WO2018135561A1

WO2018135561A1 - Method for determining therapeutic efficacy of allergen immunotherapy

Info

Publication number: WO2018135561A1
Application number: PCT/JP2018/001327
Authority: WO
Inventors: 史英伊原; 美孝岡本; 俊憲中山; 大樹櫻井
Original assignee: 国立大学法人千葉大学
Priority date: 2017-01-23
Filing date: 2018-01-18
Publication date: 2018-07-26
Also published as: JPWO2018135561A1

Abstract

The present invention addresses the problem of providing: a method for determining the therapeutic efficacy of an allergen immunotherapy on allergic rhinitis with high accuracy; and others. The fluctuation in the number of ST2⁺ CD4⁺ T cells and the fluctuation in the number of at least one type of CD4⁺ T cells selected from the group consisting of CD27^- CD4⁺ T cells, CD161⁺ CD4⁺ T cells, IL-5⁺ CD4⁺ T cells and IL-13⁺ CD4⁺ T cells before and after the start of an allergen immunotherapy on allergic rhinitis are analyzed, and it is determined that the immunotherapy using an allergen is effective for an allergic rhinitis patient when both of the cells decrease.

Description

Therapeutic effect evaluation method of allergen immunotherapy

The present invention relates to a method for determining the therapeutic effect of allergen immunotherapy for allergic rhinitis, a determination kit, and a determination biomarker.

Allergic rhinitis is an allergic disease in the nasal mucosa such as sneezing, nasal congestion, and nasal congestion caused by excessive reaction of the immune system to a foreign antigen that should be harmless. In recent decades, with changes in lifestyle and living environment, the number of patients suffering from allergic rhinitis has increased rapidly, and there has been a problem of a decrease in quality of life (QOL) and an increase in the burden of medical expenses. In particular, patients with allergic rhinitis caused by house dust such as ticks are increasing year by year, as are hay fever patients such as cedar pollinosis.

Sublingual immunotherapy is attracting attention as a treatment that can be expected to cure allergic rhinitis. The therapeutic effect of sublingual immunotherapy is usually determined based on a questionnaire based on subjective symptoms of patients with allergic rhinitis, but the subjective symptoms are thought to include a placebo effect, and the lack of objectivity is a problem. It was.

It is known that when allergic rhinitis is treated, the balance of T cells changes. For example, it has been reported that when allergen immunotherapy is performed on an allergic airway disease model mouse induced by oocyte albumin (OVA) of T cells, the number of ST2-positive CD4-positive T cells decreases (non-patented). Reference 1). Moreover, it was reported that when sublingual immunotherapy was performed on grass pollen patients, the proportion of CD27-positive and CD161-positive CD4-positive T cells was different from that in the case of administering a control placebo. (Patent Document 1).

International Publication No. 2012/148549 Pamphlet

An object of the present invention is to provide a method for accurately determining the therapeutic effect of allergen immunotherapy for allergic rhinitis.

The inventors of the present invention have been intensively researched to solve the above problems. In the process, a peripheral blood mononuclear cell-containing sample from an allergic rhinitis patient after allergen immunotherapy is cultured in the presence of the allergen to prepare a T cell-containing sample containing allergen-specific T cells. ( ⁺ ) It consists of two types of cells included in the T cell group, namely, ST2 ⁺ CD4 ⁺ T cells (i), CD27 negative ( ^- ), CD161 ⁺ , IL-5 ⁺ , and IL-13 ⁺ One or two or more types of CD4 ⁺ T cells (ii) selected from the group were detected and the change in the number of CD4 ⁺ T cells before and after treatment was analyzed. As a result, the CD4 ⁺ T cells (i) and If CD4 + ^T cell count of cells (ii) decreases both immunotherapy with allergens, found that it can be determined accurately to be effective against allergic rhinitis patients, the present onset The has been completed.

