KR101964610B1 - Novel Oncolytic Virus and Uses Thereof - Google Patents

Abstract

The present invention relates to a novel tumor-soluble virus and a use thereof, wherein the tumor-soluble virus is a novel Newcastle disease virus isolate, and the tumor-soluble virus is not only non-pathogenic but also exhibits cancer cell solubility specifically against tumor cells Can be effectively used for cancer treatment and cancer treatment.

Description

Novel Oncolytic Virus and Uses Thereof < RTI ID = 0.0 >

The present invention relates to novel tumor-soluble viruses and uses thereof.

Newcastle disease virus (NDV) is avian paramyxovirus type 1 (APMV-1) in Avulavirus belonging to the family Paramyxoviridae. The virus is a pathogenic agent causing a very deadly Newcastle disease in the poultry.

If a Newcastle disease occurs on a poultry farm that has not been vaccinated, most of the chickens on the farm are deadly to death within two weeks. If this disease is caught, the chicken may develop severe respiratory symptoms, digestive symptoms, and neurological symptoms if recovered. In the case of chicken eggs, low level of vaccine immunity causes severe egg laying rate and abnormal anomalies, resulting in poor productivity of poultry.

NDV is classified as velogenic, mesogenic, and lentogenic NDV depending on the degree of pathogenicity in chickens. Pathogenicity is measured by intracerebral pathogencity index (ICPI), mean fetal death rate (MDT), and amino acid sequence analysis of F protein gene segments at 1-day-old specific pathogen chicks . Inducible NDV is highly pathogenic to chickens and has a high mortality rate in infected chickens. According to the pathologic manifestations, the lesions are divided into viscerotropic velogenic NDV and nuerotropic velogenic NDV which severely cause gastrointestinal disease. The medium-dose NDV has a lower mortality rate than the read-only type, but it causes severe respiratory symptoms. Infectious NDV has little or no mortality rate of chicken, and the disease is very weak. Some of the virulent NDV strains are used as vaccines to prevent Newcastle disease in poultry farms.

NDV considers only a single serotype present worldwide. Viral genotypes are made through genetic analysis based on the gene sequence of the viral fusion (F) protein gene. NDV genotypes are largely classified into class 1 and class 2 types. Class 1 is mainly isolated from wild birds, ducks, and traditional markets and has no pathogenicity to chickens. Class 2 type has more than 16 genotypes to date. Both genotypes I and II are mostly virulent NDVs that do not cause damage to chickens, but the rest of the NDV genotypes are viruses that cause Newcastle disease in chickens.

These NDVs have an envelope and envelope contains HN (Haemagglutinin-Neuraminidase), which allows the virus to bind to host cells, and F (fusion) protein, which causes envelope and host cell fusion. The F and HN proteins are glycoproteins distributed on the surface of the envelope.

The F protein forms a trimeric structure as type I membrane glycoprotein. F proteins are made in the form of an inactive precursor (F0) and are cleaved by the active forms F1 and F2 during migration through the Golgi membranes. This process exposes a hydrophobic domain at the amino terminus of the F1 subunit and plays an important role in the biological activity of the mature protein. The hydrophobic domain, called the fusion peptide, is highly conserved in the paramyxovirus F protein and is thought to be directly involved in mediating membrane fusion. Paramyxovirus F protein has several common structural forms, including two regions that contain seven repeats (heptad repeats) and are likely to form an alpha-helical structure. The longest of the two repeats A (heptad repeat A) is adjacent to the hydrophobic fusion peptide at the amino terminus of F1, and the seven repeats B are closely attached to the top of the transmembrane region. Seven iterations B consist of a series of very conserved leucine or isoleucine residues every 7 residues.

HN protein is a type II membrane glycoprotein that forms a tetramer on the surface of the virus envelope and penetrates the cell membrane (Gorman et al., 1988; Ng et al., 1989). The HN protein functions to position the virion on the host cell surface by binding to sialic acids of glycoconjugates. The HN protein is divided into three regions: a transmembrane domain, a stalk domain, and a globular domain. Both the antigenic receptor binding and the neuraminidase active site are located in the spherical domain. Fusion induction activity is located in the stem domain, which interacts with the F protein (Sergei et al., 1993). The stem domain has two seven repeat regions A (positions 74-88) and seven repeat regions B (positions 96-110) with α-helices in the expected form. In addition, there are reports that any mutation that destroys the structure is responsible for the receptor binding and neuraminidase activity.

On the other hand, general cancer treatment is done through chemotherapy, radiation therapy and surgery. Except for some early cancers, however, there are side effects that are difficult to completely remove by surgery or due to cytotoxicity of chemotherapy or radiotherapy. Among the various technologies to supplement this, the strategy of attacking and killing only cancer cells is one of the most popular technologies. Among them, "oncolytic virus" is a cancer treatment technology that has been attracting attention for a long time as a virus that infects cancer cells and destroys cancer cells based on the ability of the virus to inherit or artificially add cancer cells.

That is, the tumor-soluble virus is a type of conditionally replication-competent virus that selectively proliferates in tumor cells and kills the tumor cells. During the period from the 1950s to the 1970s, the tumor-killing effect of the virus was confirmed by studies using adenovirus or mumps virus, but eventually due to transient efficacy and toxic side effects, I did not receive it. However, in the 1990s, the thymidine kinase (TK) -deficient mutant of Herpes simplex virus-1 (HSV-1) and the adenovirus mutant dl1520 selectively proliferating in p53- (ONYX-015), there has been rapid progress in the study of tumor-killing viruses (Jagus R, et al., Int. J. Biochem. Cell Biol. , (1999) 31: 123-138; Bischoff JR, et al., Science, (1996) 274 (5286): 373-376). To date, more than 80 tumor-killing viruses have been developed and are expected to be promising tools for chemotherapy.

