop codons. When a cyclokaryotic cell is used as the host cell in the present invention, the vector may also contain a ribosome binding site (RBC) and / or a transcription / translation termination sequence to initiate translation into a peptide. The expression vector of the present invention may also contain a nucleic acid sequence encoding a protease or peptidase cleavage site and one or more leader sequences, each of which encodes a signal peptide. The expression vector of the present invention may also contain one or more linker or tag sequences, which may encode a hemagglutinin (HA) tag.

[0101] In the present invention, either constitutive or inducible promoters can be used in the present invention, depending on the needs of the particular situation as determined by those skilled in the art of the present invention. Multiple promoters recognized by various host cells are widely known. The selected promoter can be operably linked to the cistron DNA encoding the peptide described herein by removing the promoter from the source DNA via a restriction enzyme recognition sequence and inserting a separate, isolated promoter sequence into the selected vector. Both the native promoter sequence and multiple heterologous promoters can be used to direct amplification and / or expression of the target gene. However, heterologous promoters generally provide transcription and expression efficiencies of the expressed target gene compared to the native target polypeptide promoter.

[0102] As an example of the present invention, when a cyclokaryotic cell is used as the host cell, it is common to include a strong promoter capable of driving transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, T7 promoter, etc.), a ribosome binding site for the initiation of translation, and a transcription / translation termination sequence. When E. coli is used as the host cell, the promoter and operator sites of the E. coli tryptophan biosynthetic pathway (Non-Patent Document 15) and the left-handed promoter of phage λ (pLλ promoter, Non-Patent Document 16) can be used as regulatory sites. Such a plasmid vector may be a plasmid commonly used in the art, such as pSC101, pBR322, pUC19, or pET-22.

[0103] In one embodiment of the present invention, when eukaryotic cells are used as host cells, a promoter derived from mammalian cells (metallothionein promoter) or a promoter derived from a mammalian virus can be used. When a virus is used as a vector of the present invention, an attenuated virus such as vaccinia virus or flowpox virus can be used to express the recombinant nucleic acid construct. Furthermore, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, herpes simplex virus, avirulent anthrax toxin vectors, etc. can also be used.

[0104] In one embodiment of the present invention, in addition to the promoters described above, promoters derived from mammalian viruses such as the CMV (cytomegalovirus) promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter, and RSV promoter, EF1 alpha promoter, metallothionein promoter, beta-actin promoter, human IL-2 gene promoter, human IFN gene promoter, human IL-4 gene promoter, human lymphotoxin gene promoter, human GM-CSF gene promoter, cancer cell-specific promoters (e.g., TERT promoter, PSA promoter, PSMA promoter, CEA promoter, E2F promoter, and AFP promoter), tissue-specific promoters (e.g., albumin promoter), and bacteriophage T7 promoter may be used, but are not limited thereto.

[0105] The expression vector of the present invention can further include an enhancer sequence to increase transcriptional activity. The enhancer sequence is a nucleic acid sequence located at various sites in the promoter that can increase transcriptional activity compared to the transcriptional activity of the promoter without the enhancer sequence. Examples of enhancers include, but are not limited to, enhancers from human actin, human myosin, human hemoglobin, and human muscle creatine, as well as viral enhancers such as those derived from CMV, SV40, RSV, and EBV.

[0106] In the present invention, an expression vector can include a sequence encoding one or more signal peptides and / or secretory signal peptides to promote and / or increase the expression or post-translational secretion of a recombinant nucleic acid construct. In the present invention, a signal sequence or peptide is a secretory signal, also known as a leader sequence or peptide or localization signal sequence, which is a short peptide located at the N-terminus of a newly synthesized protein to be secreted. Signal peptides generally stimulate cells to translocate proteins across the cell membrane. The efficiency of protein secretion is strongly determined by the signal peptide. Therefore, the immunostimulatory bacterium of the present invention contains a plasmid that can include a signal peptide and / or secretory signal peptide to promote and / or increase the expression or secretion of an encoded therapeutic agent (or agents). In the present invention, the signal sequence can be an Hly signal sequence, an ActA signal sequence, a PhoA signal sequence, an OmpA signal sequence, an α-amylase signal sequence, an LLO signal sequence, a subtilisin signal sequence, an MF-α signal sequence, a SUC2 signal sequence, an insulin signal sequence, an α-interferon signal sequence, an antibody molecule signal sequence, or any other known signal sequence.

[0107] The expression vector of the present invention may contain a transcription termination sequence to increase transcript stability or facilitate cytoplasmic transport, including, but not limited to, a polyadenylation sequence (e.g., bovine growth hormone terminator and SV40-derived polyadenylation sequence).

[0108] Furthermore, when the expression vector of the present invention is a replicable expression vector, it may contain a replication origin, which is a specific nucleic acid sequence from which replication is initiated.

[0109] The expression vectors of the present invention may contain a DNA nuclear targeting sequence (DTS). DNA nuclear targeting sequences (DTS), such as the SV40 DTS, mediate translocation of DNA sequences through the nuclear pore complex. This transport mechanism is reported to depend on the binding of DNA-binding proteins containing nuclear localization sequences. The inclusion of DTS in expression vectors to increase nuclear transport and expression has been demonstrated (see, e.g., Non-Patent Document 17) and has been used to increase gene expression from plasmids delivered by S. typhimurium (see, e.g., Non-Patent Document 18).

[0110] In one embodiment of the present invention, the DNA nuclear targeting sequence may include, but is not limited to, the base sequence shown in SEQ ID NO:27.

[0111] The expression vector of the present invention may further contain a selection marker to facilitate purification of the expressed peptide. The selection marker is used to select cells transformed with the vector, and may confer a selectable phenotype, such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface protein. Examples of such markers include, but are not limited to, glutathione S-transferase (Pharmacia, USA), maltose-binding protein (NEB, USA), FLAG (IBI, USA), and 6xHis (hexahistidine, Qiagen, USA).

[0112] A variety of in vitro amplification techniques are known for amplifying sequences cloned into the expression vectors of the invention, including polymerase chain reaction (PCR), ligase chain reaction (LCR), Qβ-replicase amplification, and other RNA polymerase-based techniques.

[0113] In the present invention, bacterial strains can be used as host cells, and bacterial strains transformed with the expression vectors of the present invention can be used to prevent, ameliorate, or treat cancer.

[0114] Therefore, the expression vector of the present invention can further comprise a polynucleotide encoding a toxin protein capable of directly or indirectly inducing the death of cancer cells. In the present invention, the toxic protein may be at least one selected from the group consisting of ricin, saporin, gelonin, momordin, debouganin, diphtheria toxin, Pseudomonas toxin, hemolysin (HlyA), FAS ligand (FASL), tumor necrosis factor-alpha (TNF-alpha), TNF-related apoptosis-inducing ligand (TRAIL), streptolysin O (SLO), pneumolysin (PLO), listeriolysin O (LLO), and cytolysin A (ClyA), but is not limited thereto.

[0115] In one embodiment of the present invention, the expression vector can further comprise a polynucleotide encoding the toxin protein listeriolysin O (LLO).

[0116] In the present invention, the "listeriolysin O (LLO)" is a hemolysin produced by the bacterium Listeria monocytogenes, a pathogenic bacterium that causes listeriosis. After lysing and destroying the phagosome, the bacterium escapes into the cytoplasm, allowing it to grow intracellularly. After escaping from the phagosome, the toxin has little activity in the cytoplasm. Listeriolysin O (LLO) is encoded by the Hly gene, which is part of the pathogenicity island LIPI-1.

[0117] In one example of the present invention, the listeriolysin O (LLO) may comprise the amino acid sequence shown in SEQ ID NO: 28, and the Hly gene may comprise the nucleotide sequence shown in SEQ ID NO: 29, but is not limited thereto.

[0118] When the expression vector of the present invention further comprises a polynucleotide encoding a toxin protein, not only the case where the recombinant nucleic acid construct and the polynucleotide encoding the toxin protein are both inserted into a single vector, but also the case where two types of vectors are included, i.e., a vector containing the recombinant nucleic acid construct and a vector containing the polynucleotide encoding the toxin protein, are included for the purposes of the present invention.

[0119] In the present invention, when a recombinant nucleic acid construct and a polynucleotide encoding a toxin protein are simultaneously contained in a single vector, they can be operably linked by a single promoter, or they can each be operably linked by two or more promoters.

[0120] FIG. 1 shows a cleavage map of a plasmid system containing a recombinant nucleic acid construct comprising polynucleotides encoding miR-138 and miR-153 initial transcripts (SEQ ID NOs: 22 and 26) in one embodiment of the present invention.

