KR20130019616A - Fusion protein includes two or more brg-1 derived bromodomains and uses thereof - Google Patents

Abstract

PURPOSE: A recombinant protein containing two or more BRG-1-derived bromodomains is provided to enhance radiosensitivity in cancer cells and cancer cell apoptosis, and to ensure anticancer effect. CONSTITUTION: A recombinant protein contains two or more BRG-1-derived bromodomains. The bromodomains are denoted by amino acid sequence 1. The recombinant protein enhances radiosensitivity in cells and additionally contains a cell internalization sequence. The cell internalization sequence contains an amino acid of polyarginine, antennapedia, TAT, HIV-Tat, penetratin, Antp-3A(Antp mutant), Buforin II, MAP(model amphiphilic peptide), K-FGF, Ku70, prion, pVEC, Pep-1, SynB1, Pep-7, HN-1, BGSC (bis-guanidinium-spermidin-cholesterol), or BGTC (bis-guanidinium-tren-cholesterol). The recombinant protein additionally contains a tumor-specific targeting sequence such as an RGD, NGR, or GSL motif. A radiosensitizer composition for radiation therapy for cancer contains the recombinant protein as an active ingredient.

Description

Fusion protein includes two or more BRG-1 derived bromodomains and uses approximately}

The present invention relates to a recombinant protein comprising two or more bromodomains derived from BRG-1, and more particularly, to a recombinant protein comprising two or more bromodomains derived from BRG-1, encoding the same. It relates to a nucleic acid, an expression vector comprising the nucleic acid, a radiation sensitive composition for cancer cell radiation therapy comprising the recombinant protein or the expression vector as an active ingredient, and a cancer cell comprising the expression vector.

In the United States, about two-thirds of patients are ultimately receiving radiation, including pain relief, while in South Korea, about one third of cancer patients receive radiation.

Radiation therapy is currently known as an essential treatment method for various types of cancers, but it has been pointed out that obtaining radiation resistance of cancer cells and damaging normal tissues in high dose radiation treatments lowers the efficiency of radiation therapy. Therefore, research on radiation therapy sensitizers to improve the efficiency of radiation therapy has been attempted.

Radiation therapy, on the other hand, is one of the most important and powerful therapies for many types of cancers, especially for local cancers that do not cause metastasis. However, only a few of the tumors to be treated by radiation therapy, such as lymphomas and normal epithelial tumors, are responsive to radiation therapy. Many other solid tumors, such as melanoma, glioma, and prostate cancer, are typically very resistant to radiation and require very large amounts of radiation treatment. The reasons why radiation therapy fails are often complex and variable. Tumor factors such as location, size and inadequate vascular supply (oxygen deficiency) can all play an important role in the lack of neoplastic responsiveness to ionizing radiation (IR). Perhaps most important is the cellular and genetic factors involved in radiation-sensitive regulation, for example differential tissue-specific gene expression, which can lead to radiation-resistant cell phenotypes. Scientists have long worked to develop various methods for increasing tumor cell sensitivity to IR. Examples include hypoxic radiation sensitive enhancers, high concentrations of oxygen, and more recently targeting many genetic factors related to radiation sensitivity. However, despite significant scientific advances in molecular biology and biochemistry over the last few decades, only limited progress has been made in the development of effective and specific radiation-sensitive enhancers in cancer genetics and molecular radiation biology.

Radiation therapy in brain tumor treatment is a method of removing abnormal cells by delaying the cell cycle (DNA damage checkpoint) or induction of apoptosis in response to DNA damage caused by radiation. However, the problem of radiation therapy is that the radiation-resistant cancer cells cause cancer recurrence due to the inherent radiation resistance of cancer cells and the increased resistance to radiation therapy, and the radiation-resistant cells are also resistant to anticancer drugs.

Therefore, there is an urgent need for the development of a radiation sensitivity enhancer for enhancing the radiation sensitivity of cancer cells having inherent radiation resistance to radiation.

On the other hand, histones are basic proteins that are commonly present in the nucleus of eukaryotic cells, ranging from multicellular organisms including humans to unicellular organisms represented by fungi (fungus, yeast), and ionically bind to genomic DNA. Histones usually consist of five components (H1, H2A, H2B, H3 and H4) and are highly similar across species. For example, in the case of histone H4, the germinating yeast histone H4 (102 amino acids in full length) and the human histone H4 (102 amino acids in full length) coincide with 92% of the amino acid sequence, and the difference is only 8 residues. Among natural proteins that are supposed to exist in tens of thousands of organisms, it is known that histones are the most highly conserved proteins among eukaryotes. Genomic DNA is superimposed by regular binding to this histone, and the complex of both forms a basic structural unit called a nucleosome. The nucleosomes aggregate to form a chromatin structure of the chromosome. Histone is subjected to modifications such as acetylation, methylation, phosphorylation, ubiquitination, and SUMOization at the N-terminal part called histonetail, and the gene expression and DNA replication by maintaining or specifically restructuring the chromatin structure. And reactions on chromosomal DNA such as DNA repair are regulated. Post-translational modifications of histones are epigenetic regulatory mechanisms and are considered essential for gene regulation in eukaryotic cells. Recent studies have altered the nucleosome structure to regulate chromatin remodeling factors, such as SWI / SNF, RSC, NURF, and NRD, which promote access to transcription factors and DNA, and regulate histone acetylation. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) have been shown to act as important regulators.

On the other hand, bromodomain is known as a domain structure of a protein that binds to histone acetylated lysine. In humans there are about 30 types of bromodomain-containing proteins. Among these, BRG1, BRD2, BRD3, etc. are known as proteins which interact with acetylated histones. Among them, BRG1 is known as a key constituent protein of the SWI / SNF complex, a chromatin remodeling factor. The mammalian SWI / SNF complex consists of 8 to 12 proteins and has a BRG-1 or hBrm as a core subunit having ATPase activity.

Gene-targeted mouse experiments have revealed that SWI / SNF acts as a tumor suppressor, and its mechanism of action is that SWI / SNF interacts with HDAC to mediate G1 / S arrest by Rb. It is known. Recent research suggests the potential for new molecular mechanisms for cancer suppression of SWI / SNF through the fact that SWI / SNF plays a role in promoting the phosphorylation and function of the cancer suppressor H2AX, which plays an important role in maintaining genome stability. This has been presented. In addition, inactivation of SWI / SNF or knockdown of BRG-1 / hBRM using siRNA significantly reduces H2AX phosphorylation and repair foci formation, making cells susceptible to DNA damage and DNA double strand break (DSB) recovery has been shown to be poor, suggesting that SWI / SNF plays a direct role in DSB recovery.

However, there has been no report on whether inhibition of the binding of acetylated histones to BRG-1 affects tumor cells.

Therefore, the inventors confirmed that bromodomain of BRG-1 (Brahma-related gene 1) can enhance cell death by increasing radiation sensitivity in radiation-resistant cancer cells as well as radiation-sensitive cancer cells. The recombinant protein comprising two or more of these BRG-1 derived bromodomains was confirmed to be more effective than the existing BRG-1 bromodomain, thus completing the present invention.

Accordingly, it is an object of the present invention to provide a recombinant protein comprising two or more bromodomains.

Another object of the present invention is to provide a nucleic acid encoding a recombinant protein comprising two or more bromodomains derived from BRG-1.

Another object of the present invention is to provide a vector having the nucleic acid.

Another object of the present invention is to provide a radiation-sensitive composition for cancer cell radiotherapy comprising a recombinant protein comprising two or more bromodomains or a vector having a nucleic acid encoding the recombinant protein as an active ingredient.

Another object of the present invention is to provide a cancer cell comprising a recombinant protein comprising two or more bromodomains derived from BRG-1.

As one aspect for achieving the above object, the present invention provides a recombinant protein comprising two or more bromodomains.

In another aspect, the invention provides nucleic acids encoding recombinant proteins comprising two or more bromodomains derived from BRG-1.

In another aspect, the present invention provides a vector having the nucleic acid.

As another aspect, the present invention provides a radiation-sensitive composition for cancer cell radiotherapy comprising a recombinant protein comprising two or more bromodomains or a vector having a nucleic acid encoding the recombinant protein as an active ingredient.

In another aspect, the present invention provides a cancer cell comprising a recombinant protein comprising two or more bromodomains derived from BRG-1. The cancer cells according to the present invention can be used as a control to confirm whether the candidate agent is a radiation sensitive agent.

Hereinafter, the present invention will be described in detail.

As used herein, the "BRG-1 protein" is a protein that interacts with acetylated histones among about 30 kinds of bromodomain-containing proteins known in humans, and is a key sub having the ATPase activity of the SWI / SNF complex, a chromatin remodeling factor. Means a protein known as a core subunit.

As used herein, “bromodomain” refers to some polypeptide sites of a protein having the activity of binding to an acetylated histone as a constituent of the protein and a nucleotide sequence encoding it. In the present invention, the bromodomain includes a polypeptide of a site having an activity that binds to acetylated K14 of histone protein H3 derived from BRG-1 protein or a nucleic acid fragment encoding the same.