Recently, the number of companion animals such as dogs and cats is increasing worldwide, and animal care and animal welfare are becoming important issues. As the breeding period of companion animals becomes longer, the cancer that occurs in companion animals is regarded as an important disease. In Europe, the incidence of tumors is estimated to be about 0.2% (200 / 100,000) per year, of which mammary tumors account for about 40% of females. In addition, mammary tumors are the third most common tumors, accounting for 10% to 12% of cervical benign diseases, and most of the developing tumors are malignant tumors. Cancer treatment strategies for dogs and cats are similar to those of humans, and it is best to apply surgery, chemotherapy, and radiation therapy alone or in parallel. Yet attempts to apply the tumor-soluble virus to dogs and cats are at an experimental level.

The ability of the virus to selectively kill cancer cells varies according to the type of cancer. Recently, studies are being conducted to apply cancer treatment viruses such as adenovirus and poxvirus, which are applied in human, for cancer treatment of companion animals. Currently, no virus has been developed targeting only mammary gland tumors. Unlike in humans, there has been no attempt to apply the Newcastle disease virus for the treatment of companion cancer. In addition, the Newcastle disease virus is known to have a higher tumor dissolving ability as the pathogenicity is higher. The efficacy evaluation is only for human cancer cells, and the efficacy data on the tumor cells of animals are extremely limited. However, it is difficult to apply indomethacin NDV to the treatment of companion animal cancer, especially in the case of companion animals that are housed in poultry farms because of the risk of spreading malignant diseases.

Therefore, it has been attempted as a realistic way to develop an enteric-type NDV that does not damage poultry farms for the treatment of companion cancer.

Under these circumstances, the present inventors have sought to develop an enteroviral NDV that maximizes the tumor killing effect more selectively in tumor cells for cancer treatment of animals. As a result, the present inventors analyzed the novel NDV isolate by isolating NDV from the domesticated poultry ducks in Korea and found that the NDV isolate has the characteristics of a pathogenic NDV and has excellent virulence against tumor cells without showing pathogenicity The present invention has been completed.

Accordingly, an object of the present invention is to provide a Newcastle disease virus (NDV) which is a tumor-cell-killing oncolytic virus.

Another object of the present invention is to provide a pharmaceutical composition for treating or preventing cancer.

It is still another object of the present invention to provide a food composition for preventing or ameliorating cancer.

Still another object of the present invention is to provide a method for treating or preventing cancer.

Hereinafter, the present invention will be described in detail.

According to one aspect of the present invention, the present invention provides a Newcastle disease virus (NDV), which is a tumor-cell-killing oncolytic virus and has the deposit number KCTC13216BP.

According to a preferred embodiment of the present invention, the Newcastle disease virus is a novel isolate of Newcastle disease virus isolated from domestic ducks infected with Newcastle disease virus.

The present inventors named the novel Newcastle disease virus isolate as "NDV QIA-CI" and deposited the NDV QIA-CI strain on the KCTC BRC on Feb. 16, 2017 and received the deposit number KCTC13216BP.

The Newcastle disease virus of the present invention comprises an F protein having an immunogenicity represented by the amino acid sequence of SEQ ID NO: 1 and equivalent thereto; And a V protein having an immunogenicity represented by the amino acid sequence of SEQ ID NO: 3 and equivalent immunogenicity.

The major surface proteins of NDV are the F and HN proteins and are important for genotyping and pathogen classification. In particular, the F protein is involved in cell surface attachment and cell fusion of viruses, and the amino acid sequence of the segment is an important factor in determining the virulence of the virus. In addition, the base sequence of the F gene is also a standard for classifying the genotype of NDV.

In addition, among the NDV internal proteins, the V protein is a secondary protein produced from the gene encoding the P protein and is known to be important for the host animal range and pathogenicity determination of NDV.

The nucleotide sequence encoding the F protein of the Newcastle disease virus of the present invention is SEQ ID NO: 2.

As used herein, a strongly pathogenic or strongly toxic Newcastle disease virus is used to include all pathogenic Newcastle disease viruses with a pathogenicity intermediate, as well as those of the river pathogenic Newcastle disease viruses, which are conventionally classified, unless otherwise stated. In the present invention, the Strong Pathogenic Newcastle Disease virus is a pathogenic disease caused by an infectious virus being produced in all cells in the body when an animal is infected. When the 113th to 116th amino acid sequences of the F protein are expressed as the following formula 1, It is cleaved by a purine or purine-like protease (hereinafter, purine) distributed in most cells, and has a structure having activity, so that the virus is infectious. Therefore, the pathogenic Newcastle disease virus is defined as having the nucleotide sequence coding for the amino acid sequence represented by the following formula 1 as the coding sequence of the 113th to 116th amino acid sequence of the F protein:

[Formula 1]

¹¹³ X ₁ X ₂ X ₃ X ₄ ¹¹⁶

In the formula

X ₁ , X ₃ and X ₄ are each independently arginine (R) or lysine (K)

X ₂ is an amino acid selected from the group consisting of alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, valine, asparagine, cysteine, glutamine, glycine, serine, threonine, tyrosine, aspartic acid, glutamic acid, arginine, histidine and lysine ,

At this time, the 112th amino acid of F is also more virulent when it is a basic amino acid such as arginine or lysine.