[0121] FIG. 2 shows a cleavage map of a plasmid system containing the recombinant nucleic acid construct and the Hly gene sequence (SEQ ID NO: 29) encoding listeriolysin O (LLO) in one embodiment of the present invention.

[0122] As an example of the present invention, DTS (SEQ ID NO: 9) may be additionally cloned into the vector shown in FIG. 1 or FIG. 2, but the present invention is not limited thereto.

[0123] 3 and 4 show, but are not limited to, cleavage maps of a plasmid system containing a recombinant nucleic acid construct and a polynucleotide encoding a toxin protein in one embodiment of the present invention.

[0124] As one example of the present invention, the expression vector may be one in which exon 1 (SEQ ID NO: 3), intron 1 (SEQ ID NO: 17), miR-138 early transcript coding sequence (SEQ ID NO: 22), miR-153 early transcript coding sequence (SEQ ID NO: 26), intron 2 (SEQ ID NO: 18), and exons 2 to 5 (SEQ ID NO: 10) of the granzyme B gene are cloned, but is not limited to this.

[0125] As one example of the present invention, the expression vector may be one into which DTS (SEQ ID NO: 27), exon 1 (SEQ ID NO: 3) of the granzyme B gene, intron 1 (SEQ ID NO: 17), miR-138 early transcript coding sequence (SEQ ID NO: 22), miR-153 early transcript coding sequence (SEQ ID NO: 26), intron 2 (SEQ ID NO: 18), and exons 2 to 5 (SEQ ID NO: 10) of the granzyme B gene are cloned, but is not limited thereto.

[0126] As one example of the present invention, the expression vector may be one in which DTS (SEQ ID NO: 27), exon 1 (SEQ ID NO: 3) of the granzyme B gene, intron 1 (SEQ ID NO: 17), miR-138 early transcript coding sequence (SEQ ID NO: 22), miR-153 early transcript coding sequence (SEQ ID NO: 26), intron 2 (SEQ ID NO: 18), and exons 2 to 5 (SEQ ID NO: 10) of the granzyme B gene are operably linked, and the Hly gene (SEQ ID NO: 29) may be additionally cloned, but is not limited to this.

[0127] Another embodiment of the present invention relates to a host cell transformed with the expression vector of the present invention. In the present invention, the host cell may be genetically engineered to express granzyme B or a fragment thereof and an interfering RNA (RNAi) having specific inhibitory activity against a tumor antigen, such as an immune checkpoint protein.

[0128] In the present invention, granzyme B may be encoded by the base sequence shown in SEQ ID NO: 1, but is not limited thereto.

[0129] In the present invention, granzyme B may include the amino acid sequence shown in SEQ ID NO: 2, but is not limited to this.

[0130] As an example of the present invention, a fragment of granzyme B may include a polypeptide encoded by at least two selected from exon 1, exon 2, exon 3, exon 4, and exon 5 of granzyme B, or fragments thereof.

[0131] In the present invention, the polypeptide encoded by exon 1 of the granzyme B gene (GZMB) may include, but is not limited to, the amino acid sequence shown in SEQ ID NO: 12.

[0132] In the present invention, the polypeptide encoded by exon 2 of the granzyme B gene (GZMB) may include, but is not limited to, the amino acid sequence shown in SEQ ID NO: 13.

[0133] In the present invention, the polypeptide encoded by exon 3 of the granzyme B gene (GZMB) may include, but is not limited to, the amino acid sequence shown in SEQ ID NO: 14.

[0134] In the present invention, the polypeptide encoded by exon 4 of the granzyme B gene (GZMB) may comprise the amino acid sequence shown in SEQ ID NO: 15, but is not limited to this.

[0135] In the present invention, the polypeptide encoded by exon 5 of the granzyme B gene (GZMB) may include, but is not limited to, the amino acid sequence shown in SEQ ID NO: 16.

[0136] In one embodiment of the present invention, the interfering RNA (RNAi) having immune checkpoint protein-specific inhibitory activity is an miRNA having inhibitory activity on the gene encoding human PD-L1 (Programmed Cell Death-Ligand-1) or human IDO (indoleamine dioxygenase), and may be preferably at least one of miR-138 and miR-153, but is not limited thereto.

[0137] In the present invention, the host cell may be genetically engineered to express miR-138 and miR-153.

[0138] In the present invention, the mature sequence of miR-138 may consist of the nucleotide sequence shown in SEQ ID NO: 19, but is not limited to this.

[0139] In the present invention, the mature sequence of miR-153 may consist of the nucleotide sequence shown in SEQ ID NO: 23, but is not limited thereto.

[0140] In addition, in the present invention, the host cell can be genetically engineered to additionally express listeriolysin O (LLO).

[0141] In the present invention, listeriolysin O (LLO) may include the amino acid sequence shown in SEQ ID NO: 28, but is not limited thereto.

[0142] In the present invention, the host cell may be transformed with the expression vector provided by the present invention.

[0143] In the present invention, a "host cell" is a cell used to house, maintain, reproduce, and / or amplify a vector. The "host cell" can also be used to express a polypeptide encoded by the vector. The nucleic acid contained in the vector is replicated when the host cell divides, amplifying the nucleic acid.

[0144] In the present invention, the host cell is preferably an immunostimulatory bacterial strain, where "immunostimulatory bacteria" are therapeutic bacteria that, when introduced into a subject, accumulate in immune-privileged tissues and cells, such as tumors, tumor microenvironments, and resident immune cells comprising tumors, and replicate and / or express products that are themselves immunostimulatory or that elicit immunostimulation.

[0145] In the present invention, the immunostimulatory bacteria can be of any suitable species. For example, Salmonella, Listeria, Rickettsia, Klebsiella, Bordetella, Neisseria, Aeromonas, Francisella, Corynebacterium, Citrobacter, Chlamydia, Haemophilus, Brucella, Mycobacterium, Mycoplasma, Legionella, Rhodococcus, Pseudomonas, Helicobacter, Vibrio, Bacillus, or Erysipelothrix The bacterial strains may include, but are not limited to, strains of Erysipelothrix. For example, Salmonella typhimurium, Salmonella choleraesuis, Salmonella enteritidis, Salmonella infantis, Salmonella paratyphi, Salmonella typhi, Listeria monocytogenes, Rickettsia rickettsiae, Rickettsia prowazekii, Rickettsia tsutsugamuchi, Rickettsia mooseri, Rickettsia sibirica, Bordetella bronchiseptica, bronchiseptica), Neisseria meningitidismeningitidis, Neisseria gonorrhoeae, Aeromonas eucrenophila, Aeromonas salmonicida, Francisella tularensis, Corynebacterium pseudotuberculosis, Citrobacter freundii, Chlamydia pneumoniae, Haemophilus somnus, Brucella abortus, Mycobacterium intracellulare, Legionella pneumophila, Rhodococcus equi The bacterial strains may include, but are not limited to, Bacillus subtilis, Erysipelothrix rhusiopathiae, Yersinia enterocolitica, Rochalimaea quintana, or Agrobacterium tumerfacium.

[0146] In one preferred embodiment of the present invention, the bacterial strain may be a Salmonella strain, and more preferably may be derived from at least one selected from the group consisting of Salmonella typhimurium, Salmonella choleraesuis, Salmonella enteritidis, Salmonella infantis, Salmonella paratyphi, and Salmonella typhi. The Salmonella strains listed are Gram-negative, facultative anaerobic rods. They are distinguished from Escherichia coli because they lack the ability to decompose lactose, form indole, and produce hydrogen sulfide. The Salmonella strains may have peritrichous hairs and be motile.

[0147] In the present invention, "Salmonella typhimurium" refers to a bacterium of the genus Salmonella that causes typhoid fever. Salmonella typhimurium is a rod-shaped bacillus with flagella and is gram-negative. Salmonella typhimurium is heat-sensitive, dying in 20 minutes at 60°C. It can be primarily contaminated by livestock, wild animals, carriers, milk, eggs, etc., and can also cause secondary infections such as salads contaminated with contaminated raw meat, which can lead to salmonellosis, a type of food poisoning. Exemplary Salmonella Typhimurium strains of the present invention include, but are not limited to, attenuated and wild-type strains, such as those derived from strains designated as AST-100, VNP20009, YS1646 (ATCC #202165), RE88, SL7207, χ8429, χ8431, and χ8468, or the wild-type strain having ATCC Accession No. 14028. Here, the VNP20009 strain is a genetically modified strain of attenuated Salmonella typhimurium harboring msbB and purI sites, as described in detail in (Non-Patent Document 19).