In general, double strand damage of DNA is caused by external environmental factors such as radiation and, if not properly repaired, can lead to chromosomal substitution, gene loss and genomic instability, which can lead to cancer. In addition, high doses of radiation treatment result in damage to normal tissue.

On the other hand, SWI / SNF is known to act as a cancer suppressor, BRG-1 is known as a key constituent protein of the SWI / SNF complex.

However, when the present inventors irradiated gamma radiation when a polypeptide corresponding to the bromodomain of BRG-1 is present in the cancer cells, the present invention enhances cell death by increasing radiation sensitivity in not only radiation-sensitive cancer cells but also radiation-resistant cancer cells. In addition, it has been found that radiation sensitivity can be more effectively increased when artificially reconstituted and expressed BRG-1 bromodomain dimer and tetrameric recombinant protein (Example 4).

In the present invention, colon cancer cell HCT116 (S, hereinafter S means radiation-sensitive cancer cells), lung cancer cell A549 (R, hereinafter R means radiation-resistant cancer cells), hepatic cancer cell HepG2 (R) with recombinant vector pCMV-BRG1-BRD. Hepat3B (R) cells were transformed to confirm that BRG-1 bromodomain was expressed. When these cancer cells expressed BRG-1 bromodomain polypeptide, the cancer cells were significantly sensitive to gamma radiation. It was confirmed that it can increase the cell death by increasing. In addition, BRG-1 bromodomain multimeric recombinant protein (pEGFP-myc-BRG1-BRD-dimer and pEGFP-myc-BRG1-BRD-tetramer) transforms HEK 293T cells, which are normal cell lines, into BRG. The expression of -1 bromodomain multimeric recombinant protein was confirmed, and the expression of these multimeric recombinant protein significantly increased the sensitivity to gamma radiation compared to the expression of BRG-1 bromodomain monomer, resulting in apoptosis. It was confirmed that it can be enhanced.

The criteria for radiation sensitivity or radiation resistance are classified by the slope value of the linear model created using the cell survival rate when irradiated with gamma radiation of 0 to 2 Gy. This classification method has been reported by Jerry R. Williams et al. (Acta Oncologica, 46, 628-638, 2007). As used herein, the term "radiation sensitivity" refers to a cell having a feature in which the slope value is in the range of 0.00 to 0.30, and "radiation resistance" refers to a cell having a feature in the range of 0.31 to 1.00. The radiation sensitive cells are known HEK 293T, SW1222, HCT116, LoVo, CBS, LS174T, 379.2, 80S4, 14-3-3σ + / +, 40-16, 14-3-3σ-/-, N6CH3 and are radiation resistant The cells are known as DLD-1, HT29, WiDR, SW480, SW116, 19S186, Caco2, Caco2-neoras, U251, U87, T98G.

Colon cancer cells HCT116 (S), lung cancer cells A549 (R), liver cancer cells HepG2 (R) and liver cancer cells Hep3B (R) are all cancer cells expressing BRG-1. Therefore, in the present invention, the cancer cells to be treated with radiation preferably express BRG-1.

It is inferred that BRG-1 binds to acetylated histones, particularly the acetylated K14 of H3 via bromodomains, thereby increasing the gamma radiation sensitivity of cancer cells and enhancing cell death.

Thus, fragments of bromodomains derived from BRG-1 have a binding activity to acetylated histones, and preferably have a binding activity to acetylated K14 of H3.

The cancer cells are preferably colon cancer, lung cancer, liver cancer cells, but are not limited thereto, and cancer cells to which bromodomain-containing proteins are expressed may be applicable.

Since the radiation sensitizer of the present invention may increase sensitivity in chemotherapy as well as radiation therapy of cancer cells, the radiation sensitizer of the present invention may also be used for the use of the sensitizer to increase the sensitivity in chemotherapy. Belongs to the category of.

One. BRG From -1 Bromodomains  Containing more than one Recombinant protein

In the present invention, non-limiting examples of BRG-1 derived bromodomains include not only polypeptides isolated from native BRG-1 protein but also cells synthesized or transformed with a nucleic acid encoding BRG-1 derived brodomains. Includes isolates and fragments thereof.

An example of a bromodomain from BRG-1 may have a polypeptide sequence of SEQ ID NO: 1.

In the present invention, as long as the fragment of BRG-1-derived bromodomain has binding activity to the acetylated histone, its length and site are not limited.

In addition, the bromodomain derived from BRG-1 in the present invention includes conservative variants and derivatives thereof having binding activity to acetylated histones.

Bromodomains derived from BRG-1 according to the present invention are amino acid sequences having at least 70% similarity to the sequences described in SEQ ID NO: 1 as well as the sequences, preferably at least 80% similarity, more preferably at least 90% similarity And even more preferably an amino acid sequence that exhibits at least 95% similarity, most preferably at least 98% similarity, and has a polypeptide that substantially binds to an acetylated histone. Also, if the sequence having such similarity is an amino acid sequence having a biological activity substantially the same as or corresponding to that of a bromodomain derived from BRG-1, protein variants having amino acid sequences in which some sequences are deleted, modified, substituted, or added are also present. It is obvious that it is included within the scope of the invention.

In the present invention, a recombinant protein comprising two or more bromodomains derived from BRG-1 constitutes a fusion protein with a polypeptide other than a natural enzymatic activity site of BRG-1, such as an ATPase active site, or further N-terminus, C- Terminal or intermediate amino acid sequences such as linkers or tags.

Hereinafter, the bromodomain derived from BRG-1 is used mixed with a polypeptide.

Protein variants and derivatives are well known to those skilled in the art and may include amino acid sequence modifications. For example, modifications of amino acid sequences typically include one or more of three classes, namely, substitution, insertion or deletion variants. Insertions include amino and / or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertion will usually be an insertion of, for example, one to four residues smaller than the insertion portion of an amino or carboxyl terminal fusion. Bromodomains derived from BRG-1 may delete N-terminal, C-terminal or intermediate amino acids that are not involved in the intrinsic activity of the polypeptide.

Amino acid substitutions typically consist of one residue, but can occur at many different gene sites at once, and insertion will usually occur at a size of about 1 to about 10 amino acid residues. Deletion or insertion is preferably in adjacent pairs, ie two residues are deleted or two residues are inserted. Substitutions, deletions, insertions or any combination thereof may be combined to form the final construct. Mutations should not occur in sequences outside the reading frame, and preferably do not create complementary regions that can produce secondary mRNA structures unless a change in the secondary structure of the mRNA is desired.

For example, substitution of one amino acid residue with another biologically and / or chemically similar is known to those skilled in the art as conservative substitutions.

For example, conservative substitutions are those in which one hydrophobic moiety is replaced with another hydrophobic moiety or a polar moiety is replaced with another polar moiety. One example of integrity substitution is shown in Table 1 below.

Such substitutions include combinations shown in Table 1 above.

Typically conservative substitutions have little effect on the biological activity of the resulting polypeptide. Peptides include one or more amino acid substitutions, for example 2 to 10 conservative substitutions, 2 to 5 conservative substitutions, 4 to 9 conservative substitutions, such as 2, 5 or 10 conservative substitutions.

As used herein, a "recombinant protein" is a fusion protein produced by linking two or more genes that encode a bromodomain. Translation of the fusion gene results in a single polypeptide having functional properties derived from each original bromodomain. Bromodomains in recombinant proteins can be linked immediately adjacent or linked through linkers. Recombinant proteins can be artificially produced by recombinant DNA technology for use in biological research or therapy. The functionality of recombinant proteins exerts a modular approach to the functional domains of many proteins.

As used herein, a "linker" can be an amino acid sequence or insertion that can be used to link or separate two distinct polypeptides or polypeptide fragments. Polypeptides provided herein can have an amino acid linker comprising, for example, amino acids GLS, ALS or LLA. As used herein, a “tag” can be a distinct amino acid sequence that can be used to detect or purify a provided polypeptide.

2. Internalization Sequence

Recombinant proteins comprising two or more bromodomains derived from BRG-1 may be linked to internalization sequences or protein transduction domains to effectively enter the cell.

Recent studies have identified several cell penetrating peptides, such as the TAT transactivating domain of the HIV virus, antennaepedia, and transportans that can easily transport molecules and small peptides across the plasma membrane. In addition, polyarginine has become an attractive tool for peptide mediated transport by transporting peptides and proteins even more efficiently across the plasma membrane. Nonarginine is one of the most efficient polyarginine base protein transduction domains and exhibits greater maximal absorption than TAT or antennaepedia. Peptide mediated cytotoxicity has also been shown to be smaller in polyarginine-based internalization sequences. R9 mediated membrane transport is facilitated through heparan sulfate proteoglycan binding and intracellular packaging. Once internalized, heparan is degraded by heparanase, resulting in release of R9 and migration to the cytoplasm. Liarginine is known as a potent cell penetrating peptide. Derivatives of polyarginine delivered full-length p53 protein to oral cancer cells, inhibiting their growth and metastasis.

