KR101236983B1 - A method for the diagnosis of cancer by identifying subtypes of peripheral blood dendritic cells - Google Patents

Abstract

The present invention relates to a method for separating dendritic cells from peripheral blood and a method for diagnosing cancer by analyzing subtypes of the isolated dendritic cells. More specifically, the method for diagnosing dendritic cells by removing the lineage cells using antibodies of the lineage cells and identifying a new cell region in addition to the plasma and myeloid dendritic cells in the isolated dendritic cells It is about.

Description

A method for the diagnosis of cancer by identifying subtypes of peripheral blood dendritic cells}

The present invention provides a new method for separating dendritic cells from peripheral blood monocytes and defining cancer patient-specific cell distribution, thereby using a small amount of peripheral blood to observe abnormalities of immune cell subtypes in cancer patients in a simple manner. And a method for diagnosing cancer using the same.

Dendritic cells were first discovered in the skin by Langerhans in 1868 and functioned as adjuvant cells by Cohn and Steinmann in 1973. In the 1990s, it was discovered that its function as a powerful professional antigen presenting cell (APC) has been found to play an important role in immune induction and immunomodulation. Dendritic cells in the body make up only about 0.3% of the total circulating leukocytes, but are composed of heterogeneous populations with a phenotype distinct from macrophages. Dendritic cells are differentiated in that they are powerful antigen presenting cells, unlike B cells or macrophages, which have relatively weak antigen presenting ability. Dendritic cells are present in the blood and are difficult to isolate, which has been a major obstacle for clinical application. However, clinical research has been actively carried out with the development of methods for differentiating dendritic cells using blood cells or stem cells in vitro. It is done. Dendritic cells are known to be derived from a variety of lineages, but usually dendritic cells are derived from IL-4 and GM-CSF stimulated unicellular or monocyte precursors (CD34 ⁺ cells) and used in vaccine protocols.

In addition, the method of separating dendritic cells from peripheral blood has been reported through several experiments. One of the most recently published methods is the use of cell surface proteins to perform several experiments on remaining cells except cells of a specific lineage. This experiment basically requires expensive equipment such as a flow cytometer and a cell separator. Do. In addition, even when using a cell separator, the distribution of dendritic cells itself is very low, so it takes a long time to separate and the separated cells die.

Research using dendritic cells has recently been in the spotlight with a new cancer treatment method called the development of cell therapy. However, the study of dendritic cells is limited to induction of differentiation and maturation using monocytes. In addition, mononuclear leukocyte differentiation dendritic cells are known to be the most common in the application of cell therapeutic agents. However, there are many problems in applying mononuclear leukocyte-induced dendritic cells to cancer treatment. The biggest problem is that dendritic cells using mononuclear leukocytes extracted from blood are rarely present in less than 10% of dendritic cells in the body.

In order to overcome the above problems, a method for separating and detecting dendritic cells present in peripheral blood rather than dendritic cells induced differentiation in vitro is required, and a study for analyzing them is urgently needed.

Accordingly, the present inventors have made a diligent effort to reduce the above problems, and developed a method of separating lineage cells from peripheral blood rather than mononuclear leukocyte-derived dendritic cells using magnetic force rather than flow cytometer. In other words, by separating monocytes from peripheral blood and removing lineage cells such as T lymphocytes, mononuclear leukocytes, B lymphocytes, and natural killer cells, we establish a method by which dendritic cells of all currently known subtypes can be separated. When the dendritic cells were isolated, the present invention was completed by confirming that specific cells were observed only in cancer patients.

Accordingly, the present invention seeks to provide a method for isolating dendritic cells from peripheral blood and analyzing subtypes of the cells.

In another aspect, the present invention is to provide a simple method for diagnosing cancer consisting of identifying a cell region that appears specifically in cancer patients.

The present invention also provides a composition for diagnosing cancer using dendritic cells composed of specific antibodies.

In order to solve the above problems, the present invention provides a method for providing information for diagnosing cancer in the dendritic cells of the family other than the plasma and myeloid dendritic cells in the dendritic cells separated from the peripheral blood.

The present invention also provides a method for separating dendritic cells by removing T lymphocytes, monocytes, B lymphocytes, and natural killer cells from peripheral blood using beads immobilized with antibodies against CD3, CD14, CD19, and CD56. do.

The present invention also provides a composition for diagnosing cancer using dendritic cells comprising antibodies to CD123 and CD11c.