[0148] In the present invention, the "Salmonella choleraesuis" is a bacterium of the genus Salmonella commonly known as the hog cholera bacterium, which infects both humans and animals. Salmonella choleraesuis is a major causative agent of acute sepsis caused by Salmonella. This bacterium is a motile, gram-negative, facultative anaerobic bacillus with peritrichous hairs. It is distinct from Escherichia coli because it lacks lactose decomposition ability, does not form indole, and does not produce hydrogen sulfide. Its optimal growth temperature is 35-37°C, its growth range is 10-43°C, and it is killed by heating at 60°C for 20 minutes. Its optimal pH is 7.2-7.4, and its size is 0.5-0.8 x 3-4 μm.

[0149] In the present invention, "Salmonella enteritidis" refers to a bacterium of the genus Salmonella that causes bacterial food poisoning and is also known as Salmonella enteritidis. Salmonella enteritidis is a representative bacterium of the Salmonella genus, capable of infecting all animals and exhibiting a high degree of host adaptability. It is a motile, gram-negative, facultative anaerobic bacillus with peritrichous hairs. It is distinct from Escherichia coli because it lacks lactose decomposition, does not form indole, and does not produce hydrogen sulfide. Its optimal growth temperature is 35-37°C, its growth temperature range is 10-43°C, and it is killed by heating at 60°C for 20 minutes. Its optimal pH is 7.2-7.4, and its size is 0.5-0.8 x 3-4 μm.

[0150] In the present invention, the "Salmonella infantis" is a bacterial strain that is transmitted through eggs or poultry, and Salmonella paratyphi and Salmonella typhi are causative strains of typhoid fever.

[0151] Another embodiment of the present invention relates to a pharmaceutical composition for preventing, ameliorating, or treating cancer, comprising the genetically engineered host cell, preferably a bacterial strain, provided herein as an active ingredient.

[0152] In the present invention, "cancer" refers to a disease characterized by the rapid and uncontrolled growth of mutant cells, and may be at least one selected from the group consisting of melanoma, laryngeal cancer, brain cancer, small intestine cancer, esophageal cancer, lymphatic cancer, gallbladder cancer, blood cancer, thyroid cancer, endocrine cancer, oral cancer, liver cancer, biliary tract cancer, colon cancer, rectal cancer, cervical cancer, ovarian cancer, kidney cancer, stomach cancer, duodenal cancer, prostate cancer, breast cancer, brain cancer, lung cancer, anaplastic thyroid cancer, uterine cancer, colon cancer, bladder cancer, ureter cancer, pancreatic cancer, bone and soft tissue sarcoma, skin cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, multiple myeloma, leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and single myeloma, but is not limited thereto.

[0153] In the present invention, "prevention" refers to any action of suppressing cancer or delaying its progression by administering the composition of the present invention.

[0154] In the present invention, "treatment" and "amelioration" refer to any action in which the symptoms of cancer are improved or beneficially altered by administering the composition of the present invention.

[0155] For administration, the pharmaceutical composition of the present invention may be formulated by adding one or more pharmaceutically acceptable carriers in addition to the active ingredients. Pharmaceutically acceptable carriers may include saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, or a mixture of one or more of these components. Other common additives, such as antioxidants, buffers, and bacteriostats, may be added as needed. Furthermore, the composition may be formulated into injectable forms such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets by adding diluents, dispersants, surfactants, binders, and lubricants. Target organ-specific antibodies or other ligands may be conjugated to the carriers to specifically act on target organs. Furthermore, formulations may be prepared according to the disease or component, using appropriate methods in the art or the methods disclosed in Remington et al. (2013).

[0156] The pharmaceutical composition of the present invention can be formulated into a solution, suspension, dispersion, emulsion, gel, injectable solution, sustained release formulation of the active compound, etc., and preferably into an injectable preparation.

[0157] When the pharmaceutical composition of the present invention is formulated into an injection, in order to ensure product stability during distribution of the injection formulation, the pH can be adjusted using an aqueous acid solution or a buffer solution such as a phosphate solution that can be used as an injection, thereby producing an injection that is very physically and chemically stable.

[0158] More specifically, the injection can be prepared by dissolving the compound in water for injection together with a stabilizer or solubilizer, followed by sterilization, particularly by high-temperature vacuum sterilization or aseptic filtration. The water for injection can be distilled water for injection or a buffer solution for injection, such as a phosphate buffer solution or sodium dihydrogen phosphate (NaH2PO4)-citrate buffer solution with a pH range of 3.5 to 7.5. The phosphate salt used can be in the form of a sodium salt or potassium salt, or in the form of an anhydrous or hydrated form, or in the form of citrate or an anhydrous or hydrated form.

[0159] Stabilizers used in the present invention include sodium pyrosulfite, sodium bisulfite (NaHSO), sodium metabisulfite (NaSO), or ethylenediaminetetraacetic acid, and solubilizers include bases such as sodium hydroxide (NaOH), sodium bicarbonate (NaHCO), sodium carbonate (NaCO), or potassium hydroxide (KOH), or acids such as hydrochloric acid (HCl) or acetic acid (CHCOOH).

[0160] The injectable preparation according to the present invention can be formulated to be bioabsorbable, biodegradable, or biocompatible. Bioabsorbable means that the injectable preparation can be dissolved in the body after initial application without degradation or decomposition of the dispersed injectable preparation. Biodegradable means that the injectable preparation can be broken down or decomposed in the body by hydrolysis or enzymatic degradation. Biosynthetic means that all of the components are non-toxic in the body.

[0161] The injection according to the present invention can be prepared using a conventional filler, weighting agent, binder, wetting agent, surfactant or other diluent or excipient.

[0162] The composition or active ingredient of the present invention can be administered in a conventional manner, such as intravenously, intraarterially, intraperitoneally, intramuscularly, intrasternally, transdermally, intracalf, subcutaneously, intrauterine durally, by inhalation, topically, rectally, orally, intraocularly, or intradermally, depending on the purpose, and is preferably administered intravenously. The composition or active ingredient of the present invention can be administered by injection or catheter.

[0163] The dosage of bacteria administered to a subject by administering the compositions of the present invention can depend on many factors, as is well known in the medical field, including the subject's dosage, the subject's species, size, body surface area, age, sex, immunocompetence, and general health, the specific bacteria administered, the duration and route of administration, the type and stage of disease, e.g., tumor size, and other compounds, e.g., drugs, being administered concomitantly. In addition to the above factors, the dosage can be determined by one skilled in the art, as it can be influenced by the infectivity and characteristics of the bacteria. In this method, an appropriate minimum dosage level of bacteria can be a level sufficient for the bacteria to survive, grow, and replicate in tumors or metastases. An exemplary minimum dosage level for administering bacteria to a 65 kg human is at least about 5 x 10 6 Colony-forming units (CFU), at least approximately 1 × 10 7 CFU, at least approximately 5 × 10 7 CFU, at least approximately 1 x 10 8 CFU, or at least about 1 x 109 In the present method, a suitable maximum dosage level of bacteria may be a level that is not toxic to the host, does not induce 3x or more splenomegaly, and does not produce colonies or plaques in normal tissues or organs after about 1 day, or about 3 days, or about 7 days. An exemplary maximum level for administering bacteria to a 65 kg human is about 5x10 11 CFU or less, approximately 1×10 11 CFU or less, approximately 5×10 10 CFU or less, approximately 1×10 10 CFU or less, or approximately 1 x 10 9 It may contain less than CFU.

[0164] The active ingredient in the pharmaceutical composition of the present invention may be contained in an amount of 0.001 to 50% by weight based on the total weight of the composition, but the content is not limited thereto.

[0165] In addition, the pharmaceutical composition of the present invention may further comprise one or more anti-cancer agents.

[0166] In the present invention, examples of the anticancer drug include nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, neratinib, lapatinib, gefitinib, vandetanib, nilotinib, semasanib, bosutinib, axitinib, cediranib, lestaurtinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, bevacizumab, cisplatin, cetuximab, viscum albumin, asparaginase, tretinoin, hydroxycarbamide, dasatinib, estramustine, gemtuzumab ozogamicin, ibritumomab tiuxetan, heptaplatin, methylaminolevulinic acid, amsacrine, alemtuzumab, procarbazine, alprostadil, and phosphatidyl nitrate. Fluorum chitosan, gemcitabine, doxifluridine, pemetrexed, tegafur, capecitabine, gimelacin, oteracil, azacitidine, methotrexate, uracil, cytarabine, fluorouracil, fludarabine, enocitabine, flutamide, decitabine, mercaptopurine, thioguanine, cladribine, carmofur, raltitrexed, docetaxel, paclitaxel, irinotecan, belotecan, topotecan, vinorelbine, etoposide, vincristine, vinblastine, teniposide, doxorubicin, idarubicin, epirubicin, mitoxantrone, mitomycin, bleomycin, daunorubicin , dactinomycin, pirarubicin, aclarubicin, peplomycin, temsirolimus, temozolomide, busulfan, ifosfamide, cyclophosphamide, melphalan, altretamine, dacarbazine, thiotepa, nimustine, chlorambucil, mitolactol, leucovorin, tretonin, exemestane, aminoglutethimide, anagrelide, navelbine, padrazole, tamoxifen, toremifene, testolactone, anastrozole, letrozole, vorozole, bicalutamide, lomustine, and carmustine, but the present invention is not limited thereto.

