JP2002365286A - Cellular immunity detecting method and its application to physic - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a simple and convenient immunity monitoring system capable of examining the frequency of T cells specific to a plurality of antigen peptides even through the use of a relatively small amount of blood. SOLUTION: In this method for detecting the antigen-specific T cells, (1) peripheral blood mononuclear cells are collected, (2) the collected peripheral blood mononuclear cells are frequently stimulated by antigens, and (3) the T cells specific to the antigens in the stimulated peripheral blood mononuclear cells are detected. In the method, antigen presenting cells are not directly used when stimulating the peripheral blood mononuclear cells.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention belongs to the field of immunotherapy and relates to a method for assaying cell-mediated immunity and treatment using the same. T cells involved in cell-mediated immunity, particularly antigen-specific T cells, play an important role in eliminating cancer cells and virus-infected cells by living organisms. It is also known that antigen-specific T cells are involved in autoimmune diseases such as multiple sclerosis and rheumatoid arthritis. Antigen-specific T cells use the T cell receptor (TCR) to bind antigen peptides displayed on the target cell surface to the major histocompatibility complex (M
HC) specifically recognizes and reacts with the complex. Thereby, cytotoxic T cells (CTLs) are thought to damage cancer cells and virus-infected cells, or autoreactive T cells cause autoimmune diseases. Therefore, to properly carry out immunotherapy aimed at enhancing cytotoxic T cells (CTL) against cancer or viral infection and suppressing autoreactive T cells against autoimmune diseases, Detection of antigen-specific T cells is essential. By detecting and monitoring it, it is possible to determine what antigenic peptide is effective for the patient before treatment, or to confirm the effect on the patient of the antigenic peptide used for treatment after treatment.

[0002]

2. Description of the Related Art Conventional methods for detecting antigen-specific T cells include limiting dilution, ELISPOT, and MHC.
The tetramer method and the like are known. In the limiting dilution method, serially diluted T cells are cultured on a plate, and then reacted with target cells that have been pulsed and presented with an antigen.
Next, statistical processing is performed based on the number of positive wells in each plate to calculate the frequency of antigen-specific T cells.
(J. Immunol. 126: 1614-1620, 1981). Although this method is excellent in quantitative performance, usually, several dilution steps are set, and one plate (60-96 well) is prepared for each dilution step.
In order to analyze one sample, a large amount of T cell samples must be prepared. In this method, the antigen-specific T
In order to increase the detection sensitivity of cells, usually, lymphocytes pulsed with an antigen peptide a plurality of times once a week are used as target cells, so the operation is complicated.

[0003] In the ELISPOT method, T cells and an antigen are added to a plate coated with an antibody against a cytokine on the bottom of a well, and cultured. The cytokine produced by the T cell is trapped. And the number of positive spots were counted to calculate the frequency of specific T cells (J.Imm
unol. Methods, 128: 65, 1990). In this method, the background is high, and it may be difficult to determine whether the spot is positive or negative.

[0004] The MHC tetramer method uses an antigen peptide, M
After terminating the end of the HLA / antigen peptide complex in which HC and β2 microglobulin are associated in vitro, the antigen-specific T cells are stained using an MHC tetramer prepared by binding to fluorescently labeled streptavidin. Analyze with flow cytometer (FACS) (Scie
nce 275: 94, 1996). Although this method can analyze a large number of specimens, it is detected only by the binding between the TCR and the MHC tetramer, and thus can be detected including non-functional T cells. In addition, it is necessary to prepare an HLA / antigen peptide complex for each peptide.

[0005]

All of the conventional methods for detecting antigen-specific T cells involve the volume of blood to be collected,
It has some problems such as simplicity and detection sensitivity. Therefore, it is desired to establish a simple immune monitoring system capable of assaying the frequency of specific T cells for a plurality of antigen peptides using a relatively small amount of blood.

[0006]

The present invention provides a new detecting method as means for solving the above-mentioned problems. That is, the present invention relates to a method for detecting antigen-specific T cells, comprising: (1) collecting peripheral blood mononuclear cells; (2) stimulating the collected peripheral blood mononuclear cells frequently with an antigen; 3) A method for detecting T cells specific to the antigen in stimulated peripheral blood mononuclear cells, wherein the method does not directly use antigen-presenting cells for the stimulation of peripheral blood mononuclear cells in step (2). . The present invention also includes a method of detecting antigen-specific T cells in the step (3) using predetermined antigen-presenting cells.

Specifically, the present invention provides a method according to the present invention, wherein the antigen is an antigenic protein or an antigenic peptide derived from the antigenic protein;
The method of the present invention, which stimulates a plurality of times at 5-day intervals; the method of the present invention in which antigen-specific T cells are detected using the amount of cytokines produced by the T cells, preferably the amount of IFN-γ as an index; The method of the present invention in which stimulation is performed in a well of a microplate, preferably a U-bottom microplate; the number of peripheral blood mononuclear cells in a 96-well microplate is 2.5 × 10 ⁴ cells / well−2 × The number of peripheral blood mononuclear cells when using 10 ⁵ cells / well or 384-well microplate was 2.5 × 10 ⁴ cells / well-1 × 10
⁵ per well.

Conventionally, to detect antigen-specific T cells,
First, an antigen is pulsed into antigen-presenting cells derived from peripheral blood mononuclear cells collected from a patient, and T cells are stimulated using the pulses.
This was performed by a predetermined detection method. On the other hand, in the present invention, peripheral blood mononuclear cells are stimulated using the antigen itself without directly using antigen presenting cells. Therefore, the amount of blood used by the patient can be reduced, and the operation is not complicated.
Many antigen peptides can be evaluated at once.
Therefore, as another aspect, the present invention relates to a method for detecting an antigen that reacts with an antigen-specific T cell, comprising: (1) collecting peripheral blood mononuclear cells; and (2) collecting a plurality of the collected peripheral blood mononuclear cells. And (3) determine which antigen candidate has induced antigen-specific T cells. (2) antigen-presenting cells upon peripheral blood mononuclear cell stimulation in step (2) Which is not directly used. The present invention also includes a method of determining the induction of antigen-specific T cells in the step (3) using predetermined antigen-presenting cells.

Specifically, the present invention provides a method according to the present invention, wherein the antigen is an antigenic protein or an antigenic peptide derived from the antigenic protein;
The method of the present invention in which stimulation is performed a plurality of times at 5-day intervals; the method of the present invention in which the determination of antigen-specific T cell induction is determined by quantifying the amount of cytokine, preferably IFN-γ produced by the T cell. A microplate for frequent stimulation,
The method of the present invention, which is preferably performed in a well of a U-bottom microplate; the number of peripheral blood mononuclear cells in a 96-well microplate is 2.5 × 10 ⁴ cells / well−2 × 10
^The number of peripheral blood mononuclear cells when using ⁵ / well or 384-well microplates was 2.5 × 10 ⁴ cells / well.
The present invention relates to the method of the present invention, wherein the number of wells is 1 × 10 ⁵ cells / well.