Thus, recombinant proteins comprising two or more bromodomains derived from BRG-1 may comprise cell internalization transporters or sequences. The cellular internalization sequence may be any internalization sequence known or newly found in the art, or a conservative variant thereof. Non-limiting examples of intracellular transporters and sequences include polyarginine (such as R9), antennapedia sequences, TAT, HIVTat, penetratin, Antp-3A (Antp mutant), Buforin II, transpotane, MAP (model amphipathic peptide), K-FGF, Ku70, prion, pVEC, Pep-1, SynB1, Pep-7, HN-1, BGSC (bis-guanidinium-spermidine-cholesterol) and BGTC (bis Guanidinium-tren-cholesterol). Also, bromodomains derived from BRG-1 or recombinant proteins comprising two or more thereof further include BGSC (bis-guanidinium-spermidine-cholesterol) or BGTC (bis-guanidinium-tren-cholesterol) can do.

3. Tumor-specific Targeting

Recombinant proteins comprising two or more bromodomains derived from BRG-1 may further comprise tumor specific targeting sequences.

The radiation sensitive agent detects only tumor cells and preferably is not introduced into normal cells. One approach to this is to utilize polypeptides (such as modified or fused proteins) that have both internalization and tumor specific targeting capabilities. Tumor vasculature is different from that of surrounding normal tissue. Biochemically, tumor vessels distinguish themselves from the rest of the vessels by expressing many angiogenesis-related molecules such as specific integrins, endothelial growth factor receptors, proteases, cell surface proteoglycans and extracellular matrix components (Ruoslahti, 2000). . In vivo selection of phage representing libraries for peptides that return to tumor vasculature when injected into mice suggests several motifs for tumor regression. It includes the RGD of the circulating peptide CDCRGDCFC, and the NGR of the circulating tumor-regressing peptide, CNGRC. NGR-containing peptides have proven useful for delivering cytotoxic drugs, pro-apoptotic peptides, and tumor necrosis factor α to tumor vasculature. The third motif, GSL, was also frequently isolated when screened for various types of tumors. Tumor regression of phage carrying RGD, NGR and GSL motif peptides is independent of tumor type, but rather regression depends on the angiogenic properties of the tumor vasculature. The receptor for the NGR tumor regression peptide is not integrin. Instead, aminopeptidase N (APN or CD13) has been identified as a receptor for NGR motif peptides in tumor vasculature. NGR-containing peptides have proven useful for delivering cytotoxic drugs, pro-apoptotic peptides, and tumor necrosis factor α to tumor vasculature. NGR peptides can bind to primary and metastatic tumors of the prostate, but not to normal prostate tissue. NGR peptides also exhibit the ability for cytosolic internalization.

Any molecule capable of targeting specific tissues can be used as the targeting molecule of the polypeptides of the invention. For example, targeting molecules include human epithelial cell mucin (Muc-1; 20 amino acid core repeats for Muc-1 glycoproteins, present in breast and prostate cancer cells), Ha-ras oncogenes, p53, cancerous antigens ( CEA), raf oncogene product, gp100 / pme17, GD2, GD3, GM2, TF, sTn, MAGE-1, MAGE-3, BAGE, GAGE, tyrosinase, gp75, melan-A / Mart-1, gp100, HER2 / neu, EBVLMP 1 & 2, HPV-F4, 6, 7, prostate-specific antigen (PSA), HPV-16, MUM, alpha-fetoprotein (AFP), CO17-1A, GA733, gp72, p53, ras tumor Gene product, HPV E7, Wilm tumor antigen-1, telomerase, melanoma ganglioside, or a molecule that interacts with a simple dural sequence.

Selective delivery of therapeutic agents to cancer cells in the living body is another area of research in which targeting of biomarkers specific to cancer is intensively studied. Immunoliposome-mediated targeting can be performed using monoclonal antibodies against the folate receptor, monoclonal antibodies against CA-125, and monoclonal antibodies against the HER2 / neu antigen.

4. Nucleic Acids

Nucleic acids encoding recombinant proteins comprising two or more bromodomains derived from BRG-1 may be, for example, nucleotides, nucleotide analogs, or nucleotide substituents.

The nucleic acid encoding the bromodomain derived from BRG-1 according to the present invention may be a nucleic acid encoding the amino acid sequence of SEQ ID NO: 1, preferably the nucleic acid of SEQ ID NO: 2, and the sequence and Substantially nucleic acid as a sequence having at least 70% similarity, preferably at least 80% similarity, more preferably at least 90% similarity, even more preferably at least 95% similarity, most preferably at least 98% similarity This encoded protein contains a nucleic acid having binding activity to an acetylated histone. In addition, a nucleic acid encoding a recombinant protein comprising two or more bromodomains derived from BRG-1 is a nucleic acid that is linked and included so that two or more nucleic acids encoding the amino acid sequence of SEQ ID NO: 1 are included.

As used herein, a “nucleic acid” refers to an mRNA molecule encoded by DNA, RNA, such as a DNA molecule, or an mRNA molecule, such as another type of RNA molecule, which is chemically synthesized, or which is substantially free or separated from at least some cellular components. Polypeptide molecules.

An isolated nucleic acid encoding a recombinant protein comprising two or more bromodomains derived from BRG-1 may be operably linked to expression control sequences. Also provided are vectors comprising one or more nucleic acids encoding a recombinant protein comprising two or more bromodomains derived from BRG-1, wherein the nucleic acids are operably linked to expression control sequences.

There are many methods that can be used to deliver nucleic acids in vivo or in vitro. These methods fall into two broad categories: one is a virus-based delivery system and the other is a virus-based delivery system. For example, nucleic acids may be genetic material using many direct delivery systems such as electroporation, lipofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phage, cosmids, or cells or carriers such as cationic liposomes. Can be delivered through

Nucleic acids encoding recombinant proteins comprising more than one bromodomain can be used with lipids such as, for example, liposomes such as cationic liposomes (eg DOTMA, DOPE, DC-cholesterol) or anionic liposomes. Liposomes may further comprise proteins to facilitate targeting specific cells, as desired. Administration of the composition comprising the compound and the cationic liposomes can be administered to the blood centered on the target organ or inhaled into the airways to target the cells of the airway.

Furthermore, the compounds can be administered as compounds of microcapsules that can be targeted to specific cell types such as macrophages, or the diffusion or compound delivery of compounds from microcapsules is designed for specific rates or unit doses.

Nucleic acid delivered to a cell to be integrated into the host cell genome typically contains an integration sequence. These sequences are sometimes virus related sequences, especially when viral based systems are used. These viral integration systems are also integrated into nucleic acids delivered using non-nucleic acid based delivery systems, such as liposomes, whereby the nucleic acids contained in the delivery system can be integrated into the host genome.

Other common techniques for incorporation into the host genome include systems designed to facilitate homologous recombination with the host genome, for example. Such systems typically rely on sequences on either side of the nucleic acid to be expressed that have sufficient homology with the target sequence in the host genome so that recombination between the vector nucleic acid and the target nucleic acid can occur to integrate the nucleic acid delivered into the host genome.

Nucleic acids encoding recombinant proteins comprising two or more bromodomains may be used in a variety of mechanisms well known in the art in vivo and / or ex vivo to a subject's cells (eg, through uptake of pure DNA, liposomal fusion, via gene guns). Intramuscular injection of a DNA, foreign cell uptake, etc.).

5. Vector

Nucleic acids encoding recombinant proteins comprising two or more bromodomains, or conservative variants thereof, can transform cancer cells in the form of a vector.

The delivery vector can be any nucleotide construct used to deliver a gene into a cell (such as a plasmid), for example as part of a recombinant retrovirus or adenovirus.

As used herein, a plasmid or viral vector is the transport of an isolated nucleic acid encoding a recombinant protein comprising two or more bromodomains in a cell without degradation, and includes a promoter that promotes expression of the gene in the cell to which it is delivered. . In one embodiment, the promoter is derived from a virus or retrovirus. Viral vectors are, for example, adenoviruses, adeno-binding viruses, herpes viruses, vaccinia viruses, polio viruses, AIDS viruses, neurotrophic viruses, Sindbis and other RNA viruses, and those viruses with an HIV backbone. Retroviruses include murine leukemia virus, MMLV, and retroviruses that express the desirable properties of MMLV as vectors. Retroviral vectors can carry larger genetic loads than other viral vectors, ie transgenes or marker genes, which is why they are commonly used. But it is not useful in cells that do not proliferate. Adenovirus vectors are relatively stable, easy to use, have high titers, can be delivered in aerosol formulations, and can transform cells that do not divide. Chickenpox virus vectors are large and have multiple sites for gene insertion, are heat stable and can be stored at room temperature. Genetically engineered viral vectors can also be used to suppress the immune response of the host organism, induced by viral antigens, and this type of vector can carry the coding region for interleukin 8 or 10.

Retroviruses are animal viruses belonging to the viral family of Retroviridae, including all types, subfamily, classes, and affinities. Retroviruses are essentially packages that package the nucleic acid contents therein. Nucleic acid content carries a packaging signal with it, which ensures that the cloned daughter molecule is effectively packaged in the packagecoat.