[0167] In the present invention, in addition to administering the composition of the present invention for the prevention, amelioration, or treatment of the above-mentioned cancers, combination therapy with other antitumor therapeutic agents and / or treatments can also be performed. For example, these can include, but are not limited to, cell therapy, e.g., administration of modified immune cells, CAR-T therapy, CRISPR therapy, immunotherapy, e.g., immune checkpoint inhibitors, e.g., antibodies and antibody fragments, chemotherapy and chemotherapeutic compounds, e.g., nucleotide analogs, surgery, oncolytic virus therapy, and radiation therapy.

[0168] Yet another embodiment of the present invention relates to a method for preventing, ameliorating, or treating cancer, comprising administering to an individual the pharmaceutical composition provided herein.

[0169] In the present invention, the individual may include, without limitation, mammals including rats, livestock, humans, birds, reptiles, farmed fish, etc. that are at risk of developing or being affected by cancer.

[0170] In the present invention, the cancer, pharmaceutical composition, and administration thereof are the same as those described above, and therefore will not be described again below. [Effects of the Invention]

[0171] The recombinant nucleic acid construct provided by the present invention is such that the first and second intron regions of the mRNA obtained after transcription are removed through the splicing process, and the first and second exon mRNAs are combined with each other, and then the polypeptide obtained through translation can express cancer cell killing activity.

[0172] Furthermore, the recombinant nucleic acid construct of the present invention may further include, between the first and second introns, a polynucleotide encoding an interfering RNA (RNAi) that targets an immune checkpoint or other tumor antigen as an inhibitor targeting an antigen present in cancer cells. In this case, the interfering RNA separated during the splicing process can be expected to synergistically enhance anti-cancer activity. In particular, by inserting miR-138 and miR-153, which target cancer cells, excellent anti-cancer activity can be expressed through their synergistic action.

[0173] Furthermore, the present invention is based on the idea that the vector expressing the recombinant nucleic acid construct is delivered via an immunostimulatory bacterial strain, thereby enabling the bacterial strain itself to exert an additional anti-cancer effect. [Brief explanation of the drawings]

[0174] [Figure 1] 1 shows a cleavage map of the pcDNA3-miRNA-Pdl1-IDO vector prepared in Example 1 of the present invention. [Figure 2] 1 shows a cleavage map of the pcDNA3-miRNA-Pdl1-IDO-Hly vector prepared in Example 2 of the present invention. [Figure 3] 1 shows a cleavage map of the pcDNA3-intron-GrB_FLAG-miRNA-Pdl1-IDO-Hly vector prepared in Example 4 of the present invention. [Figure 4] 1 shows a cleavage map of the pcDNA3-DTS-intron-GrB_FLAG-miRNA-Pdl1-IDO-Hly vector prepared in Example 5 of the present invention. [Figure 5] 1 shows the results of electrophoresis of a slice obtained after treating the pcDNA3-miRNA-Pdl1-IDO vector produced in Example 1 of the present invention with a restriction enzyme. [Figure 6] 1 shows the results of electrophoresis of a slice obtained after treating the pcDNA3-miRNA-Pdl1-IDO-Hly vector produced in Example 2 of the present invention with a restriction enzyme. [Figure 7] 1 shows the results of Western blot analysis of LLO expression levels in cell lysates of Salmonella strains transformed with the pcDNA3-miRNA-Pdl1-IDO-Hly vector prepared in Example 2 of the present invention. [Figure 8] 1 shows the results of electrophoresis of a slice obtained after treating the pcDNA3-GrB vector produced in Example 4 of the present invention with a restriction enzyme. [Figure 9]1 shows the results of electrophoresis of a slice obtained after treating the pcDNA3-intron-GrB_FLAG-miRNA-Pdl1-IDO-Hly vector produced in Example 4 of the present invention with a restriction enzyme. [Figure 10] 1 shows the results of electrophoresis of a slice obtained after treating the pcDNA3-DTS-intron-GrB_FLAG-miRNA-Pdl1-IDO-Hly vector produced in Example 5 of the present invention with a restriction enzyme. [Figure 11] 1 shows the results of Western blotting to examine the expression levels of PD-L1 and IDO in cell lysates after infection of SW480 colon cancer cells with a Salmonella strain transformed with a vector in Example 7 of the present invention. [Figure 12] FIG. 10 shows the results of Western blotting to examine the expression levels of PD-L1 and IDO in cell lysates after infection of HT29 colon cancer cells with a Salmonella strain transformed with a vector in Example 7 of the present invention. [Figure 13] 1 shows the results of Western blotting to examine the expression levels of PD-L1 and IDO in cell lysates after infection of Huh7 hepatocarcinoma cells with a Salmonella strain transformed with a vector in Example 7 of the present invention. [Figure 14] 1 shows the results of Western blotting to examine the expression levels of PD-L1 and IDO in cell lysates after infection of breast cancer MDA-MB-231 cells with a Salmonella strain transformed with a vector in Example 7 of the present invention. [Figure 15] FIG. 1 shows the results of Western blotting to examine the expression levels of PD-L1 and IDO in cell lysates after infection of pancreatic cancer Pan1 cells with a Salmonella strain transformed with a vector in Example 7 of the present invention. [Figure 16]In Example 8 of the present invention, colon cancer cells were infected with Salmonella strains transformed with vectors, and the expression levels of PD-L1 (target A) and IDO (target B) in the cell lysates were examined by Western blotting. The graph shows the percentage change in PD-L1 and IDO expression levels in colon cancer cells treated with the Salmonella strains compared to untreated colon cancer cells. [Figure 17] 1 shows the percentage change in PD-L1 and IDO expression levels in hepatoma cells treated with Salmonella strains compared to untreated hepatoma cells after infection of hepatoma cells with Salmonella strains transformed with vectors in Example 8 of the present invention and analysis of the expression levels of PD-L1 (target A) and IDO (target B) in the cell lysates by Western blotting. [Figure 18] In Example 8 of the present invention, breast cancer cells were infected with Salmonella strains transformed with vectors, and the expression levels of PD-L1 (target A) and IDO (target B) in the cell lysates were examined by Western blotting. The graph shows the percentage change in PD-L1 and IDO expression levels in breast cancer cells treated with the Salmonella strains compared to untreated breast cancer cells. [Figure 19] In Example 8 of the present invention, pancreatic cancer cells were infected with a Salmonella strain transformed with a vector, and the expression levels of PD-L1 (target A) and IDO (target B) in the cell lysate were determined by Western blotting. Graphs showing the percentage changes in PD-L1 and IDO expression levels in pancreatic cancer cells treated with the Salmonella strain compared to untreated pancreatic cancer cells are shown. [Figure 20]1 shows photographs of the expression level of granzyme B protein in SW480 colon cancer cells, observed under a fluorescence microscope after infection with a Salmonella strain transformed with a vector in Example 9 of the present invention. In each figure, "Neg" or "Neg. no Hly" indicates the results of treatment with a Salmonella strain transformed with a pcDNA3 mock vector in which the Hly gene was not cloned, "Neg. Hly" indicates the results of treatment with a Salmonella strain transformed with a pcDNA3 vector in which only the Hly gene was cloned, "P / I" or "PI" indicates the results of treatment with a Salmonella strain transformed with the pcDNA3-miRNA-Pdl1-IDO-Hly vector in Example 2, and "Int.GP / I" indicates the results of treatment with a Salmonella strain transformed with the pcDNA3-intron-GrB_FLAG-miRNA-Pdl1-IDO-Hly vector in Example 4. "DP / I" or "DPI" refers to the results of treating a Salmonella strain transformed with the pcDNA3-DTS-miRNA-Pdl1-IDO-Hly vector, which is the vector of Example 2 in which DTS was additionally cloned. "DGP / I" or "DGPI" refers to the results of treating a Salmonella strain transformed with the pcDNA3-DTS-intron-GrB_FLAG-miRNA-Pdl1-IDO-Hly vector of Example 5. "GPI" refers to the results of treating a Salmonella strain transformed with a vector in which the GrB CDS, hsa-mir-138, and hsa-mir-153 were sequentially cloned into the pcDNA3 vector and the Hly gene was additionally cloned. "Gzm" is the result of treating a Salmonella strain transformed with a vector in which only the GrB CDS was cloned into the pcDNA3 vector, and "DGzm" is the result of treating a Salmonella strain transformed with a vector in which the DTS and GrB CDS were cloned into the pcDNA3 vector. DETAILED DESCRIPTION OF THE INVENTION

[0175] In one embodiment, the present invention relates to a recombinant nucleic acid construct comprising, from the 5' to 3' positions, a first exon, a first intron, a second intron, and a second exon of the granzyme B (GZMB) gene, and comprising nucleotides between the first intron and the second intron that encode at least one interfering RNA (RNAi).