[0010] If an antigen that reacts with antigen-specific T cells can be easily detected, a useful means for treating cancer, AIDS, and the like can be provided. DNA encoding such an antigen is referred to as DNA
It has been shown that initial immunization using a vaccine followed by a booster immunization successfully protected the AIDS virus infection (Science 292; 69-74, 2001). However, this only shows efficacy as a protective vaccine and does not prove that the DNA vaccine is useful as a therapeutic vaccine. In another aspect, the present invention provides a bespoke pharmaceutical composition specific for a disease of interest in a patient for treating the disease, comprising an antigen associated with the disease of interest in the patient.

[0011] In this aspect, preferably, an antigen associated with a target disease in a patient, wherein (1) peripheral blood mononuclear cells are collected from the patient, and (2) the collected peripheral blood mononuclear cells are used as the antigen. A peripheral blood mononucleus in step (2), wherein each of them is frequently stimulated with a plurality of antigen candidates suspected to be associated with the disease, and (3) which antigen candidate has induced antigen-specific T cells is determined. A tailor-made pharmaceutical composition specific to the disease of interest in the patient for treating the disease, comprising one or more antigens selected by a method that does not use antigen presenting cells directly in stimulating the spheres is there. Specifically, the composition of the present invention, wherein the antigen is an antigen protein or an antigen peptide derived from the antigen protein; the composition of the present invention, wherein the disease is cancer; the frequency of frequent stimulation of 2 or more times or 2-5 The composition of the present invention, which is stimulated a plurality of times at daily intervals; the determination of antigen-specific T cell induction is determined by the amount of cytokines produced by the T cells, preferably IFN
The present invention relates to a composition of the present invention which is performed by quantifying the amount of γ.

In a further aspect, the present invention is characterized in that a therapeutically effective amount of an antigen associated with the disease of interest in the patient is administered, wherein the patient can carry out personalized medical treatment specific to the disease of interest. It relates to a method for treating a disease. Alternatively, a plurality of antigens related to the target disease in the patient, wherein (1) peripheral blood mononuclear cells are collected from the patient, and (2) the collected peripheral blood mononuclear cells are considered to be related to the target disease. And (3) determine which antigen candidate has induced antigen-specific T cells. (2) antigen-presenting cells upon peripheral blood mononuclear cell stimulation in step (2) Administering one or more antigens selected by a method that does not directly use a method for treating the disease, which enables personalized medical treatment specific to the disease of interest in the patient. About. Preferably, in this aspect, it relates to a method of administering a therapeutically effective amount.

Specifically, the present invention relates to the method of the present invention, wherein the antigen is an antigenic protein or an antigenic peptide derived from the antigenic protein; the method of the present invention, wherein the disease is cancer; Or a method of the present invention in which stimulation is performed a plurality of times at intervals of 2 to 5 days; a method in which antigen-specific T cell induction is determined by quantifying the amount of cytokine, preferably IFN-γ produced by the T cell. Invention method.

[0014]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Antigen-specific T
A cell is a T cell that specifically recognizes and reacts with a complex of a major histocompatibility complex (MHC) and an antigen peptide presented on the surface of a target cell by using a TCR.
TL, helper T cells and the like. Hereinafter, a method for detecting antigen-specific T cells and a treatment method will be described. (1) Collection of peripheral blood mononuclear cells Collection of peripheral blood mononuclear cells was performed using Ficoll Concentration from collected peripheral blood.
This is performed by a specific gravity centrifugation method using a ray solution or the like. The collected peripheral blood mononuclear cells are cultured in a normal lymphocyte culture solution. Cultured peripheral blood mononuclear cells have the appropriate concentration in the lymphocyte culture solution,
Preferably, the cells are suspended at 5 × 10 ⁵ cells / ml and seeded on a microplate. The microplate used here is preferably a U-bottom plate. To each well, a culture solution is added in such an amount that the number of peripheral blood mononuclear cells becomes 2.5 × 10 ⁴ cells / well−2 × 10 ⁵ cells / well.

(2) Frequent stimulation of peripheral blood mononuclear cells by antigen The antigen that stimulates peripheral blood mononuclear cells includes the antigen itself or a part thereof. For example, when the antigen is a tumor antigen, a tumor-specific protein, that is, a tumor antigen protein or an antigen peptide derived from the antigen protein is included. As tumor antigen proteins, MAGE (Science
, 254: 1643, 1991), gp100 (J. Exp. Med., 179: 1).
005, 1994), MART-1 (Proc. Natl. Acad. Sci. US
A, 91: 3515, 1994), tyrosinase (J. Exp. Med., 17).
8: 489, 1993), MAGE-related proteins (J. Exp. Med.,
179: 921, 1994), β-catenin (J. Exp. Med., 183: 11).
85, 1996), CDK4 (Science, 269: 1281, 199).
5), HER2 / neu (J. Exp. Med., 181: 2109, 199)
5), p53 (mutant) (Proc. Natl. Acad. Sci. USA, 93:
14704, 1996), CEA (J. Natl. Cancer. Inst., 87:98).
2, 1995), PSA (J. Natl. Cancer. Inst., 89: 293,
1997), HPV (J. Immunol., 154: 5934, 1995), EB
V (Int. Immunol., 7: 653, 1995).

The tumor antigen peptide is produced by synthesizing a tumor antigen protein in a cell and then decomposing the tumor antigen protein in the cell by a proteasome. The generated tumor antigen peptide is converted into MHC class I antigen (HL) in the endoplasmic reticulum.
A antigen) to form a complex, which is carried to the cell surface and presented as an antigen. SAR as a tumor antigen peptide
T-1 (J. Exp. Med., 187: 277, 1998, WO 97/46)
676), SART-3 (WO 00/12701) and the like. If the amino acid sequence of an antigen peptide is known,
It can be synthesized by a usual method, for example, the Fmoc method.

"Stimulating peripheral blood mononuclear cells with an antigen"
In this specification, it means that an antigen is added to peripheral blood mononuclear cells and cultured to react with lymphocytes. Stimulation of peripheral blood mononuclear cells is usually performed in wells of a microplate, preferably a U-bottom microplate. The method of the present invention is characterized in that antigen presenting cells are not directly used for stimulating peripheral blood mononuclear cells. "Not used directly" means that antigen-presenting cells are not added directly to peripheral blood mononuclear cells when stimulating peripheral blood mononuclear cells.