Furthermore, the present invention provides a kit for cancer diagnosis using dendritic cells comprising the composition.

Advantages and features of the present invention, and methods of achieving the same will become apparent with reference to the following detailed description and embodiments in conjunction with the accompanying drawings. However, the present invention is not limited to the examples disclosed below, but may be implemented in various different forms.

As described above, according to the present invention has laid a foundation for effectively separating dendritic cells from peripheral blood. In addition, the present invention can confirm the change of the peripheral blood dendritic cells of cancer patients can be a basis for studying the tumor immune cells abnormalities in cancer patients, a new method has been established inexpensive and simple in the diagnosis of cancer.

Therefore, the present invention is not only based on the study of peripheral blood dendritic cells but also expected to be a great force in the study of peripheral blood dendritic cell subtypes of cancer patients, and can be used as a simple and useful method for cancer diagnosis. There will be.

Figure 1 shows the cells corresponding to CD3 ⁺ (T lymphocytes), CD14 ⁺ (monocytes), CD19 ⁺ (B lymphocytes), CD56 ⁺ (natural killer cells) by separating monuclear cells from blood provided from healthy donors. The results of partitioning dendritic cells by confirming the distribution of and identifying the cells with high expression of HLA-DR, the antigens involved in the rejection of primary tissue grafts, were shown. In addition, the results showed that plasma dendritic cells with high CD123 expression and bone marrow dendritic cells with high CD11c expression were shown. Myeloid lineage dendritic cells were confirmed to be further divided into CD1c ⁺ , CD141 ⁺ , CD16 ⁺ .
Figure 2 removes the cells by binding microbeads to the respective antibodies corresponding to CD3 ⁺ (T lymphocytes), CD14 ⁺ (monocytes), CD19 ⁺ (B lymphocytes), CD56 ⁺ (natural killer cells) The distribution of dendritic cells is then measured. It was confirmed that the distribution of cells with high HLA-DR expression and the distribution of plasma-based dendritic cells or bone marrow-based dendritic cells were similar to those of FIG. to be.
Figure 3 is a result of confirming the distribution of dendritic cells in the blood provided from healthy men in their 60s by the method of FIG.
4 is a result of confirming the distribution of dendritic cells in the blood provided from cancer patients in their 60s by the method of FIG.
5 is a result of confirming the distribution of dendritic cells in the blood provided from cancer patients in their 60s by the method of FIG.
Figure 6 is a result of confirming the distribution of dendritic cells in the peripheral blood provided 3 months after surgery of cancer patients measured in Figure 5 by the method of FIG.
FIG. 7 is a graph comparing and analyzing changes of dendritic cells in peripheral blood provided from 40 healthy men and 80 cancer patients.

The present invention relates to a method for providing information for diagnosing cancer comprising the steps of identifying new cell regions in addition to plasma and bone marrow dendritic cells in dendritic cells isolated from peripheral blood.

The dendritic cells may be isolated by partitioning cells with high expression of the major tissue rejection protein, except for T lymphocytes, B lymphocytes, mononuclear leukocytes, and natural killer cells present in monocytes in peripheral blood using flow cytometry.

Preferably, the dendritic cells separated from the peripheral blood are separated by removing the lineage cells using beads immobilized with the antibodies of CD3, CD14, CD19 and CD56 in peripheral blood monocytes.

The antibody of CD3 is used to remove T lymphocytes in peripheral blood, the antibody of CD14 to remove B lymphocytes, the antibody of CD19 to remove mononuclear leukocytes, and the antibody of CD56 to remove natural killer cells. do. The cells after removal of the lineage cells are cells with high expression of HLA-DR, an antigen involved in rejection of main tissue graft, and are dendritic cells.

The study of dendritic cells is limited to induction of differentiation and maturation using mononuclear leukocytes. In most cases, dendritic cells are derived from mononuclear leukocytes. However, dendritic cells using mononuclear leukocytes are rarely present in 10% or less of dendritic cells in the body, and a large amount of blood is required to differentiate into dendritic cells. In addition, mononuclear leukocyte-induced dendritic cells show a great difference in cell surface protein factors of peripheral blood dendritic cells in the body.

In order to solve the problems of the above mononuclear leukocyte-induced dendritic cells, the present invention isolated the dendritic cells present in the peripheral blood rather than the differentiation-induced dendritic cells in vitro.