Also, in this specification, a pharmacologically or toxic Newcastle disease virus is used to mean a non-toxic or nontoxic Newcastle disease virus, as well as a pharmacokinetic Newcastle virus class normally classified unless otherwise stated.

In the present invention, the phagocytic Newcastle disease virus is activated only by some extracellular proteases in the body and intracellular proteases of the gastrointestinal tract and respiratory tract at the time of animal infection, resulting in localized virus propagation resulting in weak pathogenicity. Th amino acid sequence is expressed as the following Equation (2). Thus, the pathogenic Newcastle disease virus is defined as having the nucleotide sequence encoding the amino acid sequence represented by the following formula 2 as the coding sequence of amino acid sequence 113 to 116 of F:

[Formula 2]

¹¹³ X ₅ X ₆ X ₇ X ₈ ¹¹⁶

In the formula

X _5, X ₆ and X ₇ are each independently selected from alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, valine, asparagine, cysteine, glutamine, glycine, serine, threonine, tyrosine, aspartic acid, glutamic acid, arginine, histidine And lysine, and X ₅ and X ₇ are not arginine (R) or lysine (K) at the same time,

X ₈ is arginine (R) or lysine (K).

Newcastle disease virus is infected only when the cleavage site located at the F protein by purine is cleaved and the fusion peptide region of the F protein fused with the cell membrane is exposed. Since purine is an enzyme distributed throughout the whole body of an animal, activation of virus infectivity by purine occurs in whole body and is pathogenic.

Therefore, the virulence of the Newcastle disease virus depends on the extent to which the purine cleavage site located in the F protein is recognized and cleaved by the purine.

When the purine recognition site (113th to 116th amino acids) of the Newcastle Disease virus F protein has at least three basic amino acids ( ¹¹³ RXK / RR ¹¹⁶ ) as in the amino acid sequence of the above formula 1, the virus infection by purine is systemically Because it occurs, it shows virulence. However, when at least one basic amino acid is substituted with a non-basic amino acid, as in the amino acid sequence of the above-mentioned formula 2, recognition and cleavage by purine hardly occur and only local or extracellular protease The pathogenicity is low because no fatal systemic infection occurs.

That is, most NDV with virulence (pathogenicity) has F (phenylalanine) at the C-terminal end of the F2 protein at position ¹¹² R / KRQK / RR ¹¹⁶ and at the N-terminal residue 117 of the F1 protein while low virulence Have the sequence of ¹¹² G / EK / RQG / ER ¹¹⁶ and L (leucine) at residue 117 in the same region.

Sequence analysis of the Newcastle Disease Virus of the present invention showed that the amino acid sequence of the segment of the F protein described above was ¹¹² ERQERL ¹¹⁷ , which corresponds to the type I vaccine and is effectively attenuated, It has excellent characteristics.

In addition, the Newcastle Disease Virus isolate of the present invention has been shown to not only help cancer cells to self-destruct but also have excellent tumor killing ability.

According to another aspect of the present invention, there is provided a pharmaceutical composition for treating or preventing cancer comprising as an active ingredient Newcastle disease virus (NDV), which is Deposit No. KCTC13216BP.

In addition, the pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier.

The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc., in addition to the above components.

Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

As used herein, the term " pharmaceutically acceptable carrier ", as is commonly used in the art, includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, calcium phosphate, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not.

Suitable carriers for this are known to those skilled in the art, and such carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous carrier include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable organic ester such as ethyl oleate. Aqueous carriers include water, alcohol / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral carriers include sodium chloride solution, Ringer ' s dextrose, dextrose and sodium chloride, lactic acid treatment linger or fixed oils. The intravenous carrier includes electrolyte supplements, liquids and nutritional supplements, such as those based on Ringer ' s dextrose. Preservatives and other additives such as antimicrobial agents, antioxidants, chelating agents, inert gases and the like may be additionally present. Preferred preservatives include formalin, thimerosal, neomycin, polyamicin B and amphotericin B.

The pharmaceutical composition of the present invention can be administered orally or parenterally. In the case of parenteral administration, the composition can be administered by intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, transdermal administration, or the like.

The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, the administration method, the age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient, Usually, a skilled physician can readily determine and prescribe dosages effective for the desired treatment or prophylaxis.

In addition, the effective amount administered to a patient can be varied by a number of factors such as the pharmacokinetic characteristics of the virus, the method of administration, the age, the health and weight of the patient, the nature and extent of the disease state, the frequency of treatment, For example, depending on the virulence and potency of the virus.

The concentration of the virus to be administered will depend on the virulence of the particular viral strain to which it is to be administered and the characteristics of the target cell. According to one embodiment, an amount in the range of 10 ² to 10 ¹³ plaque forming units (" pfu ") may be administered. According to various embodiments, 10 ² to 10 ⁹ pfu, 10 ² to 10 ⁷ pfu, 10 ³ to 10 ⁶ pfu or 10 ⁴ to 10 ⁵ pfu may be administered in a single dose.