The advantage of using replication-deficient adenoviruses as vectors is that they are limited to the extent that they can spread to other cell types. Because they can replicate in early infected cells, they cannot form new infectious virus particles. Recombinant adenoviruses have been found to achieve high efficiency gene delivery after being delivered directly to the airway endothelial cells, hepatocytes, vascular epithelial cells, CNS milk tissues and many other tissue sites in vivo. Recombinant adenoviruses achieve genetic transformation by binding to specific cell surface receptors, and the virus is then internalized by receptor-mediated cellular foreign body uptake in the same way as wild-type or replication-deficient adenoviruses.

Viral vectors are based on adenoviruses with the E1 gene removed, and these virions are produced in cell lines such as human 293 cell lines. In one aspect, both the E1 and E3 genes are removed from the adenovirus genome.

Another type of viral vector is based on adeno-binding virus (AAV). This deficiency parvovirus can infect many cell types but does not cause illness in humans. AAV, a type of vector can transport about 4 to 5 kbp and wild type AAV is known to stably insert into chromosome 19. By way of example, such a vector may be a P4.1 C vector produced by Avigen (San Francisco, Calif.), Which may be a herpes simplex virus thymidine kinase gene, HSV-tk, and / or marker genes such as green fluorescent protein, May contain a gene encoding GFP.

In another type of AAV virus, AAV contains a pair of inverted terminal repeats (ITRs) on either side of at least one cassette containing a promoter, which is cell-specific expression operably linked to a heterologous gene. To indicate. Heterologous in this context refers to any nucleotide sequence or gene that is not native to AAV or B19 parvovirus.

Typically the AAV and B19 coding regions have been deleted, resulting in a vector that is safe and noncytotoxic. AAV ITRs or modifications thereof confer infectious and site-specific integration but do not exhibit cytotoxicity, and promoters direct cell-specific expression.

The disclosed vectors can provide DNA molecules that can be integrated into mammalian chromosomes without substantial toxicity.

Genes inserted into viruses and retroviruses usually contain promoters and / or enhancers to help regulate the expression of the desired gene product. A promoter is generally a sequence or sequences of DNA that function when in a relatively fixed position relative to the transcription start site. The promoter contains the core elements necessary for the basic interaction of RNA polymerase and transcription factors, and may contain upstream elements and reaction elements.

Molecular genetic experiments performed with large human herpes viruses provided a means by which large heterologous DNA fragments could be cloned, propagated and established in cells susceptible to herpes virus infection. These large DNA viruses (simple herpes virus, HSV; Epstein-Barr virus, EBV) have the potential to deliver fragments of human heterologous DNA greater than 150 kbp for specific cells. EBV recombinants can maintain large pieces of DNA in infected B-cells, such as episomal DNA. Individual clones carried human gene inserts up to 330 kbp that appeared genetically stable. Maintaining these episomes requires EBNA1, a specific EBV nuclear protein that is structurally expressed during infection with EBV. In addition, these vectors can be used for transformation, where a large amount of protein can be produced temporarily in vitro. The herpesvirus amplicon system is also used to package cells larger than 220 kbp and infect cells that can keep DNA stable as episomes.

Other useful systems are, for example, replication and host-limited non-replicating vaccinia virus vectors.

Promoters that regulate transcription from vectors of mammalian host cells include a variety of sources, such as from the genomes of viruses such as polyoma virus, apes virus 40 (SV40), adenoviruses, retroviruses, hepatitis B virus, cytomegalovirus, Or from heterologous mammalian promoters such as the beta actin promoter.

The early and late promoters of the SV40 virus are conveniently obtained as SV40 restriction fragments which also contain the origin of SV40 virus replication. The immediate early promoter of human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment. Of course, promoters obtained from host cells or related species are also useful herein.

The promoter also contains response elements that mediate transcriptional regulation. Enhancers sometimes determine the regulation of gene expression. While many enhancer sequences are known from mammalian genes (globin, elastase, albumin, α-fetoprotein and insulin), typically anyone will use enhancers from eukaryotic viruses for general expression. Examples thereof are the SV40 enhancer (100-270 bp), which acts later in the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma virus enhancer used later in the origin of replication, and adenovirus enhancers.

Promoters and / or enhancers are specifically activated by unusual chemical events or light causing their function. The system can be controlled by reagents such as tetracycline and dexamethasone. There are also methods of enhancing viral vector gene expression by exposure to radiation, such as gamma radiation, or alkylated chemotherapeutic drugs.

In certain embodiments, the promoter and / or enhancer region may act as a structural promoter and / or enhancer to maximize expression of the region of the transcription unit to be transcribed. In some constructs the promoter and / or enhancer region is active in all eukaryotic cell types, even if it is expressed only in a particular type of cell at a particular time. This type of promoter is a CMV promoter (650 bp). Such other promoters include the SV49 promoter, cytomegalovirus (full length promoter), and retroviral vector LTR.

Expression vectors used in eukaryotic host cells (yeast, fungi, insects, plants, animals, humans or nucleated cells) may also contain sequences necessary for the termination of transcription that may affect mRNA expression. These regions are transcribed as polyadenylated fragments in the untranslated portion of mRNA encoding tissue factor proteins. The 3 'untranslated region also includes a transcription termination site. The transfer unit may also contain a polyadenylation region. One advantage of this region is that it increases the likelihood that transcribed units will be processed and transported like mRNA. The identification and use of polyadenylation signals in expression constructs is well established. In some transcription units, the polyadenylation region is derived from the SV40 initial polyadenylation signal and consists of about 400 bp.

The viral vector may comprise nucleic acid sequences encoding the marker product. This marker product is used to determine whether a gene has been delivered to a cell and expressed once delivered. Examples of suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analogs G418, hydromycin, and puromycin. When a selectable marker is successfully delivered to a mammalian host cell, the transformed mammalian host cell can survive as if under selective pressure.

The vector used in one embodiment of the present invention encodes three nuclear localization signals (NLS; SEQ ID NO: 3), myc epitope (SEQ ID NO: 4), and the bromodomain of the BRG-1 gene of the BRG-1 gene. Containing one or more sequences (bromodomain, BRD; SEQ ID NO: 1 or 2). For example, recombinant vectors pCMV-BRG1-BRD, pEGFP-myc-BRG1-BRD (-dimer or -tetramer) having the cleavage map of FIGS.

6. Pharmaceutically acceptable carrier

The radiation sensitive composition according to the invention may further comprise a carrier to assist in the administration, delivery or use of the effective drug. The carrier can be, for example, small molecules, pharmaceutical drugs, fatty acids, detectable markers, inclusion tags, nanoparticles or enzymes.

The carrier can be selected to naturally minimize any degradation of the active ingredient and to minimize any harmful side effects in the subject, as is well known to those skilled in the art.

Typically, an appropriate amount of pharmaceutically acceptable salt is used in the formulation to make the formulation isotonic. Non-limiting examples of pharmaceutically acceptable carriers include saline, Ringer's solution and dextrose solution. The pH of the solution is preferably about 5 to about 8, more preferably about 7 to about 7.5. Carriers also include sustained-release preparations, such as semipermeable matrices of solid hydrophobic polymers containing the antibody, wherein the matrices are in the form of shaped articles, for example films, liposomes or microparticles.

Pharmaceutical carriers will most often be standard carriers for drugs administered to humans, such as sterile water, saline, and buffered solutions of physiological pH.

The radiation sensitive composition according to the present invention may include carriers, thickeners, diluents, buffers, preservatives, surface active agents, etc., in addition to the molecules selected. It may also include one or more active ingredients such as antibacterial agents, anti-inflammatory agents, anesthetics and the like.

Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, such as saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous vehicles include fluid and nutrient supplements, electrolyte supplements (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may be present, such as antimicrobials, antioxidants, chelating agents, inert gases, and the like.

Formulations for topical administration include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickening agents and the like may also be preferred.

Formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, disposable bags, or tablets. Thickeners, flavors, diluents, emulsifiers, dispersing aids or binders are also preferred.

Radiation sensitive compositions according to the invention are potentially used for inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid. , By action with malonic acid, succinic acid, maleic acid, and fumaric acid, or inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and ethanolamine substituted with mono-, di-, trialkyl and aryl amines It can be administered as a pharmaceutically acceptable acid- or base-addition salt formed by reaction with an organic base.

Radiation compositions according to the invention may be in the form of solutions, suspensions (eg integrated into microparticles, liposomes, or cells). These can target particular cell types via antibodies, receptors, or receptor ligands. Vehicles such as stealth and other antibody conjugated liposomes (including lipid mediated drug targeting for colonic carcinoma), receptor mediated targeting of DNA via cell specific ligands, lymphocyte directed tumor targeting, and biomarkers An example is the very specific therapeutic retrovirus targeting of murine glial cells within. In general, receptors may be structurally or ligand-induced in the pathway of cellular foreign uptake. These receptors dense into a clathrin-coated seed, enter the cell via the clathrin-coated endoplasmic reticulum, pass through the acidified endosome where the receptor is sorted, and then recycle to the cell surface and intracellularly. Stored or degraded in lysosomes.