By "antibody" is meant herein a specific protein molecule directed against the antigenic site. Antibodies used in the present invention include monoclonal or polyclonal antibodies, immunologically active fragments (eg, Fab or (Fab) ₂ fragments), antibody heavy chains, humanized antibodies, antibody light chains, genetically engineered single chain Fv molecules, Chimeric antibodies and the like. In addition, since CD3, CD14, CD19, and CD56 expressed in the cells of the present invention are known proteins, the antibodies used in the present invention can be prepared by conventional methods well known in the field of immunology using the known proteins as antigens. .

For example, polyclonal antibodies can be produced by the method of injecting a protein antigen into an animal and collecting blood from the animal to obtain a serum comprising the antibody. Such antibodies can be prepared using various warm-blooded animals such as horses, cattle, goats, sheep, dogs, chickens, turkeys, rabbits, mice or rats.

Monoclonal antibodies can also be prepared using known fusion methods, recombinant DNA methods, and phage antibody library techniques.

In one embodiment of the invention, the antibody (antibodies of CD3, CD14, CD19 and CD56) can be immobilized on magnetic beads by a known method to remove lineage cells (Example 2).

The magnetic beads are not particularly limited as long as they have magnetic properties. For example, various metal oxides including silver, palladium, platinum, magnesium oxide, various metal oxides including indium oxide, silicon oxide, glass beads, and the like may be used. In the present invention, the cells of the lineage adhere to the magnet, and the dendritic cells pass through the magnet so as not to stimulate the magnetic force on the dendritic cells.

More specifically, T cells, B lymphocytes, mononuclear leukocytes, and natural killer cells, which are cells of a specific lineage other than dendritic cells among the monocytes present in the blood, may be treated using a single cell-specific surface protein factor. Antibody cocktails can be made. At this time, the antibody uses a biotin bound, and the antibody cocktail and monocytes can be incubated at 4 ° C. for 45 minutes. Antibodies that remain unbound can be washed away, anti-biotin beads can be combined, and passed through magnets to magnetically filter out cells of the lineage. The remaining cells after passing through the magnet can be subtyped by HLA-DR, CD11c, CD123, CD16, CD1c and CD141, which are cell-specific surface protein factors of dendritic cells.

The present invention also provides a method for further subdividing the types of dendritic cells in peripheral blood separated by the above method to analyze and identify subtypes of dendritic cells.

More specifically, it provides a method for separating the dendritic cells into plasma-based dendritic cells and bone marrow-based dendritic cells using the antibodies of CD123 and CD11c to confirm the presence of dendritic cells of the family other than plasma-based and dendritic cells. .

The method may further comprise analyzing the subtypes of the bone marrow-based dendritic cells obtained by the method by using the antibodies of CD1c, CD141 and CD16. When analyzing subtypes of dendritic cells, antibodies of cell-specific protein factors were used. The dendritic cells were identified using HLA-DR, which is highly expressed in all dendritic cells, and CD11c and CD123 were divided using different fluorescent antibodies in the compartment. In addition, myeloid-derived dendritic cells with high expression of CD11c were further subdivided using different fluorescent antibodies of CD1c, CD16, and CD141.

In the diagnosis method of cancer of the present invention, the subtypes of the dendritic cells are analyzed by flow cytometry to determine whether there are new cell regions other than the plasma and myeloid dendritic cells in the dendritic cells. Can be.

The new cell region is a region where CD123 and CD11c are not expressed.

More specifically, in an embodiment of the present invention, dendritic cells are separated by removing lineage cells using beads immobilized with CD3, CD14, CD19, and CD56 antibodies in human peripheral blood monocytes, and CD123 and the isolated dendritic cells. Plasma-based dendritic cells and myeloid-derived dendritic cells showing high expression of CD11c were isolated. Of the total dendritic cells, plasma-based dendritic cells is average 25% bone marrow series dendritic cells was found to be 75% is present, bone marrow-based dendritic cells are again divided into CD1c, CD141, CD16 CD1c ⁺ is 36%, CD141 ⁺ 5 % And CD16 ⁺ were found to account for 55% (Example 2, FIG. 2).