An effective amount of the virus can be repeatedly administered according to the effect of the first therapeutic regimen. In general, administration is periodically administered during monitoring of all reactions. Those skilled in the art will readily be able to ascertain that doses lower or higher than those indicated above may be administered depending on the dosing schedule and the selected route.

The virus may be administered alone or in combination with other therapies, including chemotherapy, radiotherapy or other anti-viral therapy. For example, the virus may be administered prior to or after removal of the primary tumor via surgery, or prior to, concurrently with, or subsequent to treatment, such as with radiotherapy or conventional chemotherapy drugs. According to one embodiment, the virus can be administered with other tumor killing viruses that are specific for various tumor cell types, or in a sequential manner.

The pharmaceutical composition of the present invention may be formulated into a pharmaceutically effective amount by using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by those having ordinary skill in the art to which the present invention belongs. May be prepared in a dosage form or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

As used herein, the term " therapeutically effective amount " means that the amount of inhibitor administered alleviates to some extent one or more symptoms of cancer, the disease to be treated. Thus, a pharmacologically effective amount means to (1) reverse the rate of progression of the cancer, (2) to inhibit further progression of the cancer to some extent, and (3) Quot; means an amount that has an effect of alleviation (preferably, elimination).

"Cancer" or "tumor", which is the target disease of the present invention, refers to an aggressive characteristic of a cell which ignores normal growth limits and divides and grows, invasive characteristics that penetrate surrounding tissues, And diseases caused by cells having metastatic characteristics spreading to other parts of the body.

According to a preferred embodiment of the present invention, the cancer is selected from the group consisting of breast cancer, colon cancer, gastric cancer, lung cancer, liver cancer, blood cancer, (Eg, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, uterine sarcoma, ovarian cancer ovarian cancer, rectal cancer, anal cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, small intestine cancer, endocrine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue tumor, urethral cancer, prostate cancer, bronchial cancer bronchogenic cancer, and bone marrow tumor. But is most preferably breast cancer.

The breast cancer refers to all malignant tumors occurring in the breast, including, but not limited to, breast malignant tumors, ductal cancer, breast cancer, and breast cancer.

According to one embodiment of the present invention, the present composition comprising the Newcastle disease virus (NDV), which is an active ingredient of the present invention, not only reduces or suppresses cancer cell growth and survival rate, but also induces apoptosis of cancer cells, .

According to still another aspect of the present invention, there is provided a food composition for preventing or ameliorating cancer comprising Newcastle disease virus (NDV), which is Deposit No. KCTC13216BP, as an active ingredient.

The composition of the present invention can be added to a health functional food for the purpose of preventing or improving cancer. In the present invention, the term " health functional food " refers to a food having a biological control function such as prevention and improvement of cancer, bio-defense, immunity, recovery after disease and the like.

When the composition of the present invention is used as a food additive, the composition can be added as it is or can be used together with other food or food ingredients, and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). In general, the composition of the present invention is added in an amount of not more than 15% by weight, preferably not more than 10% by weight based on the raw material, in the production of food or beverage. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

There is no particular limitation on the kind of the food. Examples of the foods to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharine and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 10 g, preferably about 0.01 to 0.1 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, A carbonating agent used in a carbonated beverage, and the like. In addition, the composition of the present invention may comprise flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. Although the ratio of such additives is not critical, it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Since the food composition of the present invention includes the above-mentioned Newcastle disease virus (NDV), the description common to both is omitted in order to avoid the excessive complexity of the present specification.

According to another aspect of the present invention, there is provided a method for treating or preventing cancer, comprising administering the pharmaceutical composition to a subject to be treated.

The present method can use the supernatant of the culture containing the Newcastle Disease Virus as the undiluted solution containing the Newcastle Disease Virus without going through a series of processes. For example, the Newcastle Disease virus expressed in the host cell may not be subjected to a separate purification process, or the cell lysate prepared by disrupting the cultured host cell may be used as it is.

Preferably, the composition of the present method is administered by any one method selected from the group consisting of oral, nasal, peritoneal, intravenous, subcutaneous, muscular, and combinations thereof.

Preferably, the subject of the method is a mammal, and the mammal may be at least one selected from the group consisting of humans, monkeys, cows, pigs, sheep, horses, dogs, and cats, and most preferably dogs Or a cat.

Since the method of the present invention uses a composition comprising the above-mentioned Newcastle Disease Virus as an active ingredient, the contents overlapping with the composition of the present invention described above are omitted in order to avoid the excessive complexity of the present invention due to the overlapping description of the contents. do.

The novel Newcastle Disease virus of the present invention is not only non-pathogenic but also can be effectively used for cancer treatment and cancer treatment because it shows the ability to specifically dissolve cancer cells against tumor cells.

FIG. 1A shows the results of comparing the specific killing effects of cancer cells against seven kinds of NDV outdoor cells in two cancer cell lines. Figure IB is a flow diagram based on the F gene nucleotide sequence of the NDV QIA-CI, showing the location of the susceptible NDV isolate QIA-CI of the present invention. Fig. 1C shows the V protein amino acid sequence of NDV QIA-CI and has an amino acid sequence that is different from NDV LaSota, an anticancer virus host known in the art.
FIG. 2 is a graph showing the cytopathic effect of NDV QIA-CI primary infection on canine mammary tumor cell lines compared with normal cells.
FIG. 3 shows the results of observing the proliferative activity of NDV QIA-CI in an open-cell tumor cell line.
FIG. 4 shows the results of observing the survival rate changes of canine mammary tumor cell lines CHMp5b, CHMp-13a and CMT14-2 induced by the NDV QIA-CI main infection of the present invention.
FIG. 5 shows the results of DAPI staining of apoptosis levels of canine mammary tumor cell lines CHMp5b and CHMp-13a caused by NDV QIA-CI primary infection of the present invention.
FIG. 6 shows the result of observing the inhibition of tumor volume growth when NDM QIA-CI was administered to a nude mouse in which a tumor derived from a canine mammary tumor cell CHMp-13a was transplanted.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention as defined by the appended claims. It will be obvious to you.