[0176] In the present invention, the first exon of the granzyme B gene may be at least one selected from the five exons contained in the granzyme B gene or fragments thereof, i.e., exon 1, exon 2, exon 3, exon 4, exon 5, or at least one selected from fragments thereof.

[0177] In the present invention, the second exon of the granzyme B gene may be at least one selected from the five exons contained in the granzyme B gene or fragments thereof, i.e., exon 1, exon 2, exon 3, exon 4, exon 5, or at least one selected from fragments thereof.

[0178] In one illustrative example of the present invention, the first exon may comprise exon 1 of the granzyme B gene (GZMB) or a fragment thereof, and the second exon may comprise exon 5 of the granzyme B gene (GZMB) or a fragment thereof, but is not limited thereto.

[0179] In one embodiment of the present invention, the first exon may include exon 1 of the granzyme B gene (GZMB) or a fragment thereof, and the second exon may include, but is not limited to, exons 2, 3, and 4 of the granzyme B gene (GZMB) and exon 5 or a fragment thereof.

[0180] In one example of the present invention, the first exon may include exon 1 or a fragment thereof and exon 2 of the granzyme B gene (GZMB), and the second exon may include, but is not limited to, exon 3 and exon 4 of the granzyme B gene (GZMB) and exon 5 or a fragment thereof.

[0181] In one example of the present invention, the first exon may include exon 1 or a fragment thereof and exons 2 and 3 of the granzyme B gene (GZMB), and the second exon may include exon 4 and exon 5 or a fragment thereof of the granzyme B gene (GZMB), but is not limited thereto.

[0182] In one example of the present invention, the first exon may include exon 1 or a fragment thereof, and exon 2, exon 3, and exon 4 of the granzyme B gene (GZMB), and the second exon may include, but is not limited to, exon 5 or a fragment thereof of the granzyme B gene (GZMB).

[0183] In one example of the present invention, the first exon may include the nucleotide sequence shown in SEQ ID NO: 3, but is not limited thereto.

[0184] In one example of the present invention, the first exon may include the nucleotide sequence shown in SEQ ID NO: 4, but is not limited thereto.

[0185] As one example of the present invention, the second exon may contain the base sequence shown in SEQ ID NO: 10 (the sequences of SEQ ID NOs: 5 to 8 linked sequentially from the 5' position to the 3' position), but is not limited thereto.

[0186] As one example of the present invention, the second exon may contain the base sequence shown in SEQ ID NO: 11 (the sequences of SEQ ID NOs: 5 to 7 and 9 linked sequentially from the 5' position to the 3' position), but is not limited thereto.

[0187] In the present invention, the first intron or a fragment thereof may contain a splice donor site.

[0188] In the present invention, the second intron or a fragment thereof may contain a splice acceptor site.

[0189] As one example of the present invention, the first intron may include a splice donor site of the base sequence shown in SEQ ID NO: 17, and preferably may consist of the base sequence shown in SEQ ID NO: 17, but is not limited thereto.

[0190] As one example of the present invention, the second intron may include a splice acceptor site of the base sequence shown in SEQ ID NO: 18, preferably consisting of the base sequence shown in SEQ ID NO: 18, but is not limited thereto.

[0191] The recombinant nucleic acid construct of the present invention may contain a polynucleotide encoding an interfering RNA (RNAi) having tumor antigen-specific inhibitory activity, such as an immune checkpoint protein, between the first and second introns.

[0192] In one example of the present invention, the interfering RNA can be, but is not limited to, miR-138.

[0193] In one example of the present invention, the interfering RNA can be, but is not limited to, miR-153.

[0194] In one example of the present invention, the interfering RNA can be, but is not limited to, miR-138 and miR-153.

[0195] In the present invention, the mature sequence of miR-138 may be hsa-miR-138 derived from humans (homo sapiens) and may be hsa-miR-138-5p consisting of the base sequence shown in SEQ ID NO: 19, but is not limited thereto.

[0196] In the present invention, the sequence encoding the mature sequence of miR-138 may include the base sequence shown in SEQ ID NO: 20, and preferably consists of the base sequence shown in SEQ ID NO: 20, but is not limited thereto.

[0197] In the present invention, the sequence encoding the precursor (pre-miRNA) of miR-138 may include, but is not limited to, the base sequence shown in SEQ ID NO: 21, and preferably consists of, the base sequence shown in SEQ ID NO: 21.

[0198] In the present invention, the sequence encoding the initial transcript (pri-miRNA) of miR-138 may include the base sequence shown in SEQ ID NO: 22, and preferably consists of the base sequence shown in SEQ ID NO: 22, but is not limited thereto.

[0199] In the present invention, the mature sequence of miR-153 may be hsa-miR-153 derived from humans (homo sapiens) and may be hsa-miR-153-5p consisting of the base sequence shown in SEQ ID NO: 23, but is not limited thereto.

[0200] In the present invention, the sequence encoding the mature sequence of miR-153 may include the base sequence shown in SEQ ID NO: 24, and preferably consists of the base sequence shown in SEQ ID NO: 24, but is not limited thereto.

[0201] In the present invention, the sequence encoding the precursor (pre-miRNA) of miR-153 may include the base sequence shown in SEQ ID NO: 25, and preferably consists of the base sequence shown in SEQ ID NO: 25, but is not limited thereto.

[0202] In the present invention, the sequence encoding the initial transcript (pri-miRNA) of miR-153 may include the base sequence shown in SEQ ID NO: 26, and preferably consists of the base sequence shown in SEQ ID NO: 26, but is not limited thereto.

[0203] As an example of the present invention, the present invention may include, but is not limited to, a polynucleotide comprising the base sequence shown in SEQ ID NO: 21 between the first intron and the second intron, and a polynucleotide comprising the base sequence shown in SEQ ID NO: 25.

[0204] As an example of the present invention, the present invention may include, but is not limited to, a polynucleotide comprising the base sequence shown in SEQ ID NO: 22 between the first intron and the second intron and a polynucleotide comprising the base sequence shown in SEQ ID NO: 26.

[0205] Another embodiment of the present invention relates to a recombinant nucleic acid construct comprising a polynucleotide encoding each of miR-138 and miR-153.

[0206] According to yet another embodiment of the present invention, the present invention relates to an expression vector comprising the recombinant nucleic acid construct.

[0207] The expression vector of the present invention may contain a DNA nuclear targeting sequence (DTS).

[0208] In one embodiment of the present invention, the DNA nuclear targeting sequence may include, but is not limited to, the base sequence shown in SEQ ID NO:27.

[0209] The expression vector of the present invention may further comprise a polynucleotide encoding a toxin protein capable of directly or indirectly inducing the death of cancer cells. In the present invention, the toxic protein may be at least one selected from the group consisting of ricin, saporin, gelonin, momordin, devouganin, diphtheria toxin, Pseudomonas toxin, hemolysin (HlyA), FAS ligand (FASL), tumor necrosis factor-alpha (TNF-alpha), TNF-related apoptosis-inducing ligand (TRAIL), streptolysin O (SLO), pneumolysin (PLO), listeriolysin O (LLO), and cytolysin A (ClyA), but is not limited thereto.

[0210] In one embodiment of the present invention, the expression vector can further comprise a polynucleotide encoding the toxin protein listeriolysin O (LLO).

[0211] As one example of the present invention, listeriolysin O (LLO) may comprise the amino acid sequence shown in SEQ ID NO: 28, and the Hly gene may comprise the nucleotide sequence shown in SEQ ID NO: 29, but is not limited thereto.

[0212] Yet another embodiment of the present invention relates to a host cell transformed with the expression vector.

