KR101521981B1 - Composition for inhibiting proliferation or engraftment of hematologic cancer stem cell - Google Patents

Abstract

The present invention relates to a composition for inhibiting proliferation or engraftment of blood cancer stem cells comprising a substance that inhibits the expression or activity of VEGFR (Vascular Endothelial Growth Factor receptor) -3 and a composition for inhibiting the proliferation or engraftment of blood cancer stem cells The present invention relates to a method for inhibiting the proliferation or engraftment of blood cancer stem cells. Further, the present invention relates to a pharmaceutical composition for treating blood cancer by inhibiting the proliferation or engraftment of Hodgkin's stem cells.

Description

TECHNICAL FIELD The present invention relates to a composition for inhibiting proliferation or engraftment of hematologic stem cells,

The present invention relates to a composition for inhibiting the proliferation or engraftment of hematopoietic stem cells comprising a substance that inhibits the expression or activity of VEGFR (Vascular Endothelial Growth Factor receptor) -3, To a method for inhibiting engraftment. Further, the present invention relates to a pharmaceutical composition for treating blood cancer by inhibiting the proliferation or engraftment of Hodgkin's stem cells.

Hematologic cancer is a cancer of the blood constituent, and refers to malignant tumors that occur in the blood, bone marrow, hematopoietic period, lymph node, lymphatic organs and lymphatic system. Recently, the incidence of multiple myeloma, lymphoma, myelodysplastic syndrome and myeloproliferative tumors has increased rapidly as well as leukemia in hematologic malignancies. According to 2010 National Cancer Registration Statistics (Ministry of Health and Welfare Central Cancer Registration Headquarters), 7937 patients with blood cancer account for about 4.6% of all cancers. In recent decades, the number of new patients with major hematologic cancer types has increased by 89% in non-Hodgkin's lymphoma, 122% in multiple myeloma, and 51% Far exceeding. In particular, acute myeloid leukemia (AML) is the most common form of acute leukemia in adults, accounting for 65% of acute leukemia. The prevalence and the mortality rate of hematologic malignancies continue to increase with the aging of the blood cancer, and the incidence of hematologic malignancies is expected to rise to 10% do.

Until now, it has been reported that the cause of cancer is mutation of normal cells caused by some external cause. Therefore, in order to treat cancer, the development and treatment of cancer-specific anticancer drugs have been conducted in search of genes specifically expressed or overexpressed in cancer cells different from normal cells. However, In patients, the above treatment alone did not completely remove the cancer cells. In recent years, researchers have been studying these patients, and cancer cells have been found to cause cancer in nature, and these cells have normal stem cell characteristics, and these cells have been reported to cause cancer, , It was named "cancer stem cell".

The paradigm of cancer stem cells is that tumors are caused by specific cell populations that exhibit various potential pluripotency and self renewal (BM Boman, MS Wicha, Cancer stem cells: a step toward the cure , J. Clin. Oncol. 26 (2008) 2795-2799). The presence of cancer stem cells was first demonstrated in acute myelogenous leukemia and in recent years it has been confirmed that stem cells are also present in solid tumor types by demonstrating the presence of cancer stem cells in general solid tumors including breast cancer (D. Bonnet, JE Dick, Oncogene 23 (2003) 7274 (1997) 730-737; M. Al-Hajj, MF Clarke, Self-renewal and tumor stem cells, Nat. Med. -7284). These cancer stem cells are also distinct groups and are also present in blood cancers such as acute myelogenous leukemia.

Conventional chemotherapy, chemotherapy, is one of the most common and effective treatment tools for treating cancer at the present time, either alone or in combination with other therapies (such as radiation therapy). The efficacy of drugs used to treat cancer in chemotherapy depends on the ability to kill cancer cells. However, there is a great limitation in the fact that these drugs can reverse not only cancer cells but also cancer cells as well as general cells due to cytotoxicity. In addition, cancer cells with resistance to drugs are generated, and cancer cells having such resistance can survive cancer treatment and eventually cause cancer recurrence. Further, chemotherapy may cause serious side effects such as nausea, vomiting, fluid retention, diarrhea, mild convulsions such as muscle spasm and bone marrow suppression with pneumonia, depression, convulsions, heart failure, thrombosis, embolism, hemorrhage, anemia, thrombocytopenia do.

Radiation therapy is an anti-cancer therapy that treats various cancers that occur in the human body by irradiating an appropriate amount of radiation to the affected part. Such a radiation therapy lowers the hematopoietic function of the human body and suppresses the immune system, . Furthermore, since cancer cells acquire resistance through repeated irradiation, such radiation therapy also has limitations in chemotherapy.

In addition, although treatments involving specific immunotherapies seem to have considerable potential in the treatment of blood cancer, such therapies are limited to a few known malignancy-associated antigens. Thus, improved methods are needed for the treatment of blood cancers such as B-cell leukemia, lymphoma and multiple myeloma.

80% to 90% of the cancer cells do not actually have a recurrence ability, but the remaining 10-20% of the cancer stem cells do not die when the cancer drug is administered and they recur. Therefore, in order to avoid recurrence and metastasis of cancer cells and to completely eliminate cancer, it is not merely to suppress and remove cancer cells, but to treat cancer stem cells without damaging normal stem cells, which is a problem of conventional chemotherapy, It is necessary to develop an anticancer agent that selectively removes cells.