Conventionally, as the number of times of stimulation, peptide stimulation is performed a plurality of times (about three times) once a week, and it is thought that it is not necessary to perform the stimulation more frequently. However, in the present invention, frequent peptide stimulation,
For example, it was clarified that antigen-specific T cells were favorably induced by performing the test multiple times at intervals of 2 to 5 days. Therefore, "frequent" in the frequent stimulation of the present invention means two times /
It means stimulating multiple times at a frequency of a week or more, or stimulating multiple times at 2-5 day intervals. Induction of antigen-specific T cells by frequent stimulation revealed that detection was possible in a relatively short period of time, that is, 日 13 days, unlike the conventional method. Therefore, “multiple times” in the present invention means that stimulation is performed at predetermined intervals within a range where the entire period of stimulation does not exceed 15 days, and preferably does not exceed 13 days. However, increasing the number of stimulations in a range exceeding 15 days does not affect the effects of the present invention. For example, if you stimulate every 3 days,
4 times including the first day stimulus. In addition, the method of performing frequent stimulation is performed by replacing a part of the culture supernatant with the same amount of a new lymphocyte culture solution containing the antigen.

(3) Detection of antigen-specific T cells in stimulated peripheral blood mononuclear cells Detection of antigen-specific T cells can be performed by various methods. For example, a method of reacting an appropriate target cell with an antigen-specific T cell after stimulation and quantifying a cytokine produced by the T cell, preferably IFN-γ. To prepare target cells, first, an HLA gene compatible with the HLA of antigen-specific T cells is introduced into various cell lines that do not express the antigen, and the cells are stably expressed. Instead of these cells, non-antigen-expressing cells into which an HLA gene cDNA expression plasmid compatible with HLA of antigen-specific T cells has been introduced can also be used as target cells. As target cells, antigen-specific T
Various cell lines that express HLA that is compatible with the HLA of the cells but do not express the antigen can also be used.

The prepared cells are pulsed with an antigen or an antigen peptide used for stimulating peripheral blood mononuclear cells to prepare antigen-presenting cells. Here, a peptide other than the antigen or antigen peptide used for stimulating peripheral blood mononuclear cells is pulsed to prepare target cells as a control. As a result, the amount of cytokine after the reaction with the cultured peripheral blood mononuclear cells was adjusted to E
It is quantified by the LISA method or the like, and the production of the target antigen-specific T cell is evaluated using the difference from the control.

(4) Determination of Whether Antigen Candidate Induced Antigen-Specific T Cells As another aspect, the present invention provides a method for detecting an antigen that reacts with antigen-specific T cells: (1) Peripheral blood Mononuclear cells were collected, (2) the collected peripheral blood mononuclear cells were stimulated frequently with a plurality of candidate antigens, respectively, and (3) which of the candidate antigens induced antigen-specific T cells was determined. The method is characterized in that antigen-presenting cells are not directly used for stimulating peripheral blood mononuclear cells in step (2). The collection of peripheral blood mononuclear cells and the frequent stimulation with the antigen candidate substance are the same as described above except that the number of stimulation operations increases by the number of candidate substances. The determination of which antigen candidate substance has induced antigen-specific T cells is performed in the same manner as described above, for example, by reacting appropriate target cells with the antigen-specific T cells after stimulation, whereby the T cells Producing cytokine, preferably IF
It is performed by a method of quantifying N-γ and comparing the amounts.

(5) Tailored Pharmaceutical Composition The tailored pharmaceutical composition of the present invention means a pharmaceutical composition capable of treating an individual patient in accordance with an individual disease to be treated. . For example,
The antigen contained in the pharmaceutical composition of the present invention may be HL in a patient.
It usually varies between patients depending on A and the effectiveness as a vaccine. According to the antigen detection method of the present invention, an antigen specific to a target disease in a patient can be detected, and thus, it is possible to prepare such a personalized pharmaceutical composition by using the antigen. In this context, the term "disease of interest in a patient" according to the present invention means such a disease in which certain antigens that differ between patients depending on the HLA in the patient can act as a vaccine and treat it.

The custom-made pharmaceutical composition of the present invention comprises:
Not only one, but a plurality of different corresponding antigens can be included. This is because not all cancer cells express the same tumor antigen in common, and taking into account that two or more different tumor antigen peptides are presented on one cancer cell. Then, another reason is that treatment using a plurality of different tumor antigen peptides is considered to be more effective. In fact, in melanoma, the effect of a peptide derived from a single tumor antigen alone was insufficient, so that the development of a cocktail preparation of multiple peptides has been attempted (Int. J. Cancer, 66: 162, 19
96, Int. J. Cancer, 67:54, 1996).

The custom-made pharmaceutical composition of the present invention comprises:
It can be a pharmaceutical composition for preventing or treating cancer, viral disease and infectious disease. The pharmaceutical composition containing the contained antigen peptide as an active ingredient can be administered together with an adjuvant so as to effectively establish cellular immunity, or can be administered in a particulate dosage form. As an adjuvant, literature (Clin. Microbiol. Rev., 7: 277-289, 1
994) can be applied. Also, a liposome preparation, a particulate preparation bonded to beads having a diameter of several μm, a preparation bonded to lipid, and the like can be considered. Examples of the administration method include intradermal administration, subcutaneous administration, and intravenous injection. The dosage of the tumor antigen peptide of the present invention or a derivative thereof in the preparation is determined according to the disease to be treated, the age of the patient,
It can be adjusted appropriately depending on the weight etc., but usually 0.0001
mg to 1000 mg, preferably 0.001 mg to 1000 mg, more preferably 0.1 mg to 10 mg, which is preferably administered once every few days to several months.

(6) Method of Treatment In a further aspect, the present invention is characterized by administering a therapeutically effective amount of an antigen associated with the disease of interest in the patient, wherein the medical treatment is specific to the disease of interest in the patient. To a method for treating said disease which enables In the present invention, personalized medicine is medicine adapted to an individual that can vary from individual to individual depending on the particular characteristics of the individual. According to the antigen detection method of the present invention, a unique antigen that is effective for treating a target disease in a patient can be detected. Therefore, a treatment method that enables such a personalized medical treatment can be performed by using the antigen. Alternatively, one or more antigens, which are antigens associated with a target disease in a patient and are selected by the detection method of the present invention, are administered, thereby enabling personalized medical treatment unique to the target disease in the patient. A method for treating the disease. By this method, more effective antigens can be clinically selected among the selected antigens. Therefore, the present invention also includes the above-mentioned treatment method, which comprises administering a therapeutically effective amount of the selected antigen. A "therapeutically effective amount" is a dose that allows treatment of the disease of interest, i.e., prevention of symptoms, or amelioration or elimination of symptoms that have developed, or reduction of the characteristic signs of such symptoms. Collection of peripheral blood mononuclear cells, frequent stimulation with the antigen candidate substance, and determination of whether the antigen candidate substance induced antigen-specific T cells are the same as described above. The dose of the antigen and the therapeutically effective amount can be appropriately adjusted depending on the disease to be treated, the patient's age, body weight, etc., but is usually 0.0001 mg to 1000 m
g, preferably 0.001 mg to 1000 mg, more preferably 0.1 mg
1010 mg, preferably administered once every few days to several months. The administration method is as described above.