[0213] In the present invention, the host cell may be genetically engineered to express granzyme B or a fragment thereof, and an interfering RNA (RNAi) having specific inhibitory activity against a tumor antigen, such as an immune barrier protein (immune checkpoint).

[0214] In the present invention, the host cell may be genetically engineered to express miR-138 and miR-153.

[0215] In the present invention, the host cell may be genetically engineered to additionally express listeriolysin O (LLO).

[0216] Preferably, the host cell according to the present invention is an immunostimulatory bacterial strain. The immunostimulatory bacteria according to the present invention can be of any suitable species. For example, Salmonella, Listeria, Rickettsia, Klebsiella, Bordetella, Neisseria, Aeromonas, Francisella, Corynebacterium, Citrobacter, Chlamydia, Haemophilus, Brucella, Mycobacterium, Mycoplasma, Legionella, Rhodococcus, Pseudomonas, Helicobacter, Vibrio, Bacillus, or Erysipelothrix The strains of Erysipelothrix can include, but are not limited to, strains of Erysipelothrix. For example, Salmonella typhimurium, Salmonella choleraesuis, Salmonella enteritidis, Salmonella infantis, Salmonella paratyphi, Salmonella typhi, Listeria monocytogenes, Rickettsia rickettsiae, Rickettsia prowazekii, Rickettsia tsutsugamuchi, Rickettsia mooseri, Rickettsia sibirica, Bordetella bronchiseptica,bronchiseptica, Neisseria meningitidis, Neisseria gonorrhoeae, Aeromonas eucrenophila, Aeromonas salmonicida, Francisella tularensis, Corynebacterium pseudotuberculosis, Citrobacter freundii, Chlamydia pneumoniae, Haemophilus somnus, Brucella abortus, Mycobacterium intracellulare, Legionella pneumophila, Rhodococcus equi, Pseudomonas aeruginosa, Helicobacter mustelae, Vibrio cholerae, Bacillus subtilis, Erysipelothrix rhusiopathiae, Yersinia enterocolitica, Rochalimaea quintana, or Agrobacterium tumerfacium.

[0217] Another embodiment of the present invention relates to a pharmaceutical composition for preventing, ameliorating, or treating cancer, comprising the genetically engineered host cell, preferably a bacterial strain, provided herein as an active ingredient.

[0218] Yet another embodiment of the present invention relates to a method for preventing, ameliorating, or treating cancer, comprising administering to an individual the pharmaceutical composition provided herein.

[0219] The present invention will be described in more detail with reference to the following examples. It will be obvious to those skilled in the art that these examples are provided merely to illustrate the present invention in more detail, and that the scope of the present invention is not limited to these examples according to the gist of the present invention. [Example]

[0220] Production of pcDNA3-miRNA-Pdl1-IDO vector To clone hsa-mir138, which suppresses expression of the EPD-L1 plasmid, and hsa-mir153, which regulates expression of IDO, into pcDNA3, oligonucleotides were synthesized containing hsa-mir138 (SEQ ID NO: 22) and hsa-mir153 (SEQ ID NO: 26) in sequential order. The oligonucleotides were inserted between the EcoRI and XhoI restriction enzyme sites of pcDNA3 so that the hsa-mir138 and hsa-mir153 genes were cloned downstream of the T7 promoter present in pcDNA3 (see the cleavage map in Figure 1).

[0221] The resulting plasmid was then digested with HindIII / XhoI restriction enzymes, and the resulting fragment was electrophoresed on a 0.9% agarose gel, revealing a band of 585 bp, as shown in Figure 5. This indicates that hsa-mir138 and hsa-mir153 were successfully cloned into the vector, resulting in the production of a recombinant plasmid. [Example]

[0222] Production of pcDNA3-miRNA-Pdl1-IDO-Hly vector To insert the Hly gene (SEQ ID NO: 29) into the plasmid prepared in Example 1, a sense primer 5'-CCCGGGCCCTCCTTTGATTAG-3' containing a restriction enzyme XmaI recognition site at the 5' end and an antisense primer 5'-TTCGAAGCTTATATTATATGGATAAACAGTC-3' containing a restriction enzyme BstBI recognition site at the 3' end were purchased from IDT. The bacterial plasmid Pt7RNAi-Hly-Inv(TRIP) was used as the gene template for amplifying the Hly gene (Non-Patent Document 20).

[0223] To amplify the Hly gene, 0.5 ng of TRIP was mixed with 2 pmoles of sense and antisense primers and Phusion DNA polymerase (Invitrogen, USA), and PCR was performed according to the manufacturer's protocol. The PCR conditions were 98°C for 30 seconds, followed by 35 cycles of 98°C for 5 seconds, 55°C for 10 seconds, and 72°C for 30 seconds, followed by an extension reaction at 72°C for 5 minutes. After PCR, the amplified Hly gene was confirmed to be of correct size by DNA electrophoresis and then extracted using a Qiagen Extraction kit.

[0224] To insert the Hly gene into the plasmid prepared in Example 1, the plasmid and the amplified Hly gene were treated with restriction enzymes XmaI and BstBI. The gene segments were ligated to prepare the pcDNA3-miR-PDL1-IDO-Hly plasmid (see the cleavage map in Figure 2).

[0225] The resulting plasmid was digested with XmaI / BstBI restriction enzymes, and the resulting fragment was electrophoresed on a 0.9% agarose gel. As a result, a band corresponding to the size of the Hly gene, 2016 bp, was confirmed, as shown in Figure 6. This indicates that the Hly gene was successfully cloned into the vector. [Example]

[0226] Preparation of Salmonella strains transformed with pcDNA3-miR-PDL1-IDO-Hly and verification of LLO production The Salmonella typhimurium VNP20009 strain was transformed with the vector prepared in Example 1 by electroporation, and the transformed strain was cultured overnight in LB medium containing 100 μg / ml ampicillin. A control strain was transformed with Pcmv-Negative. After culturing for 24 hours in fresh LB medium containing 50 μg / ml ampicillin, whole cell lysates were obtained. An equal volume of 2X SDS-PAGE sample solution was added to the whole cell lysate and boiled for 5 minutes. 10 μl of the boiled solution was electrophoresed on a 4-20% gradient SDS-PAGE gel with protein size markers at 120 V for approximately 1 hour, followed by transfer to a nitrocellulose membrane at 100 V for approximately 50 minutes. After primary reaction with anti-Hly rabbit monoclonal antibody (Abcam), LLO expression was confirmed at the protein level using HRP-conjugated anti-rabbit mouse polyclonal antibody (Sigma), and the results are shown in Figure 7. For the same sample, loading controls were confirmed with anti-DnaK mouse monoclonal antibody (Abcam) and HRP-conjugated anti-rabbit mouse polyclonal antibody (Sigma).

[0227] As shown in Figure 7, it was confirmed that LLO was expressed in the Salmonella strain transformed with the pcDNA3-miR-PDL1-IDO-Hly vector. [Example]

[0228] Production of pcDNA3-intron-GrB_FLAG-miRNA-Pdl1-IDO-Hly vector To prepare a vector that simultaneously expresses Granzyme B, hsa-miR-138, and hsa-miR-153, the vector was designed and prepared as follows.

[0229] 1. Preparation of pcDNA3-GrB vector To clone the Gzmb CDS gene (SEQ ID NOs: 3 and 5-8) into the pcDNA3 vector, we purchased a human Granzyme B cDNA ORF clone from Synbiological. To amplify the Gzmb gene, we purchased a sense primer (5'-GGTACCGAGATGGTGCAAC-3') containing a KpnI restriction enzyme sequence at the 5' end and an antisense primer (5'-GGATCCTTACTTATCGTCGTCATCCTTGTAATCACTTCCGCCACCGTAGCGTTTCATGGT-3') containing a BamHI restriction enzyme recognition site with a FLAG tag inserted (IDT). The PCR-amplified Gzmb gene was inserted into the KpnI and BamHI restriction enzyme sites of pcDNA3.

[0230] The resulting plasmid was digested with KpnI / BstBI restriction enzymes, and the resulting fragment was subjected to electrophoresis on a 0.9% agarose gel. As shown in Figure 8, a band corresponding to 798 bp, the size of the Gzmb gene containing a Flag tag, was observed. This indicates that the Gzmb gene was cloned into the vector.