Example  1. The present invention New Newcastle disease  Viruses (Newcastle disease virus, NDV ) Separation and selection

To obtain a novel tumor-soluble virus, the present inventors isolated a variety of NDV outdoor strains from domestic ducks and selected and identified novel NDV isolates showing the best cancer-solubility among them.

1-1. variety Newcastle disease  Viruses (Newcastle disease virus, NDV ) Outdoor states  detach

The present inventors suspended the duck feces collected from duck farms in the Jeollabuk-do region of Korea in phosphate buffered saline (PBS) and then inoculated the supernatant with SPF on 9-11 days of age and cultured for 3-5 days. Thus, a total of 7 virus outdoors were obtained. The outbreaks were collected from the umbilical sulcus of the cultured developmental cells to confirm the coagulability of chicken erythrocytes. Genetic tests were performed to confirm that the NDV was pathogenic.

1-2. The New Newcastle disease  Viruses (Newcastle disease virus, NDV ) Selection

The present inventors compared and evaluated the killing effect on tumor cells in order to select a novel NDV isolate exhibiting the best cancer solubility among the seven NDV outdoor strains obtained in Example 1-1.

First, the human mammary tumor-derived cell line BT474 and the canine mammary tumor-derived cell line CMT 14-2 were cultured in a 96 well plate at a concentration of 10 ⁵ / ml, respectively. After 24 hours, 7 different NDV isolates were transfected with 10 ~ 20 MOI Lt; / RTI > After 72 hours, WST-1 was added to the culture medium to measure the absorbance of the cells at a wavelength of 235 nm within 30 minutes to 1 hour, and the survival rate of cancer cells was confirmed.

As a result, as shown in Fig. 1A, in both cancer cell lines, the cell death rate of NDV QIA-CI in seven kinds of NDV open field tested in the test was significantly lower than that of Ulster 2C strain (positive control group ).

The present inventors named the novel Newcastle disease virus isolate as " NDV QIA-CI ", and deposited the NDV QIA-CI strain in the 16th letter of February, 2017 to the KCTC BRC and received the deposit number KCTC13216BP.

Example  2. The present invention New Newcastle disease  Viruses (Newcastle disease virus, NDV ) Biological characteristics of

The present inventors analyzed the molecular biological properties of the novel NDV isolate NDV QIA-CI of the present invention obtained in Example 1-2.

In order to confirm the pathogenicity of chicken and chick, viruses were inoculated into SPF developmental bolls at 9-11 days of age, and the presence or absence of developmental follicles was observed for one week. In addition, viruses were inoculated into the brain of 10 - day - old SPF chicks and clinical symptoms and mortality were observed.

As a result, the mortality was not confirmed in both the SPF developmental field and the chick, and the mean death time (MDT) due to the virus was 120 hours or more, and the intracerebral pathogenicity index (ICPI) was 0.0.

In addition, we analyzed the gene sequence of the segment of the F gene that determines the virulence of NDV. For this purpose, RNA was extracted from the viral solution and RT-PCR was carried out so as to contain the segment of the F gene, and the nucleotide sequence of the amplified gene product was analyzed.

Based on the amino acid sequence (SEQ ID NO: 1) of the analyzed F protein (SEQ ID NO: 1) and the nucleotide sequence of the F gene (SEQ ID NO: 2), the F segment amino acid sequence of the F segment of the vulnerable NDV isolate QIA- The amino acid sequence was found to be ¹¹² ERQERL ¹¹⁷ , indicating that the novel NDV isolate of this invention is a pathogenic NDV.

In order to compare the nucleotide sequence of the analyzed F gene with the previously known NDV virus, a schematic diagram (FIG. 1B) was prepared.

As a result, as shown in FIG. 1B, NDV isolate QIA-CI of the present invention belonged to genotype 2 belonging to NDV of Class I.

In addition, the present inventors have further found that the NDV isolate of the present invention is a novel isolate of the amino acid sequence of the V protein that inhibits the interferon activity of the host cell and plays an important role in limiting the virus propagation, pathogenicity and host region SEQ ID NO: 3) was compared and compared with NDV LaSota strain for anti-cancer which is known in the art.

As a result, the NDV isolate of the present invention showed an amino acid sequence (sequence matching ratio 62.8%) different from that of NDV LaSota (Fig. 1C).

Thus, the NDV splitting site of the present invention can be seen as a novel NDV splitting site that is not known.

Example  3. The New Newcastle disease  Viruses (Newcastle disease virus, NDV ) To evaluate cancer cell-specific killing effect

The present inventors confirmed the killing effect on the tumor cells of NDV QIA-CI, a novel NDV isolate of the present invention obtained in Example 1-2.

First, NDM QIA-CI strain was infected with CMM-13 and CMM-13, which were isolated from canine malignant mammary tumors, and cytotoxic effect was observed for 5 days. Respectively.