[0026]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited thereto. Example 1 Antigen peptide specificity using antigenic peptide derived from Epstein-Barr virus
Detection of target T cells Many healthy individuals have a relatively high frequency of EB virus-specific T cell precursors due to past EB virus infection. Therefore, we attempted to detect EB virus-specific T cells from peripheral blood mononuclear cells (PBMCs) of blood-positive anti-EB virus antibody-positive healthy persons who had a history of EB virus infection. PBMC were separated from HLA-A24-positive healthy human peripheral blood by specific gravity centrifugation using Ficoll Conray solution. PBMC contains 45% RPMI1640 medium, 45% AIM-
V (manufactured by GIBCO BRL) and 10% FCS to which 100 U / ml interleukin-2 and 0.1 mM NEAA (manufactured by GIBCO BRL) were added. PBMC were suspended at 5 × 10 ⁵ cells / ml in a lymphocyte culture solution, and seeded on a 96-well U-bottom plate at 200 μl / well. The well contains an HLA-A24-restricted antigen peptide derived from Epstein-Barr virus (amino acid sequence: TYG
(PVFMCL) [J. Immunol. 158: 3325-3334, 1997] was added to a concentration of 10 μg / ml to stimulate the antigen peptide. Well 3
I prepared a point. PBMC in an incubator (37 ° C, 5% CO ₂ )
And cultured. Three days after the start of the culture, 100 µl of the culture supernatant was removed by suction, and 100 µl / well of a new lymphocyte culture solution containing 20 µg / ml of the antigen peptide was added to perform restimulation. Six days after the start of the culture and nine days after the start of the culture, the same culture medium was exchanged and restimulation was performed. Eleven days after the start of the culture, antigen peptide-specific T cell activity was detected. The HLA-A24 gene was introduced into the B1 cell lymphoblastoid cell line C1R cells and the HLA-A24 gene was stably expressed in the C1R-A24 cells. Antigen peptide (amino acid sequence: RYLRDQQLLGI) [J. Immunol.
159: 6242-6252, 1997] was pulsed at a concentration of 10 μg / ml for 2 hours, and antigen-presented cells were seeded at 1 × 10 ⁴ cells / well in a 96-well plate to obtain target cells. Half of the cultured PBMC was added to target cells pulsed with each peptide,
Mixed culture was performed for 18 hours. Collect the culture supernatant, and
The amount of IFN-γ was quantified by ELISA. As a result, the reactivity of PBMC to target cells pulsed with the EB virus-derived HLA-A24-restricted antigen peptide and the HIV-derived HLA-A24 as a negative control
PBMC against target cells pulsed with restricted antigenic peptide
The difference from the reactivity was 300 pg / ml or more in IFN-γ production. The antigen peptide used in this study was prepared by the Fmoc method. Based on the above considerations, a 96-hole U-bottom plate is
0 ⁵ cells / well of PBMC were injected with EBV-derived HLA-
It was shown that antigen peptide-specific T cells were successfully induced by culturing while stimulating with an A24-restricted antigen peptide.

Example 2 Analysis of EB Virus-Derived Antigen Peptide-Specific T Cell Frequency By the method of Example 1, it was confirmed by a limiting dilution method that the frequency of EB virus-derived antigen peptide-specific T cells was increased. PBMC were prepared from the same healthy person as in Example 1 by the same method, and the following antigen-specific T cell frequency was measured without partially stimulating the antigen peptide. Also, some PBMC
In the same manner as in Example 1, 1 × 10 ⁵ cells / well were seeded on a 96-well U-bottom plate, and cultured three times every three days while stimulating with EB virus-derived HLA-A24-restricted antigen peptide. Was measured for the frequency of antigen-specific T cells.

The frequency of the antigen-specific T cell was measured by the following method. PBMC with 400, 200, 100, 5 cells per well
96-well plates seeded with 0, 25, 12.5
I prepared one by one. At this time, feeder cel
l) Irradiated homologous (allogenic) PBMC (2
× 10 ⁵ cells / well), and add 20% FC to the cloning medium (25% RPMI1640 and 55% AIM-V mixed base).
S, 1% NEAA, 100 U / ml IL-2, and 10 μg / ml PHA). After 13 days of culture, the presence of antigen-specific T cells in each well was determined by
The amount of IFNγ produced by reacting with virus-derived HLA-A24-restricted antigen peptide-pulsed target cells and the HIV-derived HLA-A2 as a negative control
4-restricted antigen peptide I produced by reacting with pulsed target cells
The difference from the FNγ amount was examined as an index. IFNγ in the culture supernatant produced by mixing and culturing the cultured PBMC and target cells for 18 hours
The amount is measured using an ELISA method and the difference between target cells is 100
Wells of pg / ml or more were defined as positive, and the others were defined as negative. The data thus obtained was analyzed by the minimum χ ² method under 95% confidence interval conditions. The antigen-specific T cell frequency was calculated by determining the cell concentration when 37% of the wells were negative (J. Immunol. 126: 1614-1620, 1981).

As a result, the frequency of HLA-A24-restricted antigen peptide-specific T cells derived from EB virus in PBMCs not stimulated with the antigen peptide was 1 / 30,000 or less of the detection limit. The frequency of HLA-A24-restricted antigen peptide-specific T cells derived from EB virus in stimulated PBMC was 1/5639. From the above examination, it was shown that the method of Example 1 satisfactorily induced antigen-specific T cells.

Example 3 Examination of Number of Stimulations of Antigen Peptide The effect of the number of stimulations of the antigen peptide on the detection of antigen-specific T cells was examined. The test was performed in the same manner as in Example 1 except that the number of stimulations, the stimulation interval, and the culture period with the EB virus-derived HLA-A24-restricted antigen peptide were changed. The following table shows the conditions and results of each experimental group. The results showed that antigen peptide-specific T cells could be detected by antigen peptide stimulation every 2-5 days.