Dendritic cells were subdivided in the blood of healthy donors in the 60s, but in the cancer patients in the 60s, the divisions of plasma-type dendritic cells and myeloid-type dendritic cells were identified and were not included in the two dendritic cells. It was confirmed that this was present, an average of 20% or more was confirmed (Example 3, Figure 4). In the case of cancer patients, which showed a significantly higher number of cells in the plasma-derived dendritic cells and the bone marrow-derived dendritic cells, the distribution of dendritic cells at 3 months postoperatively was observed in healthy donors. Similar results were obtained with dendritic cell distribution (Example 4, Figure 6).

Based on the above experimental results, the present invention is to analyze the subtypes of dendritic cells isolated from peripheral blood monocytes, and as a result, if there is a region of cells that do not contain plasma-based dendritic cells and bone marrow-based dendritic cells diagnosed as cancer To provide a way.

There are many methods for diagnosing cancer, but it is very complicated, including having to undergo various tests to diagnose cancer. That is, a simple blood test or urine test is very difficult to diagnose cancer. However, the present invention can easily diagnose cancer even with a small amount of peripheral blood.

That is, the peripheral blood used for diagnosing cancer in the present invention is a very small amount of 25 to 35 ml. Preferably, 30 ml can accurately diagnose cancer.

Therefore, cancer patients can be easily diagnosed, and the progress of cancer treatment can be diagnosed by observing changes in immune cells after surgery or after cancer treatment.

In the present invention, all types of cancer can be diagnosed. Conventional cancer diagnostic methods are limited to the type of cancer that can be diagnosed by using a gene or protein expressed in a specific cancer cell, or by reacting with a specific compound, the present invention changes the immune cells that appear in the body when cancer occurs Invented by observing the invention is applicable to all cancers.

The present invention also relates to a cancer diagnostic composition using dendritic cells comprising the antibodies of CD123 and CD11c.

The diagnostic composition of the present invention may further comprise reagents known in the art for use in immunological assays in addition to antibodies specific for the protein. Reagents used in immunological analysis include labels, solubilizers, and detergents capable of producing a detectable signal. In addition, when the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator.

The label capable of generating a detectable signal enables qualitatively or quantitatively measuring the formation of an antigen-antibody complex, examples of which include enzymes, fluorescent materials, ligands, luminescent materials, microparticles, and redox molecules. And radioisotopes can be used. Enzymes include β-glucuronidase, β-D-glucosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, glycosidase, hexokinase, malate dehydrogenase, and glucose-6 Phosphate dehydrogenase, invertase and the like can be used. As the fluorescent substance, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, fluorine isothiocyanate and the like can be used. Examples of ligands include biotin derivatives, and light emitting materials include acridinium esters, luciferin, and luciferase. The microparticles include colloidal gold and colored latex, and the redox molecules include ferrocene, ruthenium complex, biologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone and hydroquinone. Radioisotopes include ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, ¹⁸⁶ Re, and the like. However, any of those that can be used in immunological assays other than those exemplified above may be used.

In another aspect, the present invention can provide a kit for diagnosing cancer using dendritic cells comprising the composition.

Hereinafter, the present invention will be described in more detail by way of examples. However, the embodiments are only for explaining the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, those of ordinary skill in the art. Will be self-evident.

Example 1: Identification of Dendritic Cell Sections and Detailed Cells in Monocytes of Peripheral Blood

Peripheral blood used in the present invention was provided from healthy donors in their 20s, healthy donors in their 60s and cancer patients in their 60s, and all experiments were performed based on 30 ml of blood.

Monocytes were isolated from the peripheral blood provided by healthy donors using density compartmentalization. Blood from peripheral blood was centrifuged at 3000 rpm for 20 minutes to separate plasma and blood cells. Prepare 5 ml of histopack-1077 (histopaque 1077) in a 15 ml tube, mix the plasma and blood cells in a 1: 1 ratio, slowly raise it, and centrifuge at 1500 rpm for 40 minutes. I could confirm it.

In monocytes, expression level was determined using T3 lymphocyte-specific cell surface protein CD3, mononuclear leukocyte-specific cell surface protein CD14, B lymphocyte-specific cell surface protein CD19, and natural killer cell-specific cell surface protein CD56. Except for these cells, a section of the region in which the expression of the main tissue transplant rejection protein was high was designated. Plasma-based dendritic cells and myeloid-derived dendritic cells showing high expression of CD123 and CD11c were isolated. Plasma-based dendritic cells were found to have an average of 20% of all dendritic cells and 80% of myeloid dendritic cells. Myeloid lineage dendritic cells were further subdivided into CD1c, CD141, and CD16. CD1c ⁺ was found to occupy an average of 33%, CD141 ⁺ 5%, CD16 ⁺ 60% (Fig. 1).