Internalization pathways perform a variety of functions such as nutrient uptake, removal of activated proteins, removal of macromolecules, opportunistic influx of viruses and toxins, binding and degradation of ligands, and receptor-level regulation. Many receptors follow one or more intracellular pathways depending on cell type, receptor concentration, ligand type, ligand valence, and ligand concentration.

The carrier molecule may be covalently linked to a recombinant protein comprising two or more bromodomains derived from BRG-1. The carrier molecule may be linked to the amino terminus of the disclosed peptides. The carrier molecule may be linked to the carboxy terminus of a recombinant protein comprising two or more bromodomains. The carrier molecule may be linked to an amino acid in a recombinant protein comprising two or more bromodomains. The radiation composition according to the present invention may further comprise a linker linking the recombinant molecule comprising two or more carrier molecules and bromodomains. Bromodomains or recombinant proteins comprising two or more thereof may also be used to coat bovine serum albumin (BSA; Tkachenko et al., (2003) for coating microparticles, nanoshell nanoparticles or with recombinant proteins comprising two or more thereof. ) J Am Chem Soc, 125, 4700-4701).

7. Administration method

The radiation sensitive composition according to the invention may be administered to a mammal. As used herein, the term "mammal" includes humans, livestock and breeding animals, and zoo, competition or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, and the like. Means all animals classified as mammals. Preferably, the mammal is a human.

The radiation sensitive composition according to the invention may be administered by topical, oral, or parenteral routes. For example, the radiation sensitive composition may be administered by in vitro, intracranial, intravaginal, intranasal, subcutaneous, intradermal, intracardiac, gastric, intravenous, intramuscular, intraperitoneal injection, transdermal, intranasal, or inhalation. Can be. "Intracranial administration" means delivery of a substance directly to the brain, including, for example, intramedullary, intravaginal, intraventricular, or butterfly-via delivery via a catheter or needle.

The exact amount of radiation sensitive composition required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder to be treated, the particular nucleic acid or vector used, the mode of administration, and the like. Therefore, it is not possible to specify the exact amount for all radiation sensitive agents. However, appropriate amounts can be determined by those skilled in the art using conventional experiments taught herein.

The radiation sensitive composition may be in the form of a solution or suspension (eg, integrated with microparticles, liposomes, or cells). These can target specific cell types by antibody, receptor, or receptor ligand. Excipients such as 'stealth' and other antibody conjugate liposomes (including lipid mediated drugs targeting colon carcinoma), receptor mediated targeting of DNA via cell specific ligands, lymphocyte directed tumor targeting, and murine glioma Highly specific therapeutic retrovirus targeting of cells is used in vivo. In general, receptors are involved in the pathway of constitutive or ligand-induced endocytosis.

These receptors cluster in the clathrin-coated recesses, enter the cell through the clathrin-coated vesicles, pass through the acidified endosomes where the receptors are aligned, and then recycle to the cell surface, or are stored intracellularly, Or in lysosomes. Internalization pathways provide a variety of functions such as nutrient uptake, activating protein removal, macromolecular clearance, opportunistic entry of viruses and toxins, dissociation and denaturation of ligands, and receptor-level regulation. Most receptors follow one or more internalization pathways depending on cell type, receptor concentration, ligand type, ligand valence, and ligand concentration.

The radiation sensitive composition can be administered in a number of ways depending on whether topical or systemic treatment is desired and on the site to be treated. Administration can be effected locally (ocular, vaginal, rectal, intranasal route), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection. .

Effective dosages and schedules of radiation sensitive compositions can be determined empirically, and such determinations are within the skill of one in the art. The dosage range of dosage of the radiation sensitive composition is wide enough to produce the desired effective effect on the symptoms of the disorder.

For example, a “therapeutically effective amount” of a radiation sensitive composition may include (1) the effect of inhibiting tumor growth to some extent, including the effect of slowing tumor growth and stopping it completely; (2) reducing the number of tumor cells; (3) effect of reducing tumor size; (4) inhibiting tumor cells from invading into peripheral organs (ie, reducing, slowing or completely stopping tumor cells from invading into peripheral organs); (5) the inhibitory effect of metastasis (ie, the reduction, alleviation or complete stopping effect of metastasis); (6) potentiating effects of anti-tumor immune responses that may, but not necessarily, inhibit or reject tumors; And / or (7) the effect of alleviating to some extent one or more symptoms associated with the tumor.

The dose should not be large enough to cause side effects such as unwanted cross-reactions, hypersensitivity reactions, etc. In general, the dosage depends on age, condition, sex and degree of disease of the patient, route of administration, or whether other drugs are included in the regimen and can be determined by one skilled in the art. The dose may be adjusted by the physician as a result of any response. Dosages may vary and may be administered in one or more dosage forms of administration per day, for several days or days. Dosage ranges are highly dependent on the use of the radiation sensitive composition, the severity of the condition, and the route of administration.

For example, in use as a research tool for irradiation, the radiation sensitive composition can be used in doses as low as 0.01% (w / v). This dose may be as low as 0.02% (w / v) and may be as high as 2% (w / v) in topical treatment. In applications such as cancer / tumor therapy or when used as an early concentrated bolus immediately after acute tissue injury, significantly higher composition concentrations may be used on their own or in combination with other compounds. Accordingly, the upper limit of a given polypeptide can be up to 2-5% (w / v or v / v), for example when delivered as an initial bolus directly to a tumor mass. The recommended upper dose limit for parenteral routes of administration, such as intramuscular, cerebral, intracardiac and spinal canal, may be 1% (w / v or v / v) depending on the severity of the injury. Such an upper dose limit may vary from agent to agent, depending on, for example, how the polypeptide (s) is combined with other agents that promote the action of the polypeptide or act in conjunction with the polypeptide (s).

For continuous delivery of a given polypeptide, for example in combination with intravenous drips, an upper limit of 0.01 g / kg body weight may be used over a time course determined by the physician based on improving the condition. As another example, the upper limit of a given nucleic acid concentration delivered locally will be between 5 and 10 μg / cm 2, depending, for example, on how the nucleic acid is combined with other agents that promote or act in concert with the nucleic acid. . This may be repeated at a frequency determined by the physician based on the situation of improvement. As another example, the upper limit of a given nucleic acid concentration delivered internally, such as intramuscular, cerebral, intracardiac, and intratracheal, is 50-100 μg / ml. In this case too, the frequency will be determined by the physician based on the situation of improvement.

In addition, the surgical site can be pre-conditioned with a polypeptide provided prior to surgery. The concentration of polypeptide can be 10-200 μM and mixed with 10-30% Pluronic gel or any such carrier which allows penetration of peptide (s) within the site and left for at least 3-6 hours prior to surgery. Conditioning as such a pre-procedure can improve the healing response to subsequent surgery, including reducing the inflammatory response.

8. Radiotherapy

The radiation sensitive composition for cancer cell radiotherapy according to the present invention can be used in combination with radiation therapy.

Radiotherapy is a topical treatment that damages the DNA of malignant cells. Normal cells have a greater ability to repair these damages than tumor cells. Radiotherapy takes advantage of this difference.

Since radiation therapy is a form of topical therapy, side effects are generally limited to the treated area. However, a common systemic symptom is fatigue. While many genetic factors are prevalent in secondary cancers in some patients, radiation also contributes to an increased risk. The radiation sensitive composition according to the present invention can reduce these side effects by reducing the required amount of radiation. In addition, radiation sensitivity can be increased not only in radiation-sensitive cancer cells, but also in radiation-resistant cancer cells.

Treatment regimens are based on treatment goals and possible side effects. The course of treatment may be a short course of about 1 day and a long course of about 10 weeks, with a typical duration of 2 to 7 weeks, usually consisting of 5 days of treatment each week. The patient receives radiation through a linear accelerator that most commonly produces electrons that produce x-rays that are used as or used as a treatment beam. Treatment is painless and usually lasts less than 5 minutes.

The therapeutic target may be healing or palliation. If radiotherapy potentially targets healing, the duration of treatment is usually longer, and usually consists of small daily doses over a long period of treatment. This approach minimizes late side effects. If the treatment is strictly aimed at remission, a short treatment schedule consisting of large daily treatment doses over a short period of time is used. In such cases, late side effects may not occur during the lifetime of the patient.

Radiotherapy has many possible specific prescriptions. It may be given as a primary tumor treatment, as a preoperative or postoperative therapy, or as a component of a combination or combination therapy.

Radiotherapy is suitable in nearly two thirds of cancer patients and is used for healing and alleviation purposes. Many tumors, such as prostate cancer, breast cancer, head and neck cancer, lung cancer, brain tumors, gastrointestinal tumors, liver cancers, soft tissue sarcomas, cervical cancers, lymphomas, etc., will receive radiotherapy as part of the treatment regimen.

In the present invention, the radiation source is preferably gamma radiation of ¹³⁷ Cs, but is not limited thereto.