[0031]

[Table 1] Stimulation interval (days) Number of stimulations (times) Culture period (days) EB virus-specific IFN-γ production (pg / ml) 1 10 11 0 2 6 11 59 3 5 13 400 4 4 13 319 5 3 11 161 6 3 13 0

Example 4 Examination of the Number of PBMC Cells Used for Stimulation of Antigen Peptide The influence of the number of PBMC cells used for culture on the sensitivity of detecting antigen-specific T cells was examined. PBMCs were added at 2 × 10 ⁵ cells per well
× 10 ⁵ , 5 × 10 ⁴ , 2.5 × 10 ⁴ , 1.25 × 10 ⁴ , 0.63 × 10 ⁴ , 0
And sowed. By adding irradiated PBMC, the culture was started at a total cell count of 2 × 10 ⁵ cells / well. Other conditions were the same as in Example 1. Figure 1 shows the results
Shown in An increase in the amount of IFN-γ was observed in proportion to the number of cultured PBMCs, and detection was possible even at 2.5 × 10 ⁴ cells / well.
As is clear from FIG. 1, the number of cultured PBMC cells and IFN-γ
It is proportional to the amount of production, which means that the final amount of IFN-γ production reflects the number of antigen-specific T cells at the start of culture. This indicates that this test is difficult to quantify the antigen-specific T cell frequency, but semi-quantitative measurement is possible.

Example 5 Detection of antigen-specific T cells from PBMCs from a plurality of healthy subjects PBMCs were collected from the peripheral blood of 10 healthy HLA-A24-positive subjects,
With the number of cells at the start of culture set to 1.5 × 10 ⁵ / well, detection of HLA-A24-restricted antigen peptide-specific T cells derived from EB virus was attempted in the same manner as in Example 1. The results are shown in FIG. Although there is a difference in the amount of IFN-γ production, EB
Virus-derived HLA-A24-restricted antigen peptide-specific T cells were detected.

Example 6 Investigation of Micro- Size Test Method In Example 1-5, a 96-well U-bottom plate was used for examination. However, in order to construct a system capable of measuring a larger number of samples, a 384-well plate was used. The study using was carried out. HLA-A24-positive healthy PBMC were seeded at 5 × 10 ⁴ cells / well in a 384-well plate with a culture solution of 80 μl / well, and the peptide was stimulated when half the culture solution was replaced (total of 5 peptide stimulations). An attempt was made to detect EB virus-derived HLA-A24-restricted antigen peptide-specific T cells in the same manner as in Example 1. As a result, the difference between the reactivity to the target cells pulsed with the EB virus-derived HLA-A24-restricted antigen peptide and the reactivity to the target cells pulsed with the HIV-derived HLA-A24-restricted antigen peptide of the negative control was the amount of IFN-γ production. Was 2775 pg / ml. This indicates that antigen-specific T cells can be detected even when the assay system is micronized.

Example 7 Cancer Vaccine Treatment for Cervical Cancer Patients (Part 1) (1) Selection of Administered Peptides Peripheral blood mononuclear cells were isolated from HLA-A2-positive cervical cancer patients.
Of the 16 types of antigen peptides shown in Table 2 by the same method as in Example 1, frequently stimulated with 13 types of antigen peptides,
The reactivity of specific T cells to each antigen was examined.

[Table 2]

Lymphocytes were cultured for each peptide in 4 wells, and cells in each well were measured at the time of measurement of IFN-γ production.
Eight samples were measured by equal division. The cultured lymphocytes were HLA-A2-positive T2 cells (ATCC number, CRL-1992) (hereinafter referred to as antigen peptide-T2 cells) pulsed with each antigen peptide and HLA-A2-restricted HIV-derived antigen peptide. (Amino acid sequence: SLYTYATL) [J. Exp. Med.
180: 1283-1293, 1994].
(Referred to as IV-T2 cells), the amount of IFN-γ produced in response to the reaction was quantified, and it was examined by a t-test whether the difference was significant. Table 3 shows the results.

[Table 3]

The amount of IFN-γ production in the table indicates the amount of IFN-γ produced by lymphocytes in response to each antigen peptide-T2 cell.
IFN-γ produced in response to HIV-T2 cells from the amount
This is the average of 2 wells after subtracting the amount. Also,
If the difference is less than 0 pg / ml, it is described as <0 pg. The criteria for selecting a peptide to be administered as a cancer vaccine are as follows. The amount of IFN-γ produced in response to each antigenic peptide-T2 cell is greater than the amount of IFN-γ produced in response to HIV-T2 cells and p
<0.05 wells present more than one antigen peptide, or antigen peptide-the amount of IFN-γ produced in response to T2 cells is greater than the amount of IFN-γ produced in response to HIV-T2 cells Then, the antigen peptide in which wells having a difference of 100 pg / ml or more were present was selected as a peptide to be administered as a cancer vaccine. In the case of this cervical cancer patient, the frequency of presence of antigen peptide-specific T cells is high, and
Types ppMAPkkk ₂₉₄ , ppMAPkkk ₄₃₂ , WHSC2 ₁₀₃ and HNRPL
₅₀₁ was selected based on the above criteria.

(2) Clinical therapeutic effect Montanide ISA51 for the above four selected peptides
A W / O emulsion was prepared using (Seppic), and 3 mg of each was subcutaneously administered to cervical cancer patients about 5 times every 2 weeks. Pre- and post-dose tumor marker carcinoembryonic antigen (CE
A) and the transition of measured values of squamous cell carcinoma-related antigen (SCC)
3, shown in FIG. FIG. 5 shows the reduction ratio of the tumor.
The administration of cancer vaccine lowers and stabilizes CEA levels,
The value tended to rise, but tended to be stable despite the high value. In addition, it was observed that the tumor was reduced by about 70% with the administration of the vaccine. From these results, it was shown that the cancer vaccine was effective for the treatment of cancer, although the antigen peptide selected by the novel cellular immunity qualitative method of the present invention was used.

Example 8 Cancer Vaccine Treatment for Cervical Cancer Patients (Part 2) (1) Selection of Administered Peptides Peripheral blood mononuclear cells were isolated from HLA-A24-positive cervical cancer patients and used in the same manner as in Example 7. The reactivity with respect to 13 kinds of antigen peptides among the 14 kinds of antigen peptides shown in Table 4 was examined by the method.

[Table 4]

Lymphocytes were cultured for each peptide in three wells, and cells in each well were measured when measuring the amount of IFN-γ production.
Nine samples were measured by dividing into three equal parts. And the cultured lymphocytes were pulsed with each antigen peptide HLA-A24
C pulsed with positive C1R-A24 cells (hereinafter referred to as antigen peptide-C1R-A24 cells) and HLA-A24 restricted HIV-derived antigen peptide (amino acid sequence: RYLRDQQLLGI) [supra]
The amount of IFN-γ produced in response to 1R-A24 cells (hereinafter referred to as HIV-C1R-A24 cells) was quantified, and the t-test was used to determine whether the difference was significant to determine the p-value. . Table 5 shows the results.