As a result, as shown in FIG. 2, the present QIA-CI strain produced cytopathic effect in all three mammary tumor cells, and its pattern was different from that of the haploid formation, which is characteristic of the general cytotoxic effect of NDV, After the protoplast formation, it was confirmed that the cell disappeared.

In addition, we confirmed that the present NDV QIA-CI strain shows proliferative activity in canine mammary tumor cells.

First, NDV QIA-CI strain was infected with two malignant mammary tumor cell lines CHMp-5b and CHMp-13 and the medium was recovered at 0, 24, 48 and 72 hours to measure the titer of virus.

As a result, as shown in Fig. 3, it is presumed that the present QIA-CI strain did not proliferate in the mesenchymal tumor cells, and that it exhibited the cytotoxic effect through the non-lytic pathway.

Example  4. The New Newcastle disease  Viruses (Newcastle disease virus, NDV ) On tumor dissolution ability oncolytic  activity

The present inventors confirmed the tumor dissolution ability of the novel NDV isolate NDV QIA-CI of the present invention obtained in Example 1-2.

First, the canine tumor-derived cell lines CHMp-5b, CHMp-13a, and CMT 14-2 were cultured at a concentration of 10 ⁵ / ml in a 96-well plate and infected with virus at 100-200 MOI after 24 hours. After 72 hours, WST-1 was added to the medium and the absorbance was measured at a wavelength of 235 nm within a period of 30 minutes to 1 hour to confirm the survival rate of the cancer cells.

As a result, as shown in Fig. 4, the cell survival rate of cancer cells due to infection with NDV QIA-CI was very low, which was less than 50%.

Example  5. The New Newcastle disease  Viruses (Newcastle disease virus, NDV ) Apoptosis ( apoptosis ) Induction ability  evaluation

The present inventors confirmed the apoptosis inducing ability of the novel NDV isolate NDV QIA-CI of the present invention obtained in Example 1-2.

First, CHMp-5b and CHMp-13a cells were infected with QIA-CI strain, respectively, and stained with DAPI for 24 hours, 48 hours, and 72 hours, and observed with a fluorescence microscope. DNA fragmentation can be observed in the nucleus of apoptotic cells through DAPI staining.

As a result, as shown in FIG. 5, DNA fragmentation in the nucleus was observed by the inoculation of NDV QIA-CI and it was confirmed that apoptosis induced by virus was induced.

Example  6. The New Newcastle disease  Viruses (Newcastle disease virus, NDV ) Tumor growth Inhibition  evaluation

The present inventors confirmed the in vivo tumor growth inhibitory effect of the novel NDV isolate NDV QIA-CI of the present invention obtained in Example 1-2.

First, NDV QIA-CI strain was administered to a nude mouse transplanted with an open-cell tumor cell line CHMp-13a to evaluate whether the tumor growth was delayed. CHMp-13a cells were transplanted into the lower abdominal skin of nude mice at a concentration of 10 ⁷ cells / 200 μl. On the 9th day after transplantation, mice were randomly classified and directly injected with the present QIA-CI virus strain into the tumor tissues. The control group received PBS instead of the virus. The change in tumor volume was recorded by measuring the length and width of the tumor for 14 days after administration.

As a result, as shown in FIG. 6, tumor growth was significantly inhibited by the NDV QIA-CI inoculation in comparison with the control group.

Therefore, the novel NDV isolate of the present invention, NDV QIA-CI, showed significant inhibition of tumor cell-specific survival and growth as well as induction of cancer cell apoptosis. Thus, the novel NDV The isolate can be used as a cancer treatment for the subject to be treated.

Institution name: Korea Biotechnology Research Institute

Accession number: KCTC13216BP

Checked on: 20170216

Hereinafter, formulation examples of the pharmaceutical composition containing the composition of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically described.

Formulation example  One. Sanje  Produce

NDV QIA-CI 10 ² to 10 ¹³ pfu

Lactose 1g

The above components were mixed and packed in airtight bags to prepare powders.

Formulation example  2. Preparation of tablets

NDV QIA-CI 10 ² to 10 ¹³ pfu

Corn starch 100 mg

Lactose 100mg

Magnesium stearate 2 mg

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

Formulation example  3. Preparation of capsules

NDV QIA-CI 10 ² to 10 ¹³ pfu

Corn starch 100 mg

Lactose 100mg

Magnesium stearate 2 mg

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

Formulation example  4. Preparation of injections

NDV QIA-CI 10 ² to 10 ¹³ pfu

Virus-carrying cells (Molecular Therapy. 2009 Vol. 17: 1667-1676)

Na ₂ HPO _{4 12} H ₂ O 26 mg

2974 mg of distilled water

After mixing the above components, an injection was prepared according to the usual production method.