However, studies on cancer stem cells have so far been limited, and its role in tumor formation and maintenance has not been clarified yet. To date, there have been few studies on anticancer drugs or natural product-derived extracts that directly target cancer stem cells.

Under these circumstances, the inventors of the present invention can inhibit the proliferation or engraftment of blood cancer stem cells specifically without affecting normal stem cells by inhibiting the expression or activity of VEGFR-3, It is possible to effectively treat blood cancer by inhibiting proliferation or engraftment of cells. Thus, the present invention has been completed.

It is an object of the present invention to provide a composition for inhibiting the proliferation or adhesion of hematopoietic stem cells, which comprises a substance that inhibits the expression or activity of VEGFR (Vascular Endothelial Growth Factor receptor) -3.

Another object of the present invention is to provide a method for inhibiting proliferation or engraftment of blood cancer stem cells, comprising the step of treating a blood cancer stem cell with a substance that inhibits the expression or activity of VEGFR (Vascular endothelial growth factor receptor) -3 .

It is another object of the present invention to provide a pharmaceutical composition for treating blood cancer, which comprises a substance that inhibits the expression or activity of VEGFR (Vascular endothelial growth factor receptor) -3.

In one aspect, the present invention relates to a composition for inhibiting the proliferation or adhesion of hematopoietic stem cells, comprising a substance that inhibits the expression or activity of VEGFR (Vascular Endothelial Growth Factor receptor) -3 .

In another embodiment, the present invention provides a method for inhibiting proliferation or engraftment of blood cancer stem cells, comprising the step of treating a blood cancer stem cell with a substance that inhibits the expression or activity of VEGFR (Vascular endothelial growth factor receptor) -3 .

Preferably, the blood cancer stem cells are leukemic stem cells (LSCs).

Also preferably, the substance that inhibits the expression of VEGFR-3 may be siRNA, shRNA or antisense nucleic acid that specifically binds to VEGFR-3.

Also preferably, the substance that inhibits the activity of VEGFR-3 may be an antibody or aptamer that specifically binds to VEGFR-3, or a compound of formula (I) or a salt thereof.

[Chemical Formula 1]

In another aspect, the present invention relates to a pharmaceutical composition for treating blood cancer, comprising a substance that inhibits the expression or activity of VEGFR (Vascular Endothelial Growth Factor receptor) -3.

Preferably, the substance that inhibits the expression of VEGFR-3 may be an siRNA, shRNA or antisense nucleic acid that specifically binds to VEGFR-3.

Also preferably, the substance that inhibits the activity of VEGFR-3 may be an antibody or aptamer that specifically binds to VEGFR-3, or a compound of formula (I) or a salt thereof.

[Chemical Formula 1]

Preferably, the blood cancer is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia , CLL), multiple myeloma (MM), or myelodysplastic syndrome (MDS).

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

The present invention is based on the fact that inhibition of the expression or activity of VEGFR-3 can inhibit proliferation or engraftment of hematopoietic stem cells, it is possible to inhibit the expression or activity of VEGFR-3 in cancer stem cells (CD34 ⁺ CD38 ^- VEGFR-3 cells) were injected to confirm the proliferation or engraftment result of cancer stem cells, it was confirmed that VEGFR-3 could be a new target for inhibiting the proliferation or engraftment of blood cancer stem cells . In addition, it has been confirmed that VEGFR-3 can be a new target for ultimately treating blood cancer through inhibition of proliferation or engraftment of cancer stem cells.

In a specific example of the present invention, VEGFR-3 overexpression was detected in cancer stem cells of patients with acute myelogenous leukemia (AML), and when the VEGFR-3 antagonist was treated, the frequency of cancer stem cells and expression of VEGFR-3 The change was confirmed. As a result, VEGFR-3 was overexpressed in cancer stem cells of patients with acute myelogenous leukemia (AML) (Fig. 1) and the expression of VEGFR-3 was decreased when treated with VEGFR-3 antagonist 2).

In another embodiment of the present invention, cancer stem cells treated with antagonist of VEGFR-3 were administered to mice in order to examine whether the inhibition of the expression or activity of VEGFR-3 could inhibit the proliferation or engraftment of blood cancer stem cells Respectively. After 18 hours of cancer stem cell injection, the frequency of cancer stem cell injected in bone marrow, peripheral blood and spleen was confirmed. In addition, the engraftment results were measured 6 weeks after cancer stem cell injection. As a result, the frequency of cancer stem cells and the expression of VEGFR-3 decreased in mice injected with cancer stem cells treated with VEGFR-3 antagonist, and the proliferation of cancer stem cells injected decreased markedly (FIG. 3). In addition, there was a significant decrease in overall cancer stem cells even in engraftment, but no significant difference was observed in normal stem cells (Fig. 4). Therefore, it was confirmed that inhibition of VEGFR-3 could inhibit proliferation or engraftment selectively on cancer stem cells without affecting normal stem cells.

The above experiments were performed on established humanized mouse models (Figure 5).

