JP2008275397A - METHOD FOR DETERMINING PROLIFERATIVE POTENTIAL PRIOR TO CULTURE OF gammadeltaT CELL AND KIT FOR THE SAME - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for easily, speedily, and precisely determining whether γδT cells can be cultured and proliferated or not. <P>SOLUTION: After γδT cells are collected and dyed from peripheral blood mononuclear cells of collected blood, the percentage of cells (CD45-positive and CD27-negative or CCR7-negative cells: TemraγδT cells, and CD45-positive and CD27-positive or CCR7-positive cells: TnaiveγδT cells) which has expressed a CD45RA marker expressed in the surfaces of γδT cells is measured. In the case where the percentage is 40% or less, it is determined that γδT cells of an amount required for treatments can be cultured. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for determining proliferative ability for γδT cell culture.

  The most common cause of death among Japanese people is malignant neoplasm (hereinafter referred to as cancer). Cancer treatment methods include surgical treatment, chemotherapy, and radiation therapy, which are said to be the three major therapies, but there are problems such as difficulty in treatment and side effects.

  In recent years, immune cell therapy has been performed as a new cancer treatment in addition to the above three major therapies, and this immune cell therapy has few problems such as treatment difficulties and side effects like the above three major therapies. Attention has been paid.

  Among these immunocell therapies, there is a therapy that activates T cells or NK cells, called LAK (lymphokine-activated killer) therapy. There are two types of T cells: αβ T cells that express αβ type TCR (T cell receptor) and γδ T cells that express γδ type TCR. The mainstream of LAK therapy that activates T cells is mainly αβ T cells Is a therapy that activates Here, αβ T cells are T cells mainly responsible for acquired immunity.

  On the other hand, γδT cells are cells responsible for innate immunity. Recently, it has been found that it has cytotoxic activity (non-specific activity) against cancer cells (for example, Non-Patent Document 1 and Non-Patent Paper 2), and γδTLAK immunity utilizing the strong antitumor activity of γδT cells. Research of the therapy is performed (for example, nonpatent literature 3, nonpatent literature 4, and nonpatent literature 5).

Human γδT cells and Tumor Immunotherapy (J. Clin. Exp. Hematopathol, 2006, 46 (1): 11-23) Vγ9Vδ2 Tcell Response to Colon Carcinoma Cells (The Journal of Immunology, 2005, 175: 5481-88) Phosphostim-Activated γδT Cells Kill Autologous Metallic Relative Cell Carcinoma (The Journal of Immunology, 2005, 174: 1338-1347) Effector γδT cells as immunotargets of zoletronic acid in multiplemyeloma (Lukemia, 2005, 19 (4): 664-70) Induction of γδ T-lymphocyte effector functions by bisphosphonate zoltronic acid in cancer patients in vivo (Blood, 2003, 102 (6)): 2310-1

  However, since γδT cells are usually present in only 1 to 5% in peripheral blood, even if a small amount of blood is collected to activate and / or proliferate γδT cells, sufficient purity and number of cells are ensured for treatment. It may not be possible. In this case, if the amount of blood collected from the patient is increased in order to ensure sufficient purity and the number of cells for treatment, there is a problem that a great burden is placed on the patient. Furthermore, γδ T cells may not grow at all due to various treatments previously received in some patients.

Therefore, in cancer patients, in order to obtain a satisfactory antitumor effect by γδTLAK immunotherapy, recovery of a sufficient amount of activated γδT cells from the patient's peripheral blood is the key to successful treatment, and a small amount of patient peripheral It is possible to receive appropriate γδTLAK immunotherapy cells that reduce the burden on the patient and can be expected to have a therapeutic effect if the adaptability of this treatment can be determined by determining the degree of proliferation of γδT cells using blood. Become.
However, at present, whether or not a treatment using γδT cells can be performed can be understood only after actually culturing, and it usually takes about 1 to 2 weeks until it can be determined whether or not the culture (treatment) is possible.
In addition, since the culture is performed by an engineer, there is a problem that the result of the culture changes depending on the technology possessed by each engineer, and there is no reliable method for determining whether or not the culture is possible. There is a problem.

  The present invention has been made in view of the above circumstances, and provides a technique capable of judging the proliferation ability of γδT cells before they are actually cultured, simply, quickly and accurately.