[Table 5]

The amount of IFN-γ produced in the table is the IFN-γ produced by lymphocytes in response to each antigen peptide-C1R-A24 cell.
I produced in response to HIV-C1R-A24 cells from -γ amount
The average of the values obtained by subtracting the amount of FN-γ for three wells is shown.
When the difference was smaller than 0 pg / ml, it was expressed as <0 pg. The criterion for selecting a peptide to be administered as a cancer vaccine is the same as that in Example 7. In the case of this cervical cancer patient, SART2 ₁₆₁ , S
ART2 _889, were selected four antigenic peptides lck ₂₀₈ and ART4 _34. Further, after administering the cancer vaccine using these antigen peptides five times or more, the peripheral blood mononuclear cells of the patient were separated, and the reactivity to the 14 types of antigen peptides in Table 4 was examined in the same manner as described above. Was. Table 6 shows the results.

[0042]

[Table 6] From these results, SART1 ₆₉₀ , SART3 ₁₀₉ , lck ₂₀₈ , lck ₄₈₈
Were selected and used for the administration of the eighth and subsequent cancer vaccines.

(2) Therapeutic effects in clinical practice Four kinds of peptides were prepared in the same manner as in Example 7 using Montanide ISA51.
A W / O emulsion was prepared using (Seppic), and 3 mg of each was subcutaneously administered to cervical cancer patients about every two weeks. Tumor markers carcinoembryonic antigen (CE) before 1 dose and up to 12 doses
The transition of the measured value of A) is shown in FIG. The administration of the cancer vaccine suppressed the tendency of the CEA level to rise, and showed a tendency to stabilize despite the high level. In addition, before the vaccine administration, after three times administration, the reactivity to the antigen peptide used for vaccine administration of patient peripheral blood mononuclear cells after six times administration, respectively
It was measured by a method according to the method of Example 1. Table 7 shows the results.

[Table 7]

Among the four types of antigenic peptides, there was no change in the reactivity to SART2 ₈₉₉ , but SART1 ₁₆₁ and lck
₂₀₈ , and increased response to ART4 ₇₅ ,
It was suggested that the vaccine administration increased the frequency of antigen peptide-specific T cells. As described above, the cell-mediated immunity detection method of the present invention not only selects an antigen peptide to be used for a cancer vaccine, but also monitors a specific T cell induction for the administered antigen peptide and a more effective antigen peptide after starting the vaccine treatment. Is also useful when acquiring data to change the antigen peptide to be administered.

Example 9 Cancer Vaccine Treatment for Prostate Cancer Patients (1) Selection of Antigen Peptide Peripheral blood mononuclear cells were isolated from HLA-A2-positive prostate cancer patients.
The reactivity to 13 of the 16 antigen peptides shown in Table 2 above was examined by the method of Example 7. Table 8 shows the results.

[0046]

[Table 8] In this case, the reactivity to the antigenic peptide was low overall, but only ppMAPkkk ₄₃₂ was selected as the antigenic peptide for administration.

(2) Therapeutic effect in clinical practice The peptide was prepared in the same manner as in Example 7 using Montanide ISA51 (Seppic
Was used to prepare a W / O emulsion, and 3 mg was administered subcutaneously to prostate cancer patients approximately every two weeks. The transition of the measured value of the prostate specific antigen (PSA), which is a tumor marker, is shown in FIG. PSA values that were on the rise tended to increase from the second dose of vaccine administration.
After the fifth administration of the vaccine, the value began to decrease and became lower than before the start of treatment. As described above, it has been shown that the novel cell-based immunity qualitative method of the present invention is a useful method for easily selecting T cells having a high frequency of occurrence in a patient from a large number of candidate peptides, which is effective for treating patients.

[0048]

The method of the present invention is not "quantitative" as in the conventional method, but is "semi-quantitative" as discussed in Example 4. However, the method of the present invention requires a small amount of blood (lymphocytes) of the patient to be used, and can evaluate many antigenic peptides in a short time. It is very effective in deciding the peptide to be used.

In particular, the method of the present invention for detecting an antigen that reacts with an antigen-specific T cell does not necessarily mean that all cancer cells commonly express the same tumor antigen. This is a very beneficial method considering that treatment with a plurality of different tumor antigen peptides is more effective because two or more different tumor antigen peptides are presented. Furthermore, it is very effective in monitoring the fluctuation of antigen-specific T cells before and after vaccine administration, that is, in examining whether the administered antigen peptide is effective. The method of the present invention enables a micro-technique to systematically detect antigen-specific T cells.

Further, according to the present invention, the frequency of CTL precursors against various cancer antigen peptides in peripheral blood T cells of cancer patients is measured by a novel cellular immunity qualitative method, and a highly reactive antigen peptide is selected. Good results can be obtained by performing treatments administered to cancer patients as cancer vaccines.

[0051]

[Sequence list]