<110> REPUBLIC OF KOREA (Animal and Plant Quarantine Agency) <120> Novel Oncolytic Virus and Uses Thereof <130> 1-75p <160> 3 <170> KoPatentin 3.0 <210> 1 <211> 553 <212> PRT <213> Artificial Sequence <220> <223> Newcastle disease virus F protein <400> 1 Met Asp Pro Lys Pro Ser Thr Ser His Leu His Ala Phe Pro Leu Ile   1 5 10 15 Phe Val Ala Ile Ser Leu Val Phe Met Ala Gly Arg Ala Ser Ala Leu              20 25 30 Asp Gly Arg Pro Leu Ala Ala Gly Ile Val Val Thr Gly Asp Lys          35 40 45 Ala Val Asn Ile Tyr Thr Ser Ser Gln Thr Gly Thr Ile Ile Ile Lys      50 55 60 Leu Leu Pro Asn Met Pro Lys Asp Lys Glu Gln Cys Ala Lys Ser Pro  65 70 75 80 Leu Asp Ala Tyr Asn Arg Thr Leu Thr Thr Leu Leu Thr Pro Leu Gly                  85 90 95 Asp Ser Ile Lys Arg Ile Gln Glu Ser Val Thr Thr Ser Gly Gly Glu             100 105 110 Arg Gln Glu Arg Leu Val Gly Ale Ile Ile Gly Gly Val Ala Leu Gly         115 120 125 Val Ala Thr Ala Ala Gln Ile Thr Ala Ala Ser Ala Leu Ile Gln Ala     130 135 140 Asn Gln Asn Ala Asn Ile Leu Lys Leu Lys Glu Ser Ile Ala Ala 145 150 155 160 Thr Asn Glu Ala Val His Glu Val Thr Ser Gly Leu Ser Gln Leu Ala                 165 170 175 Val Ala Val Gly Lys Met Gln Gln Phe Val Asn Asp Gln Phe Asn Lys             180 185 190 Thr Ala Gln Glu Ile Asp Cys Ile Lys Ile Thr Gln Gln Val Gly Val         195 200 205 Glu Leu Asn Leu Tyr Leu Thr Glu Leu Thr Thr Val Phe Gly Pro Gln     210 215 220 Ile Thr Ser Pro Ala Leu Thr Gln Leu Thr Ile Gln Ala Leu Tyr Asn 225 230 235 240 Leu Ala Gly Gly Asn Met Asp Tyr Met Leu Thr Lys Leu Gly Val Gly                 245 250 255 Asn Asn Gln Leu Ser Ser Leu Ile Ser Ser Gly Leu Ile Ser Gly Asn             260 265 270 Pro Ile Leu Tyr Asp Ser Gln Thr Gln Leu Leu Gly Ile Gln Val Thr         275 280 285 Leu Pro Ser Val Gly Asn Leu Asn Asn Met Arg Ala Thr Tyr Leu Glu     290 295 300 Thr Leu Ser Val Ser Thr Asn Lys Gly Phe Ala Ser Ala Leu Val Pro 305 310 315 320 Lys Val Val Thr Gln Val Gly Ser Val Ile Glu Glu Leu Asp Thr Ser                 325 330 335 Tyr Cys Ile Glu Thr Asp Leu Asp Leu Tyr Cys Thr Arg Ile Val Thr             340 345 350 Phe Pro Met Ser Pro Gly Ile Phe Ser Cys Leu Gly Gly Asn Thr Ser         355 360 365 Ala Cys Met Tyr Ser Lys Thr Glu Gly Ala Leu Thr Thr Pro Tyr Met     370 375 380 Thr Leu Lys Gly Ser Val Ile Ala Asn Cys Lys Met Thr Thr Cys Arg 385 390 395 400 Cys Thr Asp Pro Pro Gly Ile Ser Ser Gln Asn Tyr Gly Glu Ala Val                 405 410 415 Ser Leu Ile Asp Lys Lys Val Cys Asn Ile Leu Thr Leu Asp Gly Ile             420 425 430 Thr Leu Arg Leu Ser Gly Glu Phe Asp Ala Thr Tyr Gln Lys Asn Ile         435 440 445 Ser Ile Gln Asp Ser Gln Val Val Ile Thr Gly Asn Leu Asp Ser Ser     450 455 460 Thr Glu Leu Gly Asn Val Asn Asn Ser Ile Ser Asn Ala Leu Asp Lys 465 470 475 480 Leu Glu Glu Ser Asn Ser Lys Leu Asp Lys Val Asn Val Arg Leu Thr                 485 490 495 Ser Thr Ser Ala Leu Ile Thr Tyr Ile Val Leu Thr Thr Ile Ala Leu             500 505 510 Ile Cys Gly Ile Val Ser Leu Val Leu Ala Cys Tyr Ile Met Tyr Lys         515 520 525 Gln Lys Ala Gln Gln Lys Thr Leu Leu Trp Leu Gly Asn Asn Thr Leu     530 535 540 Asp Gln Met Arg Ala Thr Thr Lys Met 545 550 <210> 2 <211> 1662 <212> DNA <213> Artificial Sequence <220> <223> Newcastle disease virus F <400> 2 atggatccca agccttctac cagccatttg catgctttcc cactgatctt cgtggctatc 60 agtttagtat tcatggctgg aagggcatcg gcactggatg ggcggccgct cgccgccgca 120 ggaatagtag tcacaggaga caaggcagtt aatatataca catcgtcgca gacagggaca 180 attatcatca aattattgcc caatatgccc aaagacaaag aacaatgtgc caagagcccc 240 ctagatgcat acaacaggac ccttacaacc ctccttaccc cgctcggcga ttcgattaag 300 agaatccaag agtcggtaac cacatcagga ggagaacgtc aggagcgttt ggtaggggca 360 ataataggag gtgtcgcatt aggtgtagcc accgcagcgc agatcacagc ggcttccgct 420 ctcatacaag ccaaccagaa tgctgcaaac atactgaaat taaaggagag