  The present inventors focused on CD (CD: cluster of differentiation; 45RA), a surface marker expressed on the surface of γδT cells, and determined whether or not γδT cells can be cultured later by the expression rate. The present invention has been completed by finding out what can be done.

Specifically, the present invention relates to the following (1) to (5).
(1) In a γδT cell population in blood, a step of measuring the ratio of cells expressing the γδT cell proliferation ability determining factor on the surface, and whether the ratio of the expressed cells is 40% or more of the whole A method for determining a pre-proliferation ability in γδ T cell culture, comprising the step of:
(2) The method for determining γδT cell proliferation ability according to (1), wherein the cell expressing the γδT cell proliferation ability determining factor is a CD45RA positive cell;
(3) a terminally differentiated memory γδ T cell that is a CD45RA positive and CD27 negative or CCR7 negative cell; Temraγδ T cell; and b) a naive γδ T cell that is a CD45RA positive and CD27 positive or CCR7 positive cell; TnaiveγδT A method for determining the proliferation ability of γδT cells according to (2), characterized by comprising:
(4) A step of collecting peripheral blood mononuclear cells from peripheral blood, and the peripheral blood mononuclear cells are stained with anti-γδTCR, anti-CD45RA, anti-CD27 and / or anti-CCR7 antibody, and surface markers on γδT cells in blood A) a terminally differentiated memory γδ T cell that is a CD45RA positive and CD27 or CCR7 negative cell; a Temraγδ T cell; and b) a naive γδ T cell that is a CD45RA positive and CD27 or CCR7 positive cell; A step of measuring the total ratio of Tnaive γδ T cells, and a step of determining that the cells can be proliferated when the ratio of the Temra γδ T cells and the Tnaive γδ T cells is 40% or less. Method for determining culture growth ability;
(5) A kit for performing the determination method according to (4).

Conventionally, whether or not γδT cells can be cultured has been determined by actually culturing over about 1 or 2 weeks, but according to the technique of the present invention, a small amount of blood is collected without culturing. It is possible to determine whether or not the cells can be proliferated on the same day of blood collection, that is, whether or not treatment with γδT cells is possible. In addition, since it is not necessary to perform culture, the cost can be reduced as a medical provider.
Further, as a patient's position, it is possible to determine whether or not γδT cell therapy can be adopted without burdening the body and wasting time due to a small amount of blood collected.

  From the above, by using the diagnostic method of the present invention, it is possible to determine the γδ T cell proliferative ability of a patient that conventionally required about 1 to 2 weeks on the day of blood collection. The determination accuracy is also greatly improved. Further, the cost can be reduced in terms of time and cost.

  Hereinafter, embodiments of the present invention will be described.

The method for determining the proliferation ability of γδT cells according to the present invention (hereinafter also referred to as the method of the present invention) measures the proportion of surface markers that are γδT cell proliferation assessment factors of γδT cells from a small amount of blood, and according to the measured value, It is determined whether or not it is possible to grow as many γδ T cells as necessary for treatment such as cancer treatment.
Here, the growth ability determining factor refers to the cell surface marker CD45RA.

  That is, the method of the present invention measures the proportion of CD45RA-positive cells in a blood γδT cell population, and determines whether or not γδT cells can be cultured thereafter by determining whether the proportion is 40% or less. Judgment.

  If the proportion of CD45RA positive cells is 40% or less, preferably about 30% or less, it can be determined that the number of cells required for treatment can be cultured, and conversely, 40% or more, preferably about 50% or more. Even if the cells are actually cultured, it is determined that the culture is poor and that γδT cell therapy cannot be performed.

  As a specific technique, peripheral blood mononuclear cells are collected from a small amount of blood collected from a specimen, and γδ T cells in the blood are detected. The expression rate of CD45RA is measured in the γδ T cell population.

Here, the γδ T cell population is divided into two populations: a CD45RA positive naive γδ T cell and a CD45RA negative memory γδ T cell.
Further, as CD45RA positive γδ T cells, CD45RA positive and CD27 negative or CCR7 negative (CD45RA + CD27− / CCR7−) cells, which are terminally differentiated memory γδT cells (hereinafter referred to as TemraγδT cells), CD45RA positive and CD27 positive or CCR7 positive ( CD45RA + · CD27 + / CCR7 +) cells are divided into two subsets of naive γδ T cells (hereinafter referred to as Tnaive γδ T cells).