Taken together, the present invention first confirmed that VEGFR-3 is overexpressed in cancer stem cells of blood cancer patients, and confirmed that VEGFR-3 affects proliferation and engraftment of cancer stem cells. Further, by confirming that the antagonist of VEGFR-3 can inhibit the proliferation and engraftment of cancer stem cells, it has been confirmed that VEGFR-3 can be a new therapeutic target of hematological cancer therapy.

Accordingly, the present invention provides a composition for inhibiting proliferation or engraftment of blood cancer stem cells, a method of using the composition, and a pharmaceutical composition for treating blood cancer, which comprises a substance that inhibits the expression or activity of VEGFR-3.

As used herein, the term "blood cancer" refers to a cancer caused by a component constituting blood, and refers collectively to malignant tumors occurring in blood, bone marrow, hematopoietic period, lymph node, lymphatic organs and lymphatic system. More specifically, the present invention relates to a method for treating or preventing leukemia, acute lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, myelofibrosis, multiple myelopathy, lymphoma, non- Aplastic anemia, and aplastic anemia. However, the present invention includes various blood cancers due to the proliferation or engraftment of blood cancer stem cells.

As used herein, the term "cancer stem cell" refers to a cancer cell that has the characteristics of normal stem cells and constantly produces cancer cells having various phenotypes. Among these, "leukemia stem cell" is one of cancer stem cells, which means stem cells that cause all leukemia such as acute lymphoblastic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia and chronic lymphoid leukemia. Cancer stem cells are a cause of recurrence of cancer, and they are distinguished from general cancer cells. In order to cure cancer, cancer stem cells must be removed. In order to remove cancer stem cells, cancer stem cells can be removed in vivo by a method of inhibiting the proliferation of cancer stem cells as well as the destruction by direct cutting, chemical or radiotherapy, and inhibiting in vivo engraftment.

The term "proliferation inhibition" refers to the inhibition of proliferation, a state in which cells divide and the number of cells increases. Preferably, the present invention can inhibit the expression or activity of VEGFR-3 to inhibit the proliferation of cancer stem cells. In a specific example of the present invention, the expression or activity of VEGFR-3 is inhibited and proliferation of blood cancer stem cells is inhibited As shown in Fig.

The term "engrafting" means that the administered cells adhere to tissues in vivo when the cells are administered in vivo. Preferably, the term "cancer stem cell" as used herein means that cancer stem cells can be established in vivo to produce cancer cells, and more preferably means that blood cancer stem cells can be fixed on bone marrow to produce blood cancer cells. In this context, the term "engraftment inhibition" as used herein means preventing cancer stem cells from settlement in vivo, and preferably preventing blood cancer stem cells from colonizing bone marrow. In a specific example of the present invention, it has been proved that the inhibition of the expression or activity of VEGFR-3 can inhibit the engraftment of blood cancer stem cells.

As used herein, the term "VEGFR-3" refers to one of the receptors for vascular endothelial growth factor, which may be membrane-bound receptors (mbVEGFR) or soluble receptors (sVEGFR) have. VEGFR-3 performs the function of lymphangiogenesis and has seven immunoglobulin-like domains, one transmembrane spanning region and an intracellular domain, the extracellular portion, the tyrosine kinase domain (tyrosine-kinase domain). The VEGFR-3 gene and its protein information can be found in National Center for Biotechnology Information (NCBI). More specifically, the base sequence of the VEGFR-3 gene is registered as NM_002020, and the amino acid sequence of the protein is registered as NP_002011. VEGFR-3 is known as one of the receptors for vascular endothelial growth factor, but its specific relationship with blood cancer stem cells is not known at all.

The substance that inhibits the expression or activity of VEGFR-3 means a substance that blocks the synthesis pathway and the reaction pathway of VEGFR-3. For example, it may be siRNA, shRNA or antisense oligonucleotide which can complementarily hybridize to mRNA of VEGFR-3 gene. It is preferable that the siRNA, shRNA or antisense nucleic acid specifically binds to the mRNA base sequence of the VEGFR-3 gene and does not specifically bind to the base sequence of the other nucleic acid material. However, the synthesis pathway and the reaction pathway of VEGFR- But are not limited to, those that block substances.

Here, complementary binding means that the antisense nucleic acid is sufficiently complementary to hybridize selectively to the mRNA target of the VEGFR-3 gene under a predetermined hybridization or annealing condition, preferably physiological condition, Quot; is meant to include both substantially complementary and perfectly complementary, and preferably means completely complementary.

For example, it may be siRNA that is sufficiently complementary to selectively hybridize to the mRNA target of the VEGFR-3 gene of the present invention. The term "siRNA" means a short double-stranded RNA capable of inducing RNAi (RNA interference) phenomenon through cleavage of a specific mRNA. A sense RNA strand having a sequence homologous to the mRNA of the target gene, and an antisense RNA strand having a complementary sequence. siRNA can be provided as an efficient gene knockdown method or as a method of gene therapy since it can inhibit the expression of a target gene.