tattgccgct 480 accaatgaag cagtacatga ggtcacaagc gggctgtccc agctggctgt tgctgtaggc 540 aagatgcagc agtttgtaaa cgaccaattc aacaagacag cacaagagat cgactgtatt 600 aagatcacac aacaggttgg ggtggagttg aatctatact tgaccgaact gacaactgtg 660 ttcggaccgc aaatcacatc ccctgcactc acgcagttga ccatccaggc cttatataat 720 ctggcgggtg ggaacatgga ttacatgctg acaaagctgg gagtgggtaa taatcagctc 780 agttctctga ttagtagtgg cctaatatct gggaatccaa ttctctatga ctcgcaaact 840 cagcttctgg gtatacaggt gactctcccc tctgtgggca atctgaacaa catgcgcgcc 900 acatatctgg aaaccctctc tgtaagtacg aacaagggat tcgcatccgc attagtgcct 960 aaagtggtaa ctcaggtagg ctccgtaatt gaagagctgg atacatcgta ttgcatagag 1020 acagacttgg atttgtactg cactagaatt gtaacattcc ccatgtctcc aggtatcttc 1080 tcctgtctcg gtggaaacac ctcagcatgc atgtactcta agactgaagg agcactcacc 1140 acaccatata tgacactcaa agggtcagtc atagcaaatt gcaagatgac aacgtgtagg 1200 tgcacagatc ccccggggat aatctcacag aattatggag aggctgtctc tctaatagac 1260 aaaaaggtat gtaacattct gactctcgac gggatcacct tgcgccttag cggggaattc 1320 gatgcaacct atcagaaaaa tatctcaatt caggattccc aggtggtgat tactgggaat 1380 ctggatatat ccactgagtt agggaatgtg aataattcaa tcagcaatgc tctagacaag 1440 ctagaagaga gtaacagcaa gctggacaaa gttaatgtga gacttaccag cacatctgcc 1500 ctaatcacat atattgtctt gacaacaata gcactgatat gcgggatagt cagcttagtc 1560 ctcgcctgct atataatgta caaacagaag gcccagcaaa aaacactgct gtggctcggg 1620 aacaacacct tagaccagat gagagcaacg acaaagatgt aa 1662 <210> 3 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> Newcastle disease virus V protein <400> 3 Met Ala Thr Phe Thr Asp Ala Glu Ile Asp Glu Leu Phe Glu Thr Ser   1 5 10 15 Gly Thr Val Ile Asp Ser Ile Ile Thr Ala Gln Gly Lys Pro Val Glu              20 25 30 Thr Val Gly Arg Ser Ala Ile Pro Gln Gly Lys Thr Lys Ala Leu Ser          35 40 45 Ile Ala Trp Glu Lys His Gly Asn Ala Asn Ala Ser Ala Val Gln Glu      50 55 60 Ser Ala Gly Glu Gln Asp Gln Pro Gly Gln Asn Gln Ala Ser Asn Ser  65 70 75 80 Asn Arg Ala Pro Gln Glu Glu Gly Pro His Ser Ser Gln Ala Gln Ala                  85 90 95 Ala Ala Gln Pro Gln Glu Asp Ala Asn Glu Thr Gln Leu Lys Thr Gly             100 105 110 Ala Ser Ser Leu Leu Ser Met Leu Asp Lys Leu Ser Asn Lys Ser         115 120 125 Ser Asn Ala Lys Lys Gly Pro Thr Ser Glu Pro Ser Thr Ala Gly Pro     130 135 140 Pro Pro Arg Glu Pro His Ser Gly Thr Ser Pro Thr Trp Gly Gln 145 150 155 160 Ser Gly Glu Gly Pro Val Gly Asp Gly Ala Pro Gly Arg Pro Arg Pro                 165 170 175 Trp Ile Pro Gly His Arg Arg Glu His Ser Ile Ser Trp Thr Met Glu             180 185 190 Gly Val Thr Thr Ile Ser Trp Cys Asn Pro Ala Cys Ser Pro Ile Arg         195 200 205 Ala Glu Pro Arg Gln Tyr Ser Cys Val Cys Gly Ser Cys Pro Ala Thr     210 215 220 Cys Arg Leu Cys Ala Gly Asp Asp Val Tyr Asp Gly Gly Asp Leu Thr 225 230 235 240 Glu Gly Lys Gln Asp                 245

Claims (8)

Oncytic viruses that have the ability to kill breast cancer cells (Oncolytic Virus)
Said tumor soluble virus is Newcastle disease virus (NDV) of Deposit No. KCTC13216BP isolated from domestic ducks;
The Newcastle Disease Virus of Accession No. KCTC13216BP comprises an F protein consisting of the amino acid sequence of SEQ ID NO: 1 and a V protein consisting of the amino acid sequence of SEQ ID NO: 3;
The nucleotide sequence encoding said F protein is SEQ ID NO: 2;
The fragment amino acid sequence of the F protein is ¹¹² ERQERL ¹¹⁷ ; And
Wherein the Newcastle Disease virus of Deposit No. KCTC13216BP is a vaccine type Class I virus. delete delete delete delete A pharmaceutical composition for treating or preventing breast cancer comprising the tumor-soluble virus of claim 1 as an active ingredient. delete A food composition for preventing or ameliorating breast cancer comprising the tumor-soluble virus of claim 1 as an active ingredient.

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