  CD45RA negative γδ T cell cells include effector memory γδ T cells (hereinafter referred to as Temγδ T cells) which are CD45RA negative and CD27 negative or CCR7 negative (CD45RA-CD27- / CCR7-) cells, CD45RA negative and CD27 positive or CCR7 positive (CD45RA). -CD27 + / CCR7 +) cells are classified into two subsets of central memory γδT cells (hereinafter referred to as TcmγδT cells).

The present inventors have found that γδT cells cannot be proliferated if the ratio of these two types of CD45RA positive cells, that is, TemraγδT cells and TnaiveγδT cells, is large in the blood γδT cell population.
As a specific criterion, when the proportion of CD45RA positive cells is 40% or more, it is determined that γδT cells cannot proliferate in the blood of the patient to an amount necessary for effective disease treatment. To do.

Examples of diseases to which treatment can be applied include intractable diseases such as cancer and viral infections.
Although it can use with respect to various cancer as a kind of cancer which can be determined by this invention, Preferably it is used suitably for solid cancer. Specific examples include the following cancers.
Melanoma, colon cancer, colon cancer, granulosa cell tumor, lung cancer, breast cancer, liver cancer, kidney cancer, cervical cancer, prostate cancer, stomach cancer, pancreatic cancer, ovarian cancer, etc.

The amount of blood used for measurement may be about 100 μL to 3 mL. The amount to be used may be about 100 μL to 500 μL, for example, when red blood cells are hemolyzed using whole blood and stained with a surface marker antibody reagent depending on the state of blood at the time of measurement. When staining and measuring using peripheral blood mononuclear cells, it may be about 2 to 3 mL.
With this amount of blood collected, the burden on the patient can be reduced. Further, according to the method of the present invention, even if a patient has a small amount of γδT cells in blood, it is possible to quickly determine whether or not the culture is possible from such a small amount of blood.

Moreover, the measurement of an expression rate can be used suitably if it is a normal technique.
That is, the determination method of the present invention can be easily performed with a flow cytometer such as FACS (Fluorescence Activated Cell Sorting) using, for example, an antibody reagent.
The determination method procedure of the present invention will be specifically described below.

A. When judging using peripheral blood mononuclear cells First, blood is collected from a patient. The blood collection method can be performed using a commonly used syringe, vacuum blood collection tube, or the like. The amount of blood collected may be about 2 to 3 mL.

  Next, peripheral blood mononuclear cells (hereinafter referred to as PBMC) are collected from the peripheral blood. A conventionally known method can be used as a sampling method, but it can be easily performed by using, for example, a density gradient centrifugation method. As the amount, it is only necessary to obtain about 1 × 10 5 to 2 × 10 5 cells.

  Next, the collected PBMC is added to an Eppendorf tube or the like in which an antibody reagent as a surface marker has been dispensed in advance and stained. As the antibody, anti-γδ T cell receptor (γδTCR), anti-CD27, anti-CCR7, and anti-CD45RA antibody reagents are used. Furthermore, staining may be performed using an anti-CD3 antibody.

  The ratio of the CD45RA positive cells of the cells stained with the antibody reagent is measured using a flow cytometer. For patients whose proportion is 40% or more, it can be predicted that γδT cells cannot be expanded to the number of cells necessary for treatment.

B. When judging from hemolysis from whole blood.
First, blood is collected from a patient in the same manner as in the case of using the PBMC. The amount of blood collected may be about 100 μL to 500 μL of whole blood. Subsequent procedures may be performed in the same manner as A described above.

  Hereinafter, the present invention will be described in detail using examples. However, it goes without saying that the present invention is not limited to this.

First, blood was collected from 15 subjects.
1. Preparation of test sample (method of separating PBMC from whole blood)
Blood was collected by heparin blood sampling, and 5 mL blood was collected.
As each sample, PBMC was separated and collected by Ficoll (Ficoll (registered trademark), manufactured by Sigma).
The following four antibody reagents were used to measure a subset of γδ T cells in each sample.
1. Anti-TCRVγ9-FITC (anti-γδTCR antibody) (manufactured by Beckman Coulter)
2. Anti-CD27-PE (manufactured by Beckman Coulter)
3. Anti-CD3-ECD (manufactured by Beckman Coulter)
4). Anti-CD45RA-PC5 (Becton Dickinson)
5 μL of 1 to 4 antibody reagents were dispensed into each Eppendorf tube. Next, 2 × 10 ^ 5 PBMCs obtained earlier were put into a tube into which the antibody had been dispensed, shaken with Vortex, and after shaking, left at 4 ° C. for 30 minutes in the dark.
Thereafter, 500 μL of PBS (phosphate buffered saline) containing 0.5% PFA (Paraformaldehyde) was added and shaken with Vortex.