The siRNA is not limited to a complete pair of double-stranded RNA portions that are paired with each other, but is paired by a mismatch (the corresponding base is not complementary), a bulge (no base corresponding to one chain) May be included. The total length is 10 to 100 bases, preferably 15 to 80 bases, more preferably 20 to 70 bases. The siRNA terminal structure is capable of blunt or cohesive termini as long as it can inhibit the expression of the target gene by the RNAi effect. The adhesive end structure can be a structure having a 3 'end protruding structure and a 5' end protruding structure. The number of protruding bases is not limited. The siRNA may be a small RNA (for example, a natural RNA molecule such as a tRNA, a rRNA, or a viral RNA, or an artificial RNA molecule) at a protruding portion at one end within a range that can maintain the effect of suppressing the expression of a target gene, . ≪ / RTI > The siRNA end structure does not need to have a truncation structure on both sides, and may be a step-loop structure in which the terminal region of the double-stranded RNA is connected by linker RNA.

The siRNA used in the present invention may be a complete form having polynucleotide pairing itself, that is, a form in which siRNA is directly synthesized in a test tube and introduced into a cell through two transformation processes, Single chain oligonucleotide fragments and their reverse-phase trefoil can be derived from single-stranded polynucleotides separated by a spacer, for example, a form of siRNA expression vector or PCR-derived siRNA prepared so that the siRNA is expressed in a cell The expression cassette may be introduced into the cell through a transformation or infection process. The determination of how to prepare siRNAs and introduce them into cells or animals may depend on the objective and the cellular biological function of the target gene product.

Alternatively, it may be shRNA that is sufficiently complementary to selectively hybridize to an mRNA target of the VEGFR-3 gene of the present invention. The term "shRNA" is intended to overcome the disadvantages of high cost biosynthetic cost of siRNA, short-term maintenance of RNA interference effect due to low cell transfection efficiency, and the like. It is known that adenovirus, lentivirus and plasmid expression vector system , And it is well known that such shRNA is converted into an siRNA having an accurate structure by the siRNA processing enzyme (Dicer or Rnase III) existing in the cell to induce silencing of the target gene .

As another example, it may be antisense nucleic acid that is sufficiently complementary to selectively hybridize to the mRNA target of the VEGFR-3 gene of the present invention. The term "antisense nucleic acid" refers to DNA or RNA or a derivative thereof containing a nucleic acid sequence complementary to the sequence of a specific mRNA, and is characterized by binding to a complementary sequence in mRNA and inhibiting translation of mRNA into a protein . The antisense sequence of the present invention refers to a DNA or RNA sequence complementary to the mRNA of the VEGFR-3 gene and capable of binding to the mRNA of the VEGFR-3 gene, and is useful for translation, translocation into the cytoplasm, , Maturation, or any other overall biological function. The length of the antisense nucleic acid is 6 to 100 bases, preferably 8 to 60 bases, more preferably 10 to 40 bases.

In the case of antisense RNA, it can be synthesized in vitro in a conventional manner and administered in vivo, or the antisense RNA can be synthesized in vivo. One example of the synthesis of antisense RNA in vitro is the use of RNA polymerase I. One example of allowing antisense RNA to be synthesized in vivo is to allow the antisense RNA to be transcribed using a vector whose recognition site (MCS) origin is in the opposite direction. Such antisense RNAs are preferably made such that translation stop codons are present in the sequence so that they are not translated into the peptide sequence.

The compositions of the present invention may comprise one or more of siRNA, shRNA or VEGFR-3 antisense nucleic acids of VEGFR-3, as well as additional substances that inhibit the proliferation of cancer stem cells in a patient, or siRNA or antisense Agents that promote the uptake of nucleic acid molecules, such as liposomes (U.S. Patent Nos. 4,897,355, 4,394,448, 4,231,877, 4,231,179, 4,753,788, 4,673,567, 4,247,411, 4,814,270) or with one lipophilic carrier among a number of sterols including cholesterol, cholate and deoxycholic acid. The antisense nucleic acid may also be conjugated to a peptide that is absorbed by the cell. Examples of useful peptides include peptide hormones, antigens or antibodies, and peptide toxins.

The composition of the present invention may be used alone, but may be used alone or in combination with other agents such as radiotherapy or chemotherapy, cell growth arrest or cytotoxic agents, antibiotics, alkylating agents, antimetabolites, hormones, An anti-EGFR agent, an anti-angiogenic agent, a paroxetine transferase inhibitor, an anti-angiogenesis inhibitor, a metallo-matrix protease inhibitor, a telomerase inhibitor, a tyrosine kinase inhibitor, an anti-growth factor receptor substance, , ras-raf signaling pathway pathway inhibitors, cell cycle inhibitors, other cdk inhibitors, tubulin binders, topoisomerase I inhibitors, topoisomerase II inhibitors, etc.).

The composition of the present invention may be administered together with a pharmaceutically acceptable carrier. In oral administration, a conjugate, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, In the case of injections, a buffer, a preservative, an anhydrous agent, a solubilizer, an isotonic agent, a stabilizer and the like may be mixed. In the case of topical administration, a base, excipient, lubricant and preservative may be used. Formulations of the compositions of the present invention may be prepared in a variety of ways by mixing with pharmaceutically acceptable carriers as described above. For example, oral administration may be in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. In the case of injections, unit dosage ampoules or multiple dose forms may be prepared.