Radiotherapy includes external beam radiation (x-rays, γ-rays, protons and neutrons), brachytherapy and radioactive material implantation. Radiotherapy can be administered by a 2-D, 3-D, confocal, intensity-modulation (IMRT) and image-guide (IGRT) approach. Standard radiotherapy suitable for most solid tumors is given at 2 Gy / day in a total dose of about 60 Gys (50-70 Gys). However, it is common for those skilled in the art to select the desired radiation dose based on the particular subject, device, and tumor type. The radiation sensitive composition according to the invention constitutes an improvement over existing radiation therapy by increasing the sensitivity of cancer cells to radiation. For example, provided methods allow cancer cells to have at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more sensitivity to radiation. As used herein, the terms "sensitivity" and "radiation sensitivity" refer to viable cell numbers based on radiation dose. Thus, an increase in radiation sensitivity can lead to a decrease in the number of cells surviving at any radiation dose, a reduction in the radiation dose required for lethal doses, or a combination thereof.

Brachytherapy, also known as sealed source radiotherapy or endocurietherapy, is a form of radiation therapy in which a radioactive source is placed inside or next to the area in need of treatment. In contrast, external beam radiotherapy, or teletherapy, is applied to radiation generated externally by a linear accelerator. Brachytherapy is commonly used for the treatment of local prostate cancer and head and neck cancer.

Brachytherapy includes mold brachytherapy, strontium plate therapy, intragap brachytherapy, intracavity brachytherapy, and vascular brachytherapy. In mold brachytherapy, tumors close to the epidermis can be treated using a sealed source placed close to the skin.

Dosimetry is primarily performed in connection with the Manchester system, which is a rule-based approach designed to ensure that doses for the entire portion of the target volume are within 10% of the prescribed dose. Surface applicators, commonly referred to as strontium plate therapy, are used for lesions very close to the epidermis, less than 1 mm thick. The plate is a hollow thin silver casing, which seals off the radioactive strontium-90 powder salt. The beta (electron) particles produced due to the radioactive decay of strontium penetrate very shallowly.

Typically, the Sr90 plate is placed on a layer of ablated pterygium. A constant dose of about 10-12 Gy is delivered at the contact timing. Since the electrons only penetrate a few millimeters of air, the radiation protection problem is slightly reduced, which is a very big difference from other radiation sources. In intragap brachytherapy, the source is inserted into the tissue. Prostate cancer treatment using iodine-125 species is also classified as intragap brachytherapy. For details of gamma emitters, refer to commonly used gamma ray emitting isotopes. Intracavity brachytherapy places the source in an existing intracorporeal cavity. Intravascular brachytherapy places a catheter with a source inside the vessel. The most common application of this method is the treatment of restenosis in the coronary stent, but this therapy can also be used for the treatment of peripheral vascular stenosis.

High dose rate (HDR) brachytherapy is a common brachytherapy. Applicators in the form of catheter are generally disposed in accordance with the Manchester or Paris system or in the patient. Next, a high dose rate source (mainly iridium 192, Ir-192) is steered along the catheter at the wire end by the machine. The source stays at a preplanned position for a predetermined time, then advances along the catheter and repeats this so that the required dose distribution is achieved.

Like high dose rate (HDR), low dose rate (LDR) involves the implantation of radioactive material, which may be implanted temporarily or permanently. LDR brachytherapy with machines works in a similar way. Another variant is that the source is in the form of active and inactive balls, which are also manipulated in the patient using a machine.

In one aspect, a radioprotectant may be administered to a healthy tissue of a subject when the radiation sensitive agent is administered. In general, radioprotectants will include compositions that trap free radicals to prevent oxidative damage.

9. Screening Method

According to the present invention, cancer cells comprising a recombinant protein containing two or more fragments thereof having a binding activity to a BRG-1 derived bromodomain or an acetylated histone may be used as a positive control when screening whether a candidate agent is a radiation sensitive agent. have.

In general, candidate agents can be identified from large libraries or chemical libraries of natural products or synthetic (or semi-synthetic) extracts according to methods known in the art. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic-, or animal-based extracts, fermentation broth, and synthetic compounds, as well as modifications of existing compounds. It can also be used for random or directed synthesis (eg, semi-synthetic or total synthesis) of many chemical compounds, including sugar-, lipid-, peptide-, polypeptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available from, for example, Brandon Associates (Merrimack, NH) and Aldrich Chemical (Milwaukee, WI). Or alternatively, bacteria, fungi, from a number of sources including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, FIa.), And PharmaMar, USA (Cambridge, Mass.). Natural compound libraries in the form of plant and animal extracts are commercially available. In addition, natural and synthetic production libraries, if desired, are produced according to methods known in the art, for example by standard extraction and fractionation. Moreover, if desired, any library or compound is readily denatured using standard chemical, physical, or biochemical methods.

Recombinant protein comprising two or more BRG-1-derived bromodomains of the present invention can increase apoptosis by increasing radiation sensitivity in radiation-resistant cancer cells as well as radiation-sensitive cancer cells during radiation treatment of cancer cells. This could be widely used for more effective chemotherapy.

Figure 1 is a schematic diagram showing the manufacturing process of the BRG-1 bromodomain expression vector.
Figure 2 is a diagram showing the configuration of the BRG-1 bromodomain multimeric expression vector.
Figure 3 shows the results of confirming the survival rate of the BRG-1 bromodomain and its multimer-expressing normal cell 293T transformant according to gamma irradiation, after irradiation with a total dose of 0 to 5 Gy, Survival (A), expression of BRG-1 bromodomain and BRG-1 bromodomain multimeric recombinant protein (B), and IC50 values (C) are shown and confirmed.
Figure 4 shows the results of confirming the survival rate of gamma-ray irradiation of colon cancer cells HT-29 transformant expressing BRG-1 bromodomain and dimer thereof, cancer cells after irradiation with a total dose of 0 to 10 Gy Survival rate (A), expression of BRG-1 bromodomain and BRG-1 bromodomain multimeric recombinant protein (B), and IC50 value (C).
Figure 5 shows the results of confirming the survival rate according to gamma irradiation of the HRG-116 transformant colon cancer cells expressing BRG-1 bromodomain and dimer thereof, the cancer cells after irradiation with a total dose of 0 to 5 Gy Survival rate (A), expression of BRG-1 bromodomain and BRG-1 bromodomain multimeric recombinant protein (B), and IC50 value (C).
Figure 6 shows the results of confirming the survival rate according to gamma irradiation of ARG transformant lung cancer cells expressing BRG-1 bromodomain and its dimers, after irradiation with a total dose of 0 to 10 Gy, survival rate of cancer cells (A), BRG-1 bromodomain and BRG-1 bromodomain multimeric recombinant protein (B), and IC50 value (C) are shown and confirmed.
Figure 7 shows the results of confirming the survival rate according to gamma irradiation of the BRG-1 bromodomain and breast cancer cell MDA-MB-231 transformant expressing a dimer thereof, after irradiation with a total dose of 0 to 10 Gy , Cancer cell viability (A), expression of BRG-1 bromodomain and BRG-1 bromodomain multimeric recombinant protein (B), and IC50 value (C).
Figure 8 shows the results of confirming the survival rate according to the gamma irradiation of BRG-1 bromodomain and breast cancer cell SK-BR3 transformant expressing a dimer thereof, the cancer cells after irradiation with a total dose of 0 to 5 Gy Survival rate (A), expression of BRG-1 bromodomain and BRG-1 bromodomain multimeric recombinant protein (B), and IC50 value (C).
Figure 9 shows the results of confirming the survival rate according to gamma irradiation of colon cancer cell HCT-116 transformant expressing the BRG-1 bromodomain dimer and tetramer, respectively, after irradiation with a total dose of 0 to 5 Gy , Cancer cell survival rate (A), BRG-1 bromodomain and BRG-1 bromodomain multimeric recombinant protein (B) is confirmed and shown.
Figure 10 shows the results of confirming the survival rate according to gamma irradiation of the lung cancer cell A549 transformant expressing the BRG-1 bromodomain dimer and tetramer, respectively, the cancer cells after irradiation with a total dose of 0 to 10 Gy Survival rate (A), BRG-1 bromodomain and BRG-1 bromodomain multimeric recombinant protein (B) is confirmed and shown.
Figure 11 shows the results of confirming the survival rate according to gamma irradiation of lung cancer cells NCI-H446 transformant expressing BRG-1 bromodomain dimer and tetramer, respectively, after irradiation with a total dose of 0 to 5 Gy , Cancer cell survival rate (A), BRG-1 bromodomain and BRG-1 bromodomain multimeric recombinant protein (B) is confirmed and shown.

Hereinafter, the present invention will be described in more detail with reference to Examples of the present invention and Comparative Examples which are not based on the present invention, but the scope of the present invention is not limited by the following Examples.

Example  1: cell culture conditions and DNA  Operation

Colon cancer cells HT-29 (R) and HCT116 (S) were cultured using McCoy's 5a medium (manufactured by WelGene) containing 10% fetal bovine serum, and lung cancer cells using F-12K medium (manufactured by WelGene). A549 (R) and NCI-H446 (S) were cultured, and normal cells 293T (S), breast cancer cells MDA-MB-231 (R) and SK-BR3 (Minimum Essential Medium Eagle medium (manufactured by ATCC) were used). S) were incubated at 37 ° C., 5% CO ₂ , respectively.