SEQUENCE LISTING <110> Itoh, Kyogo <120> Assay based on cellular immunity and therape
utic application there <130> 178241 <140><141> 2001-09-18 <160> 32 <170> PatentIn Ver. 2.1 <210> 1 <211> 9 <212> PRT <213> Homo sapiens <300><303> Int.J. Cancer <304> 88 <306> 633-639 <307> 2000 <400> 1 Leu Leu Gln Ala Glu Ala Pro Arg Leu 1 5 <210> 2 <211> 9 <212> PRT <213 > Homo sapiens <300><303> Int.J. Cancer <304> 88 <306> 633-639 <307> 2000 <400> 2 Arg Leu Ala Glu Tyr Gln Ala Tyr Ile 1 5 <210> 3 <211> 10 <212> PRT <213> Homo sapiens <300><303> Jpn.J. Cancer Res. <304> 92 <306> 762-767 <307> 2001 <400> 3 Lys Leu Lys His Tyr Gly Pro Gly Trp Val 1 5 10 <210> 4 <211> 8 <212> PRT <213> Homo sapiens <300><303> Jpn.J. Cancer Res. <304> 92 <306> 762-767 <307> 2001 <300 ><303> Jpn. J. Cancer Res. <304> 92 <306> 762-767 <400> 4 Val Leu Glu Gly Met Glu Val Val 1 5 <210> 5 <211> 9 <212> PRT <213> Homo sapiens <300><303> Int.J. Cancer <307> 2001 <400> 5 Lys Leu Val Glu Arg Leu Gly Ala Ala 1 5 <210> 6 <211> 9 <212> PRT <213> Homo sapiens <300><303> Int. J. Cancer <307> 2001 <400> 6 Asp Val Trp Ser Phe Gly Ile Leu Leu 1 5 <210> 7 <211> 9 <212> PRT <213> Homo sapiens <300><303> Cancer Res. <304> 61 <306> 2038-2046 <307> 2001 <400> 7 Arg Ile Ile Tyr Asp Arg Lys Phe Leu 1 5 <210> 8 <211> 9 <212> PRT <213> Homo sapiens <300><303> Cancer Res. <304> 61 <306> 2038 -2046 <307> 2001 <400> 8 Gly Leu Leu Phe Leu His Thr Arg Thr 15 <210> 9 <211> 9 <212> PRT <213> Homo sapiens <300><303> Cancer Res. <304> 61 <306> 2038-2046 <307> 2001 <400> 9 Asp Leu Leu Ser His Ala Phe Phe Ala 1 5 <210> 10 <211> 9 <212> PRT <213> Homo sapiens <300><303> Cancer Res. <304> 61 <306> 2038-2046 <307> 2001 <400> 10 Ala Ser Leu Asp Ser Asp Pro Trp Val 1 5 <210> 11 <211> 9 <212> PRT <213> Homo sapiens <300 ><303> Cancer Res. <304> 61 <306> 2038-2046 <307> 2001 <400> 11 Ile Leu Gly Glu Leu Arg Glu Lys Val 1 5 <210> 12 <211> 9 <212> PRT <213 > Homo sapiens <300><303> Cancer Res. <304> 61 <306> 2038-2046 <307> 2001 <400> 12 Arg Leu Gln Glu Trp Cys Ser Val Ile 1 5 <210> 13 <211> 9 <212> PRT <213> Ho mo sapiens <300><303> Cancer Res. <304> 61 <306> 2038-2046 <307> 2001 <400> 13 Leu Ile Ala Asp Phe Leu Ser Gly Leu 1 5 <210> 14 <211> 9 <212 > PRT <213> Homo sapiens <300><303> Cancer Res. <304> 61 <306> 2038-2046 <307> 2001 <400> 14 Ile Leu Pro Arg Lys His His Arg Ile 1 5 <210> 15 <211> 9 <212> PRT <213> Homo sapiens <300><303> Cancer Res. <304> 61 <306> 2038-2046 <307> 2001 <400> 15 Ala Leu Val Glu Phe Glu Asp Val Leu 1 5 <210> 16 <211> 10 <212> PRT <213> Homo sapiens <300><303> Cancer Res. <304> 61 <306> 2038-2046 <307> 2001 <400> 16 Asn Val Leu His Phe Phe Asn Ala Pro Leu 1 5 10 <210> 17 <211> 9 <212> PRT <213> Homo sapiens <300><303> Int. J. Cancer <304> 81 <306> 459-466 <307> 1999 <400> 17 Glu Tyr Arg Gly Phe Thr Gln Asp Phe 1 5 <210> 18 <211> 9 <212> PRT <213> Homo sapiens <300><303> J. Immunol. <304> 164 <306> 2565- 2574 <307> 2000 <400> 18 Asp Tyr Ser Ala Arg Trp Asn Glu Ile 1 5 <210> 19 <211> 9 <212> PRT <213> Homo sapiens <300><303> J. Immunol. <304> 164 <306> 2565-2574 <307> 2000 <400> 19 Ala Tyr Asp Phe Leu Tyr Asn Tyr Leu 1 5 <210> 20 <211> 9 <212> PRT <213> Homo sapiens <300><303> J. Immunol. <304> 164 <306> 2565-2574 <400> 20 Ser Tyr Thr Arg Leu Phe Leu Ile Leu 1 5 <210> 21 <211> 10 <212> PRT <213> Homo sapiens <300><303> Cancer Res. <304> 59 <306> 4056-4063 <307> 1999 <400> 21 Val Tyr Asp Tyr Asn Cys His Val Asp Leu 1 5 10 <210> 22 <211> 9 <212> PRT <213> Homo sapiens <300><303> Cancer Res. <304> 59 <306> 4056-4063 <307> 1999 <400> 22 Ala Tyr Ile Asp Phe Glu Met Lys Ile 1 5 <210> 23 <211> 9 <212> PRT <213> Homo sapiens <300><303> J. Immunol. <304> 163 <306> 4994-5004 <307> 1999 <400> 23 Lys Phe His Arg Val Ile Lys Asp Phe 1 5 <210> 24 <211> 9 <212> PRT <213> Homo sapiens <300><303> J. Immunol. <304> 163 <306> 4994-5004 <307> 1999 <400> 24 Asp Phe Met Ile Gln Gly Gly Asp Phe 1 5 <210> 25 <211> 9 <212> PRT <213> Homo sapiens <300><303> Eur. J. Immunol. <304> 31 <306> 323-332 <307> 2001 <400> 25 His Tyr Thr Asn Ala Ser Asp Gly Leu 1 5 <210> 26 <211> 9 <212> PRT <213> Homo sapiens <300><303> Eur. J. Immunol. <304> 31 <306> 323-332 <307> 2001 <400> 26 Thr Phe Asp Tyr Leu Arg Ser Val Leu 1 5 <210> 27 <211> 10 <212> PRT <213> Homo sapiens <300><303> Eur. J. Immunol. <304> 31 <306> 323-332 <307> 2001 <400 > 27 Asp Tyr Leu Arg Ser Val Leu Glu Asp Phe 1 5 10 <210> 28 <211> 8 <212> PRT <213> Homo sapiens <300><303> Cancer Res. <304> 60 <306> 3550- 3558 <307> 2000 <400> 28 Ala Phe Leu Arg His Ala Ala Leu 1 5 <210> 29 <211> 10 <212> PRT <213> Homo sapiens <300><303> Cancer Res. <304> 60 <306> 3550-3558 <307> 2000 <400> 29 Asp Tyr Pro Ser Leu Ser Ala Thr Asp Ile 1 5 10 <210> 30 <211> 9 <212> PRT <213> Homo sapiens <300><303> Int J. Cancer <304> 88 <306> 633-639 <307> 2000 <400> 30 Glu Tyr Cys Leu Lys Phe Thr Lys Leu 1 5 <210> 31 <211> 9 <212> PRT <213> EB Virus <300><303> J. Immunol. <304> 158 <306> 3325-3334 <307> 1997 <400> 31 Thr Tyr Gly Pro Val Phe Met Cys Leu 1 5 <210> 32 <211> 11 <212> PRT <213> Human immunodeficiency vir us <300><303> J. Immunol. <304> 159 <306> 6242-6252 <307> 1997 <400> 32 Arg Tyr Leu Arg Asp Gln Gln Leu Leu Gly Ile 1 5 10

[Brief description of the drawings]

FIG. 1 is a graph showing the effect of antigen-specific T cell detection sensitivity on the number of peripheral blood mononuclear cells used for antigen stimulation.