[0231] 2. Preparation of pcDNA3-intron-GrB_FLAG-miRNA-Pdl1-IDO-Hly vector (see cleavage map in Figure 3) To clone intron-containing hsa-mir138 and hsa-mir153 into the constructed pcDNA3-GrB vector, gene amplification was performed using the pcDNA-miR-Pdl1-IDO-Hly vector constructed in Example 2 as a template. The sense primer used was 5'-GTAAGAGTCGATCGCTTCCCGGTTCTCACTTCTG-3', which contains the 3' end of the first Gzmb exon (CDS of exon 1, SEQ ID NO: 3), the intron 1 gene sequence (SEQ ID NO: 17), and a portion of the 5' end of hsc-miRNA-138. The antisense primer used was 5'-CTGCAGGATATCAAAAAAAAAAGAAGAGGTACCAGTTAGTAACGCGTCGGGCGAGAG-3', which contains a portion of the 3' end of hsc-miRNA-153 and the intron 2 gene sequence (SEQ ID NO: 18) followed by a portion of the 5' end of the second Gzmb exon (CDS of exons 2-4 and exon 5, SEQ ID NO: 10). Both primers were purchased from IDT.

[0232] To amplify the intron-miRNA gene, 0.2 ng of the pcDNA-miR-Pdl1-IDO-Hly expression vector prepared in Example 2, 2 pmoles of sense and antisense primers, and Phusion DNA polymerase (Invitrogen, USA) were mixed and PCR was performed according to the manufacturer's protocol. The PCR reaction conditions were 98°C for 30 seconds, followed by 35 cycles of 98°C for 5 seconds, 68°C for 10 seconds, and 72°C for 8 seconds, followed by a 5-minute extension at 72°C. To use the amplified gene for assembly, the assembly-intron-miRNA gene was amplified using gene assembly primers. The PCR reaction conditions were 98°C for 30 seconds, followed by 35 cycles of 98°C for 5 seconds, 71°C for 30 seconds, and 72°C for 8 seconds, followed by a 5-minute extension at 72°C.

[0233] Gene amplification was then performed to obtain the assembly-Gzmb vector gene for assembly with the amplified assembly-intron-miRNA gene. The sense primer used was 5'-GGGAGATCATCGGGGGACATG-3', which corresponds to the 5' end of Gzmb exon 5, and the antisense primer was 5'-CTGCATCTGCCCTGGGCAGCA-3', which corresponds to the 3' end of Gzmb exon 1, both purchased from IDT. PCR was performed using 0.2 ng of Pcmv-Gzmb as the template, 0.2 pmoles of the sense and antisense primers, and Phusion DNA polymerase (Invitrogen, USA), followed by amplification according to the manufacturer's recommendations. The PCR reaction conditions were 98°C for 30 seconds, followed by 35 cycles of 98°C for 5 seconds, 63°C for 30 seconds, and 72°C for 118 seconds, followed by a 5-minute extension at 72°C.

[0234] To synthesize the amplified assembly-intron-miRNA and assembly-GZMB vector into a single vector, we used a method developed by Daniel G. Gibson (21). Approximately 100 ng of the 7918 bp assembly-GZMB vector and 15 ng of assembly-intron-miRNA were used per reaction using the NEBuilder HiFi DNA Assembly kit provided by New England BioLabs according to the manufacturer's recommended method. The two gene fragments were incubated at 50°C for 30 minutes.

[0235] To confirm that the GZMB (SEQ ID NOs: 3 and 10), hsa-mir-138 (SEQ ID NO: 22), and hsa-mir-153 (SEQ ID NO: 26) genes were cloned into a single vector, the resulting vector was cleaved with KpnI and BamHI restriction enzymes, and the resulting fragment was subjected to electrophoresis on a 0.9% agarose gel. As a result, the expected 686 bp band was observed, as shown in Figure 9. This indicates that the GZMB, hsa-miR-138, and hsa-miR-153 genes were successfully cloned into the vector. [Example]

[0236] Preparation of pcDNA3-DTS-intron-GrB_FLAG-miRNA-Pdl1-IDO-Hly vector To clone a DNA nuclear targeting sequence (DTS) into the vectors prepared in Examples 2 and 4, a DTS template (SEQ ID NO: 27) was purchased from IDT and used as a PCR template. The sense primer used for PCR was 5'-AGGCGTTTTGCGCTGCTTCG-3', and the antisense primer was 5'-TATATCTGGCCCGTACATCGGGAAAGTC-3'. PCR was performed using Phusion DNA polymerase (Invitrogen, USA) according to the manufacturer's protocol. The PCR reaction conditions were 98°C for 30 seconds, followed by 35 cycles of 98°C for 5 seconds, 68°C for 10 seconds, and 72°C for 2 seconds, followed by a 5-minute extension at 72°C.

[0237] To prepare the amplified DTS gene assembly, the vectors prepared in Examples 2 and 4 were treated with NruI restriction enzyme at 37°C for 30 minutes to obtain linear vectors. Next, the amplified DTS gene fragments and the linear vector were combined using the NEBuilder HiFi DNA Assembly kit provided by New England BioLabs according to the method recommended by the manufacturer to prepare gene fragments. The two gene fragments were reacted at 50°C for 30 minutes.

[0238] Subsequently, to confirm that the two genes had been ligated into one vector, the resulting section obtained after treatment with BglII and NdeI was subjected to electrophoresis on a 0.9% agarose gel. As a result, the expected 541 bp band was observed, as shown in Figure 10. [Example]

[0239] Production of vector-transformed Salmonella strains To transform the Salmonella typhimurium VNP20009 strain with the vectors prepared in Examples 1, 2, 3, 4, and 5, the VNP20009 strain cultured on agar medium was washed with 1 ml of cold 10 mM HEPES and centrifuged at 3,000 rpm. After removing the supernatant, the centrifuged bacterial cells were washed again with 1 ml of 10 mM HEPES and cooled on ice for 10 minutes. This washing process was repeated three times, and the remaining centrifuged bacterial cells were mixed with 100 ng of the transformation vector (the vector prepared in Examples 1, 2, 3, 4, or 5) in 40 μl of cold 10% glycerol and left on ice for 15 minutes. The cells were then transferred to a 1 mm cuvette for electroporation, and the vector was transferred into the strain using an Eppendorf Eporator at 1,700 V. The cells were then incubated in 250 μl of SOC medium at 37°C in a shaking incubator at 200 rpm for 1 hour, and then cultured on agar medium containing ampicillin at 37°C for at least 24 hours. Recombinant Salmonella colonies cultured on agar medium were selected and transferred to 10 ml of BHI medium containing ampicillin, and then cultured again at 37°C. The recombinant Salmonella cultured in BHI medium was centrifuged at 3000 rpm for 10 minutes, and the resulting bacterial cells were washed with 5 ml of serum-free Dulbecco's Modified Eagles Media (DMEM), a process that was repeated three times. [Example]

[0240] Inhibition of PD-L1 and IDO protein expression in cancer cells treated with transformed Salmonella strains (1) To confirm the inhibitory effect of the vectors prepared in the Examples on PD-L1 and IDO protein expression in tumor cells, SW480 colon cancer cells, HT29 colon cancer cells, Huh7 liver cancer cells, MBA-MB-231 breast cancer cells, and Panc1 pancreatic cancer cells were infected with the recombinant Salmonella strains of Example 6 transformed with the respective vectors. Specifically, tumor cells cultured in 12-well culture plates were infected for 2 hours with 1 ml of DMEM medium adjusted to the desired cell:recombinant Salmonella ratio. Salmonella strains that had not penetrated into the tumor cells were then removed for 1 hour with DMEM containing 200 μl / ml gentamicin. The recombinant Salmonella-infected tumor cells were cultured in fresh DMEM medium for 48 hours, then lysed in RIPA hemolysis buffer at ice temperature. After 30 minutes, the cells were centrifuged and the supernatant was collected and mixed with an equal volume of 2X SDS sample. 20µg of protein was quantified and separated according to molecular weight on an SDS-PAGE gel at 100V for 1 hour using the SDS-PAGE method described above, then transferred to a nitrocellulose membrane at 100V for 50 minutes. PD-L1 rabbit monoclonal antibody (R&D Systems) was used as the primary antibody to detect PD-L1 protein expression, and IDO rabbit monoclonal antibody (Cell Signaling) was used as the primary antibody to detect IDO protein expression. GAPDH mouse monoclonal antibody (Abcam) was used as a control. HRP-conjugated mouse anti-rabbit polyclonal antibody and rabbit anti-mouse polyclonal antibody were used as secondary antibodies.