Example  2: Bromodomain  Preparation of monomer expression vectors and Intracellular  Introduction

In order to prepare a bromodomain expression vector (pCMV-BRG1-BRD), a gene cloned by fusion of three nuclear localization signals (NLS) and a myc epitope portion to a GFP-N1 vector having a high expression rate was cloned into the nucleus. It was made to be able to function. At this time, the three nuclear position signal and myc epitope portion was used 693 ~ 833 base portion of the pCMV / myc / nuc vector. The bromodomain (BRD) portion of the BRG-1 gene was obtained by using SEQ ID NOs: 5 and 6 as primers through polymerase chain reation (PCR). The obtained fragment was cloned into a GFP-N1 vector in which three nuclear position signals and a myc epitope portion were fused (Fig. 1). The cloned pCMV-BRG1-BRD vector was cultured by the above culture method. Normal cell 293T (S), Colon cancer cell HT-29 (R), Colon cancer cell HCT116 (S), Lung cancer cell A549 (R), Lung cancer cell NCI Transformants were prepared by transducing H446 (S), breast cancer cell MDA-MB-231 (R), and breast cancer cell SK-BR3 (S) using the calcium phosphate method.

SEQ ID NO: 5 (forward): 5'-gctcctcgagatggccgagaaactctcccct -3 '

SEQ ID NO: 6 (reverse): 5'-gctcaagcttctcctcgccttcactgtc -3 '

Example  3: Bromodomain Multimer Recombinant protein  Preparation of expression vectors and Intracellular  Introduction

In order to prepare a bromodomain multimeric recombinant protein expression vector, using a primer of SEQ ID NOS: 7 and 8 without an enzyme recognition site, a single-stranded end of the monomer of the BRG1-BRD prepared as described above was carried out to perform a linkage reaction ( blunt end ligation). Among the results obtained through the ligation reaction, a gel of a size expected to obtain only a dimer without a restriction enzyme recognition site was cut, and DNA was purified on the gel. Then, DNA fragments encoding bromodomain dimers were obtained through polymerase chain reaction using primers of SEQ ID NOs: 5 and 6 having Xho1 and Hind3 restriction enzyme recognition sites, respectively, as a template. The obtained DNA fragment was cloned into pEGFP-myc vector according to the method of Example 2 to prepare a bromodomain dimer expression vector (pEGFP-myc-BRG1-BRD-dimer). The cloned pEGFP-myc-BRG1-BRD-dimer vector was cultured by the above culture method. Normal cells 293T (S), colon cancer cells HT-29 (R), colon cancer cells HCT116 (S), lung cancer cells A549 (R) , Transformed into breast cancer cells MDA-MB-231 (R) and breast cancer cells SK-BR3 (S).

SEQ ID NO: 7 (forward): 5'-gccgagaaactctcccct-3 '

SEQ ID NO: 8 (reverse): 5'-ctcctcgccttcactgtc -3 '

In addition, by using the primers of SEQ ID NOs: 9 and 10 having the Sal1 and BamH1 restriction enzyme recognition sites, respectively, using DNA fragments encoding the bromodomain dimers prepared as described above, other restriction enzymes were subjected to polymerase chain reaction. A DNA fragment encoding a bromodomain dimer having a recognition site was obtained. The obtained DNA fragment was inserted into another restriction enzyme recognition site of the bromodomain dimer expression vector thus prepared and cloned to prepare a bromodomain tetramer expression vector (pEGFP-myc-BRG1-BRD-tetramer) (FIG. 2). . The transformed vector was transfected into normal cells 293T (S), colon cancer cells HCT116 (S), lung cancer cells A549 (R), and lung cancer cells NCI-H446 (S) according to the method of Example 2. Got it.

SEQ ID NO: 9 (forward): 5'-gctcgtcgacaagccgagaaactctcccct -3 '

SEQ ID NO: 10 (reverse): 5'-gctcggatcccctcctcgccttcactgtc -3 '

Example  4: Survival rate survey by gamma radiation

Example  4-1: Survival Rate by Gamma Irradiation in Normal Cells

In order to confirm the survival rate of the transformed normal cells by gamma irradiation, transformed 293T cells (S) were dispensed into 2 x 105 cells in a 100 mm culture dish and irradiated with radiation the next day. The source was γ-ray (137Cs; Cell Irradiation System, GC 3000 Elan-Model b; MDS Nordion, Ontario, Canada) and after 10 days after the total dose of 0 to 5 Gy to check the cell survival rate in Figure 3 Indicated.

Bromodomain expression vector (pEGFP-myc-BRG1-BRD) or bromodomain multimeric expression vector (pEGFP-myc-BRG1-BRD-dimer and pEGFP-myc-BRG1-BRD-) in normal hepatocyte HEK 293T (S) It was confirmed that BRG1 bromodomain or BRG1 bromodomain multimeric recombinant protein is expressed in cells transformed with tetramer) (FIG. 3B). In this case, the survival rate of 10% or less was observed at 3 Gy intensity irradiation. As a control that does not express bromodomains, the cells transformed with the pEGFP-myc vector killed cancer cells even at a radiation intensity weaker than the result of about 10% survival at 4 Gy (FIG. 3A), and their IC50 values. As the BRG1 bromodomain multimerized to 1.60, 1.20 and 0.97, respectively, it was confirmed that the radiation sensitivity of the cells was increased (FIG. 3C).

These results indicate that expression of bromodomains using the bromodomain expression vectors according to the present invention can increase radiation sensitivity, meaning that the effect can be increased as the bromodomains expressed are multimerized. do.

Example 4-2 Survival Rate by Gamma Irradiation by BRG1 Bromodomain and Dimer Expression in Cancer Cells

In order to confirm the survival rate of cancer cells transformed to express BRG1 bromodomain or dimer thereof according to gamma irradiation, the survival rate of the transformed cancer cells was confirmed using the method of Example 4-1. To 8 are shown.

Unlike 293T cells, which are normal cells, there are also cells that are not well transduced by the usual calcium phosphate method. Thus, each cancer cell has a viral vector that is easy to express bromodomain or dimer thereof. The experiment was carried out by fabrication.

BRG-1 in cells transformed with a bromodomain expression vector (pMX-myc-BRG1-BRD) or a bromodomain dimer expression vector (pMX-myc-BRG1-BRD-dimer) in colorectal cancer cells HT29 (R) It was confirmed that bromodomain is expressed (B of FIG. 4). In this case, when gamma radiation of the same intensity was irradiated, it was confirmed that the control group which does not express bromodomains showed a lower survival rate than that of cells transformed with the pMX-myc vector (FIG. 4A). In addition, the IC50 values of 2.81, 2.40 and 2.01, respectively, as BRG1 bromodomains were multimerized, it was confirmed that the radiation sensitivity of the cells increased (FIG. 4C).

BRG-1 in cells transformed with a bromodomain expression vector (pMX-myc-BRG1-BRD) or a bromodomain dimer expression vector (pMX-myc-BRG1-BRD-dimer) in colorectal cancer cells HCT116 (S) It was confirmed that bromodomain is expressed (B of FIG. 5). Also, in this case, a cell transformed with the pMX-myc vector as a control that does not express bromodomains with a survival rate of about 10% at 3 Gy intensity irradiation was weaker than a result of survival rate of about 10% at 4 Gy. Cancer cells were also killed (A in FIG. 5), and their IC50 values were 1.88, 1.12, and 1.02, respectively. As the BRG1 bromodomain multimerized, the radiation sensitivity of the cells was increased (FIG. 5C).

BRG-1 in cells transformed with a bromodomain expression vector (pMX-myc-BRG1-BRD) or bromodomain dimer expression vector (pMX-myc-BRG1-BRD-dimer) in lung cancer cells A549 (R) It was confirmed that bromodomain is expressed (B of FIG. 6). Also, in this case, the cells transformed with the pMX-myc vector as a control that do not express bromodomains showed a survival rate of about 10% at 6 Gy intensity irradiation, which was weaker than the result of survival rate of about 10% at 8 Gy. Cancer cells were also killed (A in FIG. 6), and their IC50 values were 2.88, 2.49, and 2.00, respectively. As the BRG1 bromodomain multimerized, the radiation sensitivity of the cells was increased (FIG. 6C).

Cells transformed with a bromodomain expression vector (pMX-myc-BRG1-BRD) or bromodomain dimer expression vector (pMX-myc-BRG1-BRD-dimer) in breast cancer cell MDA-MB-231 (R) It was confirmed that BRG-1 bromodomain is expressed in (B of FIG. 7). In addition, in this case, when gamma radiation of the same intensity was irradiated, it was confirmed that the control group which does not express bromodomains showed a lower survival rate than the survival rate of cells transformed with the pMX-myc vector (FIG. 7A). And, their IC50 values were 3.01, 2.61 and 2.46, respectively, and as the BRG1 bromodomain multimerized, it was confirmed that the radiation sensitivity of the cells was increased (FIG. 7C).