As used herein, the term administration refers to the introduction of a composition of the present invention to a patient in any suitable manner, and the route of administration of the composition of the present invention may be administered via any conventional route so long as it can reach the target tissue have. But are not limited to, oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, intranasal administration, intrapulmonary administration, rectal administration, intraperitoneal administration, intraperitoneal administration, Do not.

The effective dose range of the composition of the present invention can be varied depending on the sex, body surface area, kind and severity of disease, age, sensitivity to the drug, administration route and discharge rate, administration time, treatment period, target cell, And may be readily determined by one of ordinary skill in the art.

As a specific example of the present invention, a composition for inhibiting the proliferation or adhesion of hematopoietic stem cells may include an antibody capable of specifically binding to the VEGFR-3 protein, a platemma or a compound of the formula (I) or a salt thereof.

[Chemical Formula 1]

The term "antibody ", as it is known in the art, refers to a specific protein molecule directed against an antigenic site. For the purpose of the present invention, an antibody refers to an antibody that specifically binds to a VEGFR-3 protein, which is a marker of the present invention. Such an antibody can be obtained by cloning VEGFR-3 gene into an expression vector according to a conventional method, To obtain a VEGFR-3 protein that is encoded by the VEGFR-3 gene and can be prepared from the obtained VEGFR-3 protein by a conventional method. Also included are partial peptides that can be made from the VEGFR-3 protein, and the partial peptides of the invention include at least 7 amino acids, preferably 9 amino acids, more preferably 12 amino acids. The form of the antibody of the present invention is not particularly limited, and a polyclonal antibody, a monoclonal antibody or a part thereof having antigen binding ability is included in the antibody of the present invention and includes all immunoglobulin antibodies. Furthermore, the antibodies of the present invention include special antibodies such as humanized antibodies.

The antibody used in the composition for inhibiting the proliferation or adhesion of hematopoietic stem cells of the present invention is not limited to a complete form having two full-length light chains and two full-length heavy chains, ≪ / RTI > A functional fragment of an antibody molecule refers to a fragment having at least an antigen-binding function, and includes Fab, F (ab ') 2, F (ab') 2 and Fv.

The term " platamer "refers to a nucleic acid molecule having binding activity to a given target molecule. The aptamer can inhibit the activity of a predetermined target molecule by binding to a predetermined target molecule. The aptamer of the present invention may be a linear or cyclic nucleic acid such as RNA, DNA, modified nucleic acid or a mixture thereof. The aptamers are such as those produced by phage display or monoclonal antibodies ("MAbs"), which are capable of specifically binding to a selected target and modulating the activity of the target, Through combination, the aptamer can block the ability of the target to function. An aptamer, as produced by an in vitro selection process from a pool of random sequence oligonucleotides, is generated over 100 or more proteins, including growth factors, transcription factors, enzymes, immunoglobulins, and receptors. A typical aptamer is 10-15 kDa in size (30-45 nucleotides), binds to its target with an affinity of sub-nanomolar, and is distinguished from closely related targets (e.g., Do not typically bind to other proteins from the same gene family). Specifically on a target selected through the same type of binding interactions (e.g., hydrogen bonding, electrostatic complementation, hydrophobic contact, steric exclusion) leading to affinity of the aptamer and specificity in the antibody-antigen complex It can bind, regulate the activity of the target, and block the ability of the target.

The "platamer" of the present invention is an platamer having a binding activity to VEGFR-3. Preferably, the aptamer of the present invention binds to VEGFR-3 and inhibits the binding of VEGF to VEGFR-3, thereby inhibiting the activity of VEGFR-3.

In one aspect, the present invention relates to a method for treating blood cancer stem cell, comprising administering a composition for inhibiting proliferation or engraftment of blood cancer stem cells, which comprises a substance that inhibits the expression or activity of VEGFR-3, A method for inhibiting proliferation or engraftment of blood cancer stem cells.

More specifically, a composition comprising a substance capable of inhibiting the expression or activity of VEGFR-3 at an mRNA level or a protein level may be treated in vitro, such as blood cancer stem cells or tissues including blood cancer stem cells, Treating the affected animal, and examining the proliferation and engraftment of blood cancer stem cells.

The term " blood cancer treatment "as used herein means treatment of blood cancer through reduction of blood cancer stem cells in the body by inhibiting proliferation or engraftment of blood cancer stem cells, or reduction of blood cancer stem cells. Preferably means inhibiting the proliferation or engraftment of blood cancer stem cells by inhibiting the expression or activity of VEGFR-3 to treat blood cancer.

In a specific example of the present invention, when the blood cancer stem cells treated with a substance that inhibits the expression or activity of VEGFR-3 were injected into a mouse, it was confirmed that the cancer stem cells were decreased and the cancer stem cells were also decreased. ≪ RTI ID = 0.0 > cancer. ≪ / RTI >

The present invention provides a composition for inhibiting the proliferation or adhesion of hematopoietic stem cells comprising a substance that inhibits the expression or activity of VEGFR (Vascular Endothelial Growth Factor receptor) -3 in order to inhibit proliferation or engraftment of blood cancer stem cells By using this, it is possible to treat blood cancer by inhibiting the proliferation or engraftment of blood cancer stem cells.