Example 2: Removal of Lineage Cells and Identification of Isolated Dendritic Cells

T lymphocytes, B lymphocytes, mononuclear leukocytes, and NK cells from the peripheral blood mononuclear cells of 20 healthy donors were effectively removed using biotin beads binding. As a result of measuring the expression, it was confirmed that the sections of T lymphocytes, B lymphocytes, monocytes, and natural killer cells were not identified. T lymphocytes, B lymphocytes, monocytes, and natural killer cells, CD3, CD19, CD14 and CD56, were incubated at 4 ° C for 45 min. At this time, the antibody was used that the biotin (biotin) is bound, and after cultivation once washed and put the anti-biotin beads (anti-biotin beads) again and incubated for 45 minutes at 4 ℃. After washing once, the cells were passed through a column maintained by magnetic force to remove cells corresponding to each lineage, and a desired cell was taken in a new tube.

As a result of confirming the high expression level of the main tissue transplant rejection protein using the flow cytometer, it was confirmed that the dendritic cell compartment was clearly entered. As a result of confirming the detail cells of dendritic cells, similar results were obtained before the removal of lineage cells. Among the total dendritic cells, 25% of the plasma-derived dendritic cells and 75% of the myeloid-derived dendritic cells were identified. The granularity of the myeloid dendritic cells was 36% for CD1c ⁺ , 5% for CD141 ⁺ and 55% for CD16 ^+. It was found to occupy% (FIG. 2).

Example 3: Comparison of Dendritic Cell Distribution between Healthy Donors in 60s and Cancer Patients in 60s

As a result of confirming the distribution of dendritic cells in the blood of healthy donors in their 60s using the method presented in Example 2, it was shown that there is no difference between the plasma dendritic cells and myeloid dendritic cells. It was also confirmed that there was no significant difference in the detail cells of myeloid dendritic cells (FIG. 3).

In patients with cancer in their 60s, the division of plasma-type dendritic cells and bone marrow-type dendritic cells was confirmed, and there were regions of cells not included in the two dendritic cells. 4).

Example 4 Comparison of Dendritic Cell Distribution and Postoperative Dendritic Cell Distribution in Cancer Patients

As a result of confirming the distribution of dendritic cells in cancer patients, a significantly higher number of cells were found in the cell region outside of plasma-type dendritic cells and bone marrow-type dendritic cells (FIG. 5), but 3 months after surgery. When the distribution of dendritic cells was again confirmed at the time point, the cells leaving the plasma-type dendritic cells and the bone marrow-type dendritic cells rapidly decreased, confirming the results similar to those of the healthy donor dendritic cell distribution (FIG. 6).

Example 5: Subtype comparison of dendritic cells in 40 healthy donors and 80 cancer patients

Subtypes of peripheral blood dendritic cells from healthy donors were analyzed for a total of 40 times. As a result, no subtype abnormalities of dendritic cells in cancer patients were found.

Subtypes of peripheral blood dendritic cells were analyzed in 80 cancer patients, and abnormalities of dendritic cell subtypes were observed in 95% or more, and the degree was also confirmed in 20% or more (FIG. 7).

Claims (9)

(a) separating dendritic cells by removing T lymphocytes, mononuclear leukocytes, B lymphocytes, and natural killer cells from peripheral blood using beads immobilized with antibodies to CD3, CD14, CD19, and CD56;
(b) identifying dendritic cells other than plasma and bone marrow lines that do not express CD123 and CD11c using the antibodies against CD123 and CD11c in the dendritic cells isolated in step (a); And
(c) identifying dendritic cells (HLA-DR + CD11c-CD123-CD16 +) in which CD123 and CD11c are not expressed and CD16 is expressed, using the antibody against CD16 in the dendritic cells isolated in step (b). Information providing method for diagnosing cancer comprising a. delete delete delete delete The method according to claim 1, wherein in the step (c), CD123 and CD11c are not expressed, and when dendritic cells (HLA-DR + CD11c-CD123-CD16 +) expressing CD16 are identified, cancer is diagnosed. To provide information for diagnosing cancer. According to claim 1, wherein in the step (a) peripheral blood is characterized in that 25 to 35 ml, information providing method for cancer diagnosis. delete delete

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