[0241] As a result, as shown in Figures 11 to 15, it was confirmed that the expression levels of PD-L1 and IDO were significantly reduced in tumor cells infected with Salmonella strains transformed with the pcDNA3-intron-GrB_FLAG-miRNA-Pdl1-IDO-Hly vector of the present invention (Example 4) compared to the control group. Furthermore, it was confirmed that the expression levels of PD-L1 and IDO were further reduced in tumor cells infected with Salmonella strains transformed with a vector (pcDNA3-DTS-intron-GrB_FLAG-miRNA-Pdl1-IDO-Hly vector of Example 5) in which a DNA nuclear targeting sequence (DTS) was cloned together with GZMB (exon 1 and exon 2), hsc-miRNA-138, and the sequence encoding hsc-miRNA-138. [Example]

[0242] Inhibition of PD-L1 and IDO protein expression in cancer cells treated with transformed Salmonella strains (2) To quantitatively confirm the inhibitory effect of the vectors prepared in the Examples on the expression of PD-L1 and IDO proteins in tumor cells, colon cancer cells, liver cancer cells, breast cancer cells, and pancreatic cancer cells were infected with the recombinant Salmonella strains transformed with each of the vectors described in Example 6, using the same method as in Example 7. The expression levels of PD-L1 (target A) and IDO (target B) expressed in the tumor cells were then confirmed by Western blotting, and the percentage change in expression level due to treatment with each strain compared to the expression level in the untreated control group was measured. The results are shown in Figures 16 to 19.

[0243] As shown in Figures 16 to 19, in all of the colon cancer cells, liver cancer cells, breast cancer cells, and pancreatic cancer cells, treatment with the Salmonella strain transformed with the pcDNA3-DTS-intron-GrB_FLAG-miRNA-Pdl1-IDO-Hly vector of the present invention (Example 5) demonstrated a significant decrease in the expression levels of PD-L1 and IDO compared to the untreated control group or the wild-type Salmonella strain. [Example]

[0244] Expression of granzyme B protein in cancer cells treated with transformed Salmonella strains Salmonella strains transformed with each vector prepared in Example 6 were applied to SW480 colon cancer cells cultured on poly-L-lysine coverslips. After 48 hours, the cells were subjected to immunocytochemistry. Specifically, SW480 cells were fixed with 4% paraformaldehyde and then delipidated with 0.5% Triton X-100 and 0.5% saponin. The cells were blocked with 5% normal donkey serum and incubated with rabbit anti-FLAG antibody (Proteintech) at 4°C for one day. The FLAG antibody was then detected using donkey anti-rabbit Alexa488 antibody (Invitrogen). To confirm alpha-tubulin, the sections were treated with mouse anti-alpha-tubulin monoclonal antibody, followed by donkey anti-mouse Alexa594 (Invitrogen) as the secondary antibody. To fluorescently stain DNA, the coverslip and slide were attached to each other using a mounting solution containing 4',6-diamidino-2-phenylindole (DAPI), ensuring that no air was trapped between them. Images of the fluorescently stained cells were taken using a Zeiss Axioimager M1 epifluorescence microscope and are shown in Figure 20. In this experiment, instead of inserting hsa-mir-138 and hsa-mir-153 together with an intron between the two exon fragments of Gzmb, we prepared the pcDNA3-GrB_FLAG-miRNA-Pdl1-IDO-Hly vector in which the Gzmb CDS (sequential sequence of SEQ ID NOs: 3 and 10), hsa-mir-138 (SEQ ID NO: 22), and hsa-mir-153 (SEQ ID NO: 26) were cloned into the pcDNA3 vector in that order, and the experiment was performed in the same manner. In addition, we also prepared a vector in which only the GrB CDS (sequential sequence of SEQ ID NOs: 3 and 10) was cloned into the pcDNA3 vector, or a vector in which the DTS (SEQ ID NO: 27) and the Gzmb CDS (sequential sequence of SEQ ID NOs: 3 and 10) were cloned into the pcDNA3 vector, and the experiment was performed in the same manner.

[0245] As shown in Figure 20, it was confirmed that granzyme B was expressed at the highest level in colon cancer cells infected with a Salmonella strain transformed with the pcDNA3-intron-GrB_FLAG-miRNA-Pdl1-IDO-Hly vector of the present invention (Example 4).It was also confirmed that the expression level of granzyme B was higher than that when treated with a Salmonella strain transformed with the pcDNA3-GrB_FLAG-miRNA-Pdl1-IDO-Hly vector (GPI), in which the CDS of GzmB, hsa-mir-138, and hsa-mir-153 are linked in sequence so that granzyme B, miR-138, and miR-153 can be expressed without splicing.

[0246] The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting. [Industrial Applicability]

[0247] The present invention relates to recombinant nucleic acid constructs useful in cancer therapy, gene expression systems containing the same, and bacterial strains transformed with the expression systems.

Claims

1. The first exon, the first intron, the second intron, and the second exon of the granzyme B (GZMB) gene are included; A recombinant nucleic acid construct comprising nucleotides encoding at least one interfering RNA (RNAi) between the first intron and the second intron.

2. The recombinant nucleic acid construct of claim 1, wherein the first exon or the second exon is at least one selected from exon 1, exon 2, exon 3, exon 4, and exon 5 of granzyme B.

3. 2. The recombinant nucleic acid construct of claim 1, wherein the first exon and the second exon are identical to or different from each other.

4. the first exon comprises exon 1 of the granzyme B gene or a fragment thereof; 2. The recombinant nucleic acid construct of claim 1, wherein the second exon comprises exon 2, exon 3, and exon 4 of the granzyme B gene, and exon 5 or a fragment thereof.

5. the first exon comprises a base sequence represented by SEQ ID NO: 3 or 4, The recombinant nucleic acid construct according to claim 1, wherein the second exon comprises a base sequence represented by SEQ ID NO: 10 or 11.

6. the first intron comprises a splice donor site; 2. The recombinant nucleic acid construct of claim 1, wherein the second intron comprises a splice acceptor site.

7. The first intron comprises a base sequence represented by SEQ ID NO: 17, The recombinant nucleic acid construct according to claim 1, wherein the second intron comprises the base sequence represented by SEQ ID NO:

18.

8. 2. The recombinant nucleic acid construct of claim 1, wherein the first intron and the second intron are the same or different from each other.

9. The recombinant nucleic acid construct according to claim 1, wherein the interfering RNA (RNAi) is shRNA, siRNA, or microRNA (miRNA) having immune checkpoint protein inhibitory activity.

10. The recombinant nucleic acid construct according to claim 9, wherein the immune barrier protein is PD-L1 (Programmed Cell Death-Ligand-1) or IDO (indolamine dioxygenase).

11. The recombinant nucleic acid construct of claim 1, wherein the interfering RNA (RNAi) is at least one of miR-138 and miR-153.

12. An expression vector comprising the recombinant nucleic acid construct of any one of claims 1 to 11.

13. The expression vector of claim 12, wherein the expression vector comprises one or more promoters, one or more introns, one or more transcription termination regions, one or more start codons, and one or more end or stop codons.

14. The expression vector of claim 12, further comprising a polynucleotide encoding a toxin protein.

15. The toxic proteins include ricin, saporin, gelonin, momordin, debouganin, diphtheria toxin, Pseudomonas toxin, hemolysin (HlyA), FAS ligand (FASL), tumor necrosis factor-alpha (TNF-alpha), TNF-related apoptosis-inducing ligand (TRAIL), and streptolysin O.

15. The expression vector of claim 14, wherein the vector is at least one selected from the group consisting of pneumolysin O (SLO), pneumolysin (PLO), listeriolysin O (LLO), and cytolysin A (ClyA).

16. The expression vector of claim 15, further comprising a polynucleotide encoding listeriolysin O (LLO).

17. The expression vector of claim 12, further comprising a DNA nuclear targeting sequence (DTS).

18. The expression vector of claim 17, wherein the DNA nuclear targeting sequence (DTS) comprises a base sequence represented by SEQ ID NO:

27.

19. A host cell transformed with the expression vector of claim 12.

20. A host cell characterized by being genetically engineered to express granzyme B or a fragment thereof, and at least one of miR-138 and miR-153.

21. 21. The host cell of claim 20, wherein the host cell is genetically engineered to additionally express listeriolysin O (LLO).

22. The host cells may be selected from the group consisting of Salmonella, Listeria, Rickettsia, Klebsiella, Bordetella, Neisseria, Aeromonas, Francisella, Corynebacterium, Citrobacter, Chlamydia, and the like.

21. The host cell of claim 20, wherein the host cell is one or more bacterial strains selected from the group consisting of strains of the genera Bacillus, Bacillus subtilis, Bacillus sp., Bacillus spp ...

23. A pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient a host cell transformed with an expression vector containing the recombinant nucleic acid construct of any one of claims 1 to 11.

24. A pharmaceutical composition for preventing or treating cancer, comprising the host cell of any one of claims 20 to 22 as an active ingredient.

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