FIG. 1A shows fluorescence activated cell sorter (FACS) analysis results of VEGFR-3 expression in mononuclear cells and cancer stem cells of normal controls and leukemia patients. "In R1" is the frequency of cancer stem cells in the mononuclear cells, "In CD3 + CD38- cells" is the number of cancer cells in the tumor, "AML" is the acute myeloid leukemia, "Normal" is the normal control, Shows VEGFR-3 expression in cancer stem cells.
FIG. 1B shows statistical results of fluorescence activated cell sorter (FACS) analysis on expression of VEGFR-3 in cancer stem cells of normal controls and leukemia patients. "In MNCs" refers to mononuclear cells, and "In CD3 + CD38-" refers to cancer stem cells.
FIG. 1c shows the result of confirming abnormal cancer cells present in blood through H & E (Hematoxylin and Eosin) staining. "Normal PB" refers to the peripheral blood in the normal control group, "AML PB" refers to the peripheral blood of patients with acute myeloid leukemia, and "M4 / M5" represents the type of leukemia.
Figure 1d shows the degree of erythropoiesis in the bone marrow (stomach) and spleen (below) of mice injected with CD34 + CD38-VEGFR-3 + cells and with CD34 + CD38-VEGFR- . &Quot; CD34 + CD38- cells "refers to cancer stem cells," VEGFR-3 + "refers to cancer stem cells with VEGFR-3, and" VEGFR- 3- "refers to cancer stem cells without VEGFR-3.
FIG. 1E is a result of confocal microscopy of fluorescence immunohistochemistry of co-expression of VEGFR-3 in cancer stem cells. "DAPI" is stained with DAPI (4 ', 6-diamidino-2-phenylindole), and a white arrow indicates coexpression.
FIG. 2 shows the frequency of monocyte and cancer stem cells and the expression of VEGFR-3 in cancer stem cells when MAZ51, a VEGFR-3 antagonist, was not treated or treated with monocyte and cancer stem cells in leukemia patients . The results of "Leukemic cells" in the CD34 + CD38-VEGFR-3 cell line derived from leukocyte patients without MAZ51 and "MAZ51 treated" in the CD34 + CD38-VEGFR-3 cell line derived from leukocyte treated with MAZ51 .
Figure 3 shows the frequency of human CD34 + cells in bone marrow, peripheral blood and spleen after 18 hours of injection into the tail vein of human CD34 + CD38-VEGFR-3 cells treated or not treated with MAZ51, homing efficiency. "In BM" represents bone marrow, "Leukemic cells" represents results in mice injected with human CD34 + CD38-VEGFR-3 cell group not treated with MAZ51, and "MAZ51 treated" + CD38-VEGFR-3 < / RTI > cells.
Figure 4 shows the engraftment results of the CD34 + CD38-VEGFR-3 cell line after 6 weeks of injecting human CD34 + CD38-VEGFR-3 cell line treated and not treated with MAZ51 into the tail vein of mice. "MAZ treated" indicates the results in mice injected with human CD34 + CD38-VEGFR-3 cells treated with MAZ51 and "Leukemic cells ≪ / RTI >
FIG. 5A shows the results of homing with bone marrow by injecting CD34 + CD38-VEGFR-3 + cells in a humanized mouse model, and confirming viability by FACS and immunostaining. "CD34 + CD38-VEGFR-3 +" represents cancer stem cells with VEGFR-3.
FIG. 5B is a DNA finger print result that confirms whether human cells derived from bone marrow are injected from a patient by injecting CD34 + CD38-VEGFR-3 + cells in a humanized mouse model. "CD34 + CD38-VEGFR-3 +" represents cancer stem cells with VEGFR-3.
FIG. 5c shows the results of hematoxylin & eosin immunostaining of the endosteal niche in the humanized mouse model to determine the extent of both mouse and human cells.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example  1. Acute Myeloid leukemia ( AML ) Overexpression of VEGFR-3 in Mononuclear Cells and Cancer Stem Cells of Patients

1-1. FACS  analysis

Monocyte and cancer stem cells (LSC) were isolated from the blood of 42 AML patients and 11 healthy volunteers and the expression of VEGFR-3 was confirmed in cancer stem cells. As a specific experimental method, the blood was mixed with DPBS (Dulbecco's Phosphate Buffered Saline) on a 15 ml solution having a density of 1.077 and capable of separating mononuclear cells, followed by centrifugation (2500 rpm, 20 minutes). The mononuclear cells in the buffy coat layer were pipetted and washed with DPBS. About 100,000 cells were placed in DPBS in 200 ul and mixed well. Then, each antibody, anti-CD34, anti-CD38, and anti-VEGFR-3 were placed in a tube and exposed in a refrigerator for 20 minutes. After exposure, the cells were washed and observed a certain degree of expression change with FACS caliber.

As a result, the frequency of CD34 + CD38-cancer stem cells among normal mononuclear cells was only 0.7%, while the frequency of CD34 + CD38-cancer stem cells among mononuclear cells in acute myeloid leukemia patients was as high as 10.9%. In addition, Expression of VEGFR-3 in CD34 + CD38- cells was only about 1.7%, whereas expression of VEGFR-3 in cancer stem cells in patients with acute myeloid leukemia was as high as 26.1% (FIGS. 1A and 1B).