BRG in breast cancer cells SK-BR3 (S) transformed with bromodomain expression vector (pMX-myc-BRG1-BRD) or bromodomain dimer expression vector (pMX-myc-BRG1-BRD-dimer) It was confirmed that -1 bromodomain is expressed (B of FIG. 8). In this case, when gamma radiation of the same intensity was irradiated, it was confirmed that the control group which does not express bromodomains showed a lower survival rate than the survival rate of cells transformed with the pMX-myc vector (FIG. 8A). In addition, their IC50 values were 1.11, 0.88, and 0.83, respectively. As the BRG1 bromodomain was multimerized, the radiation sensitivity of the cells was increased (FIG. 8C).

These results indicate that the overexpressed bromodomains can increase the radiation sensitivity and effectively kill cancer cells, and that the effect can be stably maintained in various cancer cells such as colon, lung and breast. In addition, as the bromodomain expressed is multimerized, the effect may be increased.

Example 4-3 Survival Rate According to Gamma Irradiation by BRG1 Bromodomain Tetramer Expression in Cancer Cells

In order to confirm the survival rate of the cancer cells transformed to express the BRG1 bromodomain dimer and tetramer according to gamma irradiation, the survival rate of the transformed cancer cells was confirmed using the method of 4-1 above. To 11 are shown.

Bromodomain expression vector (pMX-myc-BRG1-BRD) or bromodomain multimeric expression vector (pMX-myc-BRG1-BRD-dimer and pMX-myc-BRG1-BRD-tetramer in colorectal cancer cells HCT116 (S) It was confirmed that BRG-1 bromodomain or a multimer is expressed in cells transformed with) (FIG. 9B). In addition, in this case, when gamma radiation of the same intensity was irradiated, the bromodomain was multimerized by showing a lower survival rate than that of a cell transformed with the pMX-myc vector as a control that does not express bromodomain. It was confirmed that the radiation sensitivity of the cells is increased (FIG. 9A).

Bromodomain expression vector (pMX-myc-BRG1-BRD) or bromodomain multimeric expression vector (pMX-myc-BRG1-BRD-dimer and pMX-myc-BRG1-BRD-tetramer in lung cancer cells A549 (R) It was confirmed that BRG-1 bromodomain or a multimer is expressed in cells transformed with) (FIG. 10B). In addition, in this case, when gamma radiation of the same intensity was irradiated, the bromodomain was multimerized by showing a lower survival rate than that of a cell transformed with the pMX-myc vector as a control that does not express bromodomain. It was confirmed that the radiation sensitivity of the cells is increased (FIG. 10A).

Bromodomain expression vector (pMX-myc-BRG1-BRD) or bromodomain multimeric expression vector (pMX-myc-BRG1-BRD-dimer and pMX-myc-BRG1-BRD in lung cancer cell NCI-H446 (R) -tetramer), it was confirmed that BRG-1 bromodomain or multimer expression in cells transformed with (tetra 11B). In addition, in this case, when gamma radiation of the same intensity was irradiated, the bromodomain was multimerized by showing a lower survival rate than that of the cells transformed with the pMX-myc vector as a control that does not express bromodomain. It was confirmed that the radiation sensitivity of the cells is increased (FIG. 11A).

These results indicate that the expression of bromodomains and their multimerization can increase the radiation sensitivity of cancer cells to effectively kill cancer cells, and that the effect can be stably maintained in various cancer cells. .

<110> Ewha University - Industry Collaboration Foundation <120> Fusion protein includes two or more BRG-1 derived bromodomains          and uses <130> PA110276KR <160> 10 <170> Kopatentin 1.71 <210> 1 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> BRD aa sequences <400> 1 Ala Glu Lys Leu Ser Pro Asn Pro Pro Asn Leu Thr Lys Lys Met Lys   1 5 10 15 Lys Ile Val Asp Ala Val Ile Lys Tyr Lys Asp Ser Ser Ser Gly Arg              20 25 30 Gln Leu Ser Glu Val Phe Ile Gln Leu Pro Ser Arg Lys Glu Leu Pro          35 40 45 Glu Tyr Tyr Glu Leu Ile Arg Lys Pro Val Asp Phe Lys Lys Ile Lys      50 55 60 Glu Arg Ile Arg Asn His Lys Tyr Arg Ser Leu Asn Asp Leu Glu Lys  65 70 75 80 Asp Val Met Leu Leu Cys Gln Asn Ala Gln Thr Phe Asn Leu Glu Gly                  85 90 95 Ser Leu Ile Tyr Glu Asp Ser Ile Val Leu Gln Ser Val Phe Thr Ser             100 105 110 Val Arg Gln Lys Ile Glu Lys Glu Asp Asp Ser Glu Gly Glu Glu         115 120 125 <210> 2 <211> 381 <212> DNA <213> Artificial Sequence <220> <223> BRD-coding DNA sequences <400> 2 gccgagaaac tctcccctaa cccacccaac ctcaccaaga agatgaagaa gattgtggat 60 gccgtgatca agtacaagga cagcagcagt ggacgtcagc tcagcgaggt cttcatccag 120 ctgccctcgc gaaaggagct gcccgagtac tacgagctca tccgcaagcc cgtggacttc 180 aagaagataa aggagcgcat tcgcaaccac aagtaccgca gcctcaacga cctagagaag 240 gacgtcatgc tcctgtgcca gaacgcacag accttcaacc tggagggctc cctgatctat 300 gaagactcca tcgtcttgca gtcggtcttc accagcgtgc ggcagaaaat cgagaaggag 360 gatgacagtg aaggcgagga g 381 <210> 3 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> 3x NLS sequences <400> 3 Asp Pro Lys Lys Lys Arg Lys Val Asp Pro Lys Lys Arg Lys Val Asp   1 5 10 15 Pro Lys Lys Arg Lys Val              20 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Myc sequences <400> 4 Glu Gln Lys Leu Ile Ser Phe Glu Asp Leu   1 5 10 <210> 5 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> forward primer sequence for PCR <400> 5 gctcctcgag atggccgaga aactctcccc t 31 <210> 6 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> reverse primer sequence for PCR <400> 6 gctcaagctt ctcctcgcct tcactgtc 28 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer sequence for PCR <400> 7 gccgagaaac tctcccct 18 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer sequence for PCR <400> 8 ctcctcgcct tcactgtc 18 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> forward primer sequence for PCR <400> 9 gctcgtcgac aagccgagaa actctcccct 30 <210> 10 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> reverse primer sequence for PCR <400> 10 gctcggatcc cctcctcgcc ttcactgtc 29

Claims (22)

A recombinant protein comprising two or more BRG-1 derived bromodomains.
The recombinant protein of claim 1, wherein the BRG-1 derived bromodomain is represented by an amino acid sequence as set forth in SEQ ID NO: 1.
The recombinant protein of claim 1, wherein the recombinant protein increases the radiation sensitivity of the cell.
The recombinant protein of claim 1, wherein the recombinant protein further comprises a cell internalization sequence.
The method according to claim 4, wherein the cell internalization sequence is polyarginine, antennapedia, TAT, HIV-Tat, penetratin, Antp-3A (Antp mutant), Buforin II, transfortan, MAP (model amphipathic peptide), K With FGF, Ku70, Prion, pVEC, Pep-1, SynB1, Pep-7, HN-1, BGSC (bis-guanidinium-spermidine-cholesterol) and BGTC (bis-guanidinium-tren-cholesterol) A recombinant protein characterized by comprising an amino acid of a protein selected from the group consisting of.
The recombinant protein of claim 1, wherein the recombinant protein further comprises a tumor specific targeting sequence.
The recombinant protein of claim 6, wherein the tumor specific targeting sequence comprises an RGD, NGR, or GSL motif.
A nucleic acid encoding the recombinant protein of any one of claims 1 to 7.
Recombinant vector comprising the nucleic acid of claim 8.
Claim 1 to claim 7, comprising a recombinant protein of any one of the active ingredient, the radiation-sensitive composition for cancer cell radiation therapy.
Claim 9 the vector as an active ingredient, cancer cell radiation therapy radiation sensitive composition.
The radiation sensitive composition of claim 10, wherein the cancer cell expresses BRG-1.
The radiation sensitive composition of claim 11, wherein the cancer cell expresses BRG-1.
The radiation sensitive composition of claim 10, wherein the cancer cells are radiation resistant cells.
The radiation sensitive composition of claim 11, wherein the cancer cells are radiation resistant cells.
The radiation sensitive composition of claim 10, wherein the radiation is gamma radiation.
The radiation sensitive composition of claim 11 wherein the radiation is gamma radiation.
A cancer cell comprising the recombinant protein of any one of claims 1 to 7.
19. The cancer cell according to claim 18, wherein the nucleic acid of claim 8 is formed by expression in the cell.
19. The cancer cell of claim 18, wherein said nucleic acid is mRNA or in the form of a vector operably linked to expression control sequences.
19. The cancer cell according to claim 18, wherein the cancer cell is used as a control to confirm whether the candidate agent is a radiation sensitive agent.
19. The cancer cell of claim 18, wherein the cancer cell is a radiation resistant cell.

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