FIG. 2 is a graph showing the detection results of EB virus-derived HLA-A24-restricted antigen peptide-specific T cells from peripheral blood mononuclear cells from 10 HLA-A24-positive healthy persons.

FIG. 3 is a graph showing changes in the measured values of carcinoembryonic antigen (CEA) before and after treatment of a cervical cancer patient with an antigen peptide selected by the method of the present invention.

FIG. 4. Squamous cell carcinoma-associated antigen (SC) before and after treatment of a cervical cancer patient with an antigenic peptide selected by the method of the present invention.
It is a graph which shows transition of the measured value of C).

FIG. 5 is a graph showing the reduction ratio of tumor before and after treatment of a cervical cancer patient with an antigen peptide selected by the method of the present invention.

FIG. 6 is a graph showing the transition of measured values of carcinoembryonic antigen (CEA) before and after treatment of a cervical cancer patient with an antigen peptide selected by the method of the present invention.

FIG. 7. Prostate specific antigen (PSA) before and after treating a prostate cancer patient with an antigenic peptide selected by the method of the present invention.
6 is a graph showing the transition of the measured values of FIG.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) C12Q 1/02 ZNA C12Q 1/02 ZNA G01N 33/53 G01N 33/53 D PY 33/574 33/574 DF term (reference) 2G045 BB20 CB01 FB03 GC22 JA01 4B063 QA19 QQ08 QQ79 QQ96 QR48 QS33 4C085 AA03 BB01 CC12

Claims (28)

[Claims] 1. A method for detecting antigen-specific T cells, comprising: (1) collecting peripheral blood mononuclear cells; (2) stimulating the collected peripheral blood mononuclear cells frequently with an antigen; A) detecting antigen-specific T cells in the stimulated peripheral blood mononuclear cells, wherein the step of (2) stimulating peripheral blood mononuclear cells does not directly use antigen-presenting cells. 2. The method according to claim 1, wherein the antigen is an antigen protein or an antigen peptide derived from the antigen protein. 3. The method according to claim 1, wherein the frequent stimulation is stimulating a plurality of times at a frequency of 2 times / week or more. 4. The method according to claim 1, wherein the repetitive stimulation is a plurality of stimulations at intervals of 2 to 5 days. 5. The method according to claim 1, wherein the detection of the antigen-specific T cell is performed using the amount of cytokine produced by the T cell as an index.
The method according to any of the above. 6. The method according to claim 5, wherein the cytokine is IFN-γ. 7. The method according to claim 1, wherein the frequent stimulation is performed in a well of a microplate. 8. The method according to claim 7, wherein the microplate is a U-bottom microplate. 9. The peripheral blood mononuclear cell count when using a 96-well microplate is 2.5 × 10 ⁴ cells / well-2.
The method according to claim 7, wherein the number is × 10 ⁵ cells / well. 10. The peripheral blood mononuclear cell count when using a 384-well microplate is 2.5 × 10 ⁴ cells / well−1 × 10 ⁵ cells / well. the method of. 11. A method for detecting an antigen that reacts with an antigen-specific T cell, comprising: (1) collecting peripheral blood mononuclear cells; and (2) collecting the collected peripheral blood mononuclear cells with a plurality of antigen candidate substances. (3) Determine which antigen candidate substance induced antigen-specific T cells. (3) Do not directly use antigen presenting cells to stimulate peripheral blood mononuclear cells in step (2). A method characterized by the following. 12. The method according to claim 11, wherein the antigen is an antigen protein or an antigen peptide derived from the antigen protein. 13. The method according to claim 11, wherein the repetitive stimulation is a multiple stimulation at a frequency of 2 times / week or more. 14. The method according to claim 11 or 13, wherein the frequent stimulation is stimulating a plurality of times at intervals of 2-5 days. 15. The method according to claim 11, wherein the determination of antigen-specific T cell induction is performed by quantifying the amount of cytokine produced by the T cell. 16. The method according to claim 15, wherein the cytokine is IFN-γ. 17. The method according to claim 11, wherein the frequent stimulation is performed in a well of a microplate. 18. The method of claim 17, wherein the microplate is a U-bottom microplate. 19. The number of peripheral blood mononuclear cells when using a 96-well microplate is 2.5 × 10 ⁴ cells / well-
Is 2 × 10 ⁵ cells / well, according to claim 17 or 18 A method according. 20. The peripheral blood mononuclear cell count when using a 384-well microplate is 2.5 × 10 ⁴ cells / well−1 × 10 ⁵ cells / well.
8. The method according to 8. 21. A tailored pharmaceutical composition specific for a disease of interest in a patient for treating the disease, comprising an antigen associated with the disease of interest in the patient. 22. An antigen associated with a target disease in a patient, comprising: (1) collecting peripheral blood mononuclear cells from the patient; and (2) determining that the collected peripheral blood mononuclear cells are related to the target disease. Stimulate frequently with each of a number of potential antigen candidates,
And (3) determining which antigen candidate substance induced antigen-specific T cells, one selected by a method not directly using antigen presenting cells when stimulating peripheral blood mononuclear cells in step (2). Or more antigens, the bespoke pharmaceutical composition specific to the disease of interest in the patient for treating the disease. 23. The pharmaceutical composition according to claim 21, wherein the antigen is an antigen protein or an antigen peptide derived from the antigen protein. 24. The method according to claim 21, wherein the disease is cancer.
3. The pharmaceutical composition according to 2. 25. A method for treating a disease in a patient, the method comprising administering a therapeutically effective amount of an antigen associated with the disease in the patient, wherein the disease enables personalized medicine specific to the disease in the patient. the method of. 26. An antigen associated with a target disease in a patient, comprising: (1) collecting peripheral blood mononuclear cells from the patient; and (2) associating the collected peripheral blood mononuclear cells with the target disease. Stimulates frequently with each of a number of potential antigen candidates,
And (3) determining which antigen candidate substance induced antigen-specific T cells, one selected by a method not directly using antigen presenting cells when stimulating peripheral blood mononuclear cells in step (2). Or more antigens for the treatment of the disease, which allows for personalized medicine specific to the disease in the patient. 27. The method according to claim 25, wherein the antigen is an antigen protein or an antigen peptide derived from the antigen protein. 28. The disease according to claim 25 or 2, wherein the disease is cancer.
6. The method according to 6.