1-2. H & E ( Hematoxylin & Eosin ) dyeing

Peripheral blood of AML patients and healthy volunteers was dropped on a glass slide, stained, dried and stained with H & E dye. Cells were observed in the ideal zone.

Abnormal cancer cells existing in blood were confirmed by H & E staining, and a large number of abnormal cancer cells corresponding to M4 / 5 of leukemia type were found in blood of AML patients (FIG. 1C).

1-3. Red blood cell formation ( erythropoiesis ) analysis

The mice injected with human CD34 + CD38-VEGFR-3 + cells and mice injected with human CD34 + CD38-VEGFR-3 cells were excised from the thigh bones, Gross examination was performed to confirm the degree of erythropoiesis.

When the cells were injected with human cells, the whites were more visible in the mouse than in the red part because the cells were injected with human cells. CD34 + CD38-VEGFR-3 + cells were injected into the bone marrow and spleen, + CD38-VEGFR-3-cells, the erythropoiesis decreased and the spleen became larger than the bone marrow of the mice injected (Fig. 1d). Therefore, it was confirmed that VEGFR-3 induces carcinogenesis of cancer stem cells.

1-4. Confocal  Microscope analysis

To confirm the coexpression of CD34 + CD38- and VEGFR-3 in cancer stem cells of AML patients as shown in the FACS analysis of Example 1-1, a rat anti-human VEGFR-3 antibody was conjugated with a FITC conjugated anti rat IgG 2nd antibody And PE conjugated anti-human CD34 antibody were simultaneously fluorescently immunostained and confirmed by Confocal microscope (confocal scanning, Carl Zeiss microscopy).

As a result, it was confirmed that VEGFR-3 was coexpressed in cancer stem cell CD34 + CD38-, and it was once again confirmed that VEGFR-3 was expressed in cancer stem cells of AML patients (FIG.

Example  2. VEGFR -3 Antagonist  In treatment, monocytic cells and cancer stem cells from leukemia patients VEGFR -3 expression changes

30 uM / ul of MAZ51 (VEGFR-3 antagonist) (Calbiochem Cat no 676492) and DMEM medium (Dulbecco's Modified Eagle Medium) were added to a tissue culture flask, And 5 × 10 ⁵ to 1 × 10 ⁶ cells / 3 μl of cancer stem cells and AML patient's mononuclear cells) were cultured in a 5% CO 2 incubator incubator at 37 ° C. for 24 hours. After suspension, cells in suspension were collected, washed twice with DPBS, and then analyzed for expression of CD34, CD38, and VEGFR-3, which are markers of cancer stem cells, by FACS analysis (same as FACS method in Example 1-1) Were compared

As a result, the treatment of MAZ51, which is the most common VEGFR-3 antagonist so far commercialized, decreased the incidence of monocyte and cancer stem cells in AML patients and decreased the expression of VEGFR-3 in cancer stem cells (Fig. 2).

Example  3. VEGFR -3 Antagonist  In treatment, the estrogenicity of cancer stem cells in mice ( homing efficiency ) Check frequency

A group of human CD34 + CD38-VEGFR-3 cells not treated with MAZ51 or a group of human CD34 + CD38-VEGFR-3 cells treated with MAZ51 were inoculated into 7-week old mice (NOD scid gamma, NSG, NOD . Cg-Prkdcscid Il2rgtm1Wjl / SzJ immunodeficient mice, JACKSON LABORATORY) (5 mice administered in 1 cage). The prepared cells (about 500,000) were placed in 200 ul DPBS, wrapped in foil, and transported. After injection, blood was checked for hemostasis, wiped with alcohol, and placed in a cage. The mice were held for 18 hours to confirm the frequency of human CD34 + cells in bone marrow, peripheral blood, and spleen.

As a result, the homing efficiency to the bone marrow of the MAZ51-treated cell group was significantly lower than that of the group not treated with MAZ51. These results demonstrate that inhibiting the expression or activity of VEGFR-3 reduces the engraftment of cancer stem cells, which may affect the proliferation or recurrence of cancerous tumor cells (FIG. 3).

Example  4. VEGFR -3 Antagonist  In treatment, the expression of cancer stem cells in mice Viability  Confirm

Each antibody against CD34, CD38, and VEGFR-3 was attached to a human CD34 + CD38-VEGFR-3 cell line that did not receive MAZ51 or a human CD34 + CD38-VEGFR-3 cell line that was cultured with MAZ51 treatment and classified using FACS moflo respectively. The sorted cells were washed well and 100,000 to 500 million cells were injected into 200 μl of DPBS and irradiated mice. After the injection, it was wiped with alcohol cotton and checked for hemostasis and placed in CAGE. The engraftment may be different depending on the condition of the patient's blood, and the engrafting ability may be different according to the frequency of the cancer stem cells even if the same patient's blood is used. Therefore, the state of the mouse should be followed up for at least 5 weeks after the injection, It was possible to confirm the engraftment of blood stably. After that, the left thigh bones of dead mice were cut, and unnecessary tissues such as muscles were discarded, and only one bone was put into 4% PFA (paraformaldehyde) solution. Two days later, the bones were placed in a formic acid solution and exposed for one week until the bones were wiggled. After that, the tap water was washed with water one day, and the tissue was put into a paraffin block and cut to confirm whether or not human cells were present (Fig. 5c). As soon as the other thigh bones were applied with muscles, the cells in them were taken out, Was cut with a knife and DPBS was put in a 1 ml syringe and the syringe was pushed out to make the water penetrate the inside of the bone to obtain cells. The collected cells were then red blood cell (RBC) lysis and red blood cells were prepared and processed for FACS.