2. Preparation of test sample (method of lysing red blood cells from whole blood)
100 μL of the whole blood obtained previously was put in a tube into which antibody had been dispensed, and shaken with Vortex. After shaking, the mixture was allowed to stand for 15 minutes in a dark place at room temperature (about 20 ° C. to 25 ° C.). Thereafter, 500 μL of Optilyse C (manufactured by Beckman Coulter) was added, and the mixture was further shaken with vortex and allowed to stand at room temperature in the dark for 10 minutes.
Subsequently, 500 μL of PBS was added and shaken with Vortex. Thereafter, centrifugation was performed at 450 G for 5 minutes using a centrifuge (manufactured by Beckman Coulter).
The supernatant after separation was discarded, and 500 μL of PBS containing 0.5% PFA was added to the cell fraction and shaken with Vortex.

3. Measurement with a flow cytometer Or 2. The surface marker (CD45, CD27, CD3, TCR) was measured using a flow cytometer for each subject sample prepared by the above method.

4). Verification by culture Thereafter, bisphosphonate-based bone metabolism improving agent zoledronic acid sodium hydrate (Zometa (registered trademark), Novartis Pharma) and IL-2 were added to PBMC in the same manner as before. Then, γδT cells were cultured.

5. Results The results of the culture are shown in Table 1.

Table 1 shows the subject group A with poor growth and the subject group B with good growth according to the culture results.
In Table 1, the proportion of CD45RA positive γδ T cells in all γδ T cells in all 9 cases in Group A is 40% or more, and the proliferation rate after culturing of γδ T cells is also low. In one case, although the proportion of CD45RA positive γδT cells is lower than 40% (16%), the proliferation rate remains low (28%), which is due to the constitutional factors of the subject. It was judged.
On the other hand, the proportion of CD45RA positive γδT cells in all γδT cells in all 8 cases in group B is 40% or less, and the proliferation rate of γδT cells is also very high.

  From the above results, in most cases, the group in which the proportion of CD45RA positive cells is 40% or more cannot grow γδT cells to the level required for treatment, and conversely, in the group of 40% or less, all It has been shown that it can proliferate efficiently and be used for therapy.

As described above, the γδ T cell proliferating ability determination method of the present invention can perform culture prediction in a much shorter period than before and has higher accuracy than before. As a result, it is possible to promptly determine whether or not a treatment using γδT cells can be performed, so that the start of treatment can be accelerated, and even if it is determined that treatment cannot be performed, treatment other than γδT cell therapy can be considered promptly. it can. Thereby, the quality of treatment can also be improved.

Claims (5)

Measuring the proportion of cells expressing the γδ T cell proliferative capacity determining factor on the surface in the γδ T cell population in the blood;
Determining whether the ratio of the expressed cells is 40% or more of the total;
A method for determining a pre-proliferation ability in γδ T cell culture. 2. The method for determining the proliferation ability of γδ T cells according to claim 1, wherein the cells expressing the γδ T cell proliferation ability determining factor are CD45RA positive cells. The CD45RA positive cells are
a) terminally differentiated memory γδ T cells that are CD45RA positive and CD27 negative or CCR7 negative cells;
b) naive γδ T cells that are CD45RA positive and CD27 positive or CCR7 positive cells; Tnaive γδ T cells;
The method for determining the proliferation ability of γδ T cells according to claim 2, comprising: Collecting peripheral blood mononuclear cells from peripheral blood;
Staining the peripheral blood mononuclear cells with anti-γδTCR, anti-CD45RA, anti-CD27 and / or anti-CCR7 antibody and measuring the proportion of surface markers in γδT cells in the blood;
In said measurement, a) terminally differentiated memory γδ T cells that are CD45RA positive and CD27 or CCR7 negative cells;
b) measuring the sum of the proportion of CD45RA positive and CD27 positive or CCR7 positive cells naive γδ T cells; Tnaive γδ T cells;
Determining that proliferation is possible when the ratio of the Temraγδ T cells to the Tnaiveγδ T cells is 40% or less;
A method for determining γδ T cell culture proliferation ability, comprising: A kit for performing the diagnostic method according to claim 4.