As a result, in the case of MAZ51 treatment, there was a significant decrease in the overall cancer stem cell count (statistically significant at 0.09) in the results after 6 weeks after injection (4.3% in MAZ51 treated & 11.0% in non MAZ51 treated In the CD45 group (known as the leukemic group), which is known to be an abnormal cancer cell, the frequency of CD34 + CD38- was significantly reduced to 22.4% in the MAZ51-treated group and 9.4% in the MAZ51-treated group. On the other hand, the CD45 group (12.4% vs. 15.9%) showed no significant difference in normal stem cells. These results demonstrate that inhibition of VEGFR-3 expression or activity is likely to act only on cancer stem cells without affecting normal stem cells (FIG. 4).

Example  5. Establishment of humanized leukemia mouse model

The humanized mouse model allows human blood cells to exist in vivo in mice. Establishment and validation of a humanized mouse model enables experiments on the engraftment of cancer stem cells and interaction with neighboring cells. Establishment of a humanized mouse model can lead to a stable advancement of cancer stem cell experiments This is a basic system that allows you to

The mice used in Examples 1-3, Examples 3 and 4 were prepared and established as follows in the human leukemia mouse model.

In order to establish a humanized leukemia mouse model, human blood cells isolated in immunodeficient NSG mouse tail vein were injected into the cells of the remaining mice by irradiation. Mice already treated with radiation have rapidly accepted human blood cells and confirmed that human cells are present in whole body after several weeks.

5-1. FACS  Analysis and Immunostaining

Expression of CD34 stem cell markers and the presence of CD45 blood cells were confirmed by FACS (same as FACS method of Example 1-1) and immunostaining in blood cells.

For immunostaining, 5 ul of FITC CONJUGATED ANTI-HUMAN CD45 antibody was placed in a 200 μl cell-incubated tube for 2 hours, followed by fluorescence microscopy with DAPI. However, APC conjugated mouse CD45 was used for mouse cells and FITC conjugated anti human CD45 was used for human cells to eliminate fluorescence interference (FLASE positive).

As a result, the injected human CD34 + CD38-VEGFR-3 + cells were homing with bone marrow and viability (Fig. 5A).

5-2. DNA finger print  Confirm

DNA fingerprinting was used to confirm whether the cells of the human bone marrow were from the patient. For this purpose, the patient's cells were collected and genomic DNA was extracted using a kit. The injected and engrafted cells were taken out of the thigh bone of the mouse and DNA was extracted using a kit. The DNA fingerprinting method is a method commonly used to identify the origin of a fingerprint, and a general PCR method is used which is interpreted as a band type by gel loading. The amount of DNA was 20 ug and the amount of loading did not exceed 2 ㎕. The gel concentration was 2%, and it was slowly dropped to 150 volts, so that the band did not clump and the difference could be confirmed.

As a result, it was confirmed that the primary cells of the patient and the cells in the bone marrow were the same patient's DNA. The universal UFP fingerprint genomic DNA kit (JK BioTech.Ltd.Cat No. JK090016) was used for primers 2, 5, and 13.

As a result, one patient showed complete agreement and one patient showed partial agreement. The reason for the partial match seems to be that it is mixed with mouse cells. However, this is a question of the frequency of engraftment, and it is a clear result that human cells are engaged. Thus, it was confirmed that the primary cells of the patient and the cells of the bone marrow were the same patient's DNA (Fig. 5B).

5-3. Immunostaining confirmation

The intramedullary part was cut directly and immunohistochemically stained to investigate the extent of both mouse and human cells.

As a result, it was confirmed that CD34 + CD38-VEGFR-3 + cells had fewer cells, whereas human cells were spread (in a yellow solid line in FIG. 5C) The bone marrow of the injected mice, on the other hand, was confirmed to have a large number of mouse cells (FIG. 5c).

Claims (12)

An siRNA, an shRNA, or an antisense nucleic acid that inhibits the expression of VEGFR (Vascular endothelial growth factor receptor) -3; or
An antibody that inhibits the activity of VEGFR-3, an abtamer, or a compound of formula (I)
Compositions for inhibiting proliferation or engraftment of blood cancer stem cells:
[Chemical Formula 1]

The composition according to claim 1, wherein the blood cancer stem cell is a leukemic stem cell.
delete delete An siRNA, an shRNA, or an antisense nucleic acid that inhibits the expression of VEGFR (Vascular endothelial growth factor receptor) -3; or
An antibody that inhibits the activity of VEGFR-3, an umbrella or a compound of formula
Treating the hematopoietic stem cell in vitro or in an animal other than human.
A method for inhibiting proliferation or engraftment of blood cancer stem cells.
[Chemical Formula 1]

[Claim 6] The method according to claim 5, wherein the hematopoietic stem cell is a leukemic stem cell.
delete delete delete delete delete delete

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