That is, the present invention is as follows.
[1] A method for determining the therapeutic effect of allergen immunotherapy for allergic rhinitis comprising the following steps (a) and (b) (hereinafter sometimes referred to as “the present determination method”).
(A) In a peripheral blood mononuclear cell-containing sample obtained from a patient with allergic rhinitis after the start of allergen immunotherapy, or a T cell-containing sample obtained by culturing the peripheral blood mononuclear cell-containing sample in the presence of allergen Measuring the number of the following CD4-positive T cells (i) and CD4-positive T cells (ii) contained in
(I) ST2-positive CD4-positive T cells (ii) one or more CD4-positive T cells selected from the group consisting of CD27-negative, CD161-positive, IL-5-positive, and IL-13-positive (b) A step of comparing the number of cells of the CD4 positive T cells (i) and the number of cells of the CD4 positive T cells (ii) with respective control values, when both are lower than the respective control values, The step (b), which shows that immunotherapy using the allergen is likely to be effective for the allergic rhinitis patient;
[2] The method according to [1] above, wherein in the step (a), the number of CD4 positive T cells (i) and CD4 positive T cells (ii) contained in the T cell-containing sample is measured. Judgment method.
[3] The determination method according to [1] or [2], wherein the allergic rhinitis is tick allergic rhinitis and the allergen immunotherapy is an allergen immunotherapy using a tick antigen.
[4] The determination method according to any one of [1] to [3], wherein the CD4 positive T cell (ii) is CD27 negative.
[5] The determination method according to [4], wherein the CD4 positive T cell (ii) is a CD27 negative, CD161 positive, IL-5 positive, and IL-13 positive CD4 positive T cell. .
[6] An antibody that specifically binds to the following CD4 positive T cells (i), an antibody that specifically binds to the following CD4 positive T cells (ii), or a label thereof: A kit for determining the therapeutic effect of allergen immunotherapy for allergic rhinitis (hereinafter sometimes referred to as “the determination kit”).
(I) ST2-positive CD4-positive T cells (ii) one or more CD4-positive T cells selected from the group consisting of CD27-negative, CD161-positive, IL-5-positive, and IL-13-positive [7] The kit according to [6] above, wherein the allergic rhinitis is mite allergic rhinitis and the allergen immunotherapy is an allergen immunotherapy using a tick antigen.
[8] The kit according to [6] or [7] above, wherein the CD4 positive T cells (ii) are CD27 negative.
[9] The kit according to [8] above, wherein the CD4 positive T cells (ii) are CD27 negative, CD161 positive, IL-5 positive and IL-13 positive CD4 positive T cells.
[10] A biomarker for determining the therapeutic effect of allergen immunotherapy for allergic rhinitis (hereinafter referred to as “for the present determination”, comprising the following CD4 positive T cells (i) and CD4 positive T cells (ii): Sometimes referred to as a “biomarker”).
(I) ST2-positive CD4-positive T cells (ii) one or more CD4-positive T cells selected from the group consisting of CD27-negative, CD161-positive, IL-5-positive, and IL-13-positive [11] The biomarker according to [10] above, wherein the allergic rhinitis is mite allergic rhinitis and the allergen immunotherapy is an allergen immunotherapy using a tick antigen.
[12] The biomarker according to [10] or [11] above, wherein the CD4 positive T cell (ii) is CD27 negative.
[13] The biomarker according to [12] above, wherein the CD4 positive T cell (ii) is a CD27 negative, CD161 positive, IL-5 positive, and IL-13 positive CD4 positive T cell. .

As another embodiment of the present invention, the method for diagnosing the therapeutic effect of allergen immunotherapy for allergic rhinitis and allergen immunotherapy for allergic rhinitis characterized by comprising the above steps (a) and (b) The CD4-positive T cells (i) and CD4-positive T cells (ii) used for the determination of the therapeutic effect of can be mentioned.

By using the present invention, it is possible to objectively and accurately evaluate the effectiveness of allergen immunotherapy such as sublingual immunotherapy, and to enable early prediction of effects, thereby reducing the burden on allergen immunotherapy patients. And is expected to reduce medical costs.

FIG. 1A is a diagram showing the results of measuring the amount of change in ST2 ⁺ / CD4 ⁺ ratio in the active drug group (n = 36) and the placebo group (n = 40) before and after treatment with sublingual immunotherapy. FIG. 1B shows CD27 ⁻ .. In the active drug group (n = 38) and placebo group (n = 35) before and after treatment with sublingual immunotherapy. CD161 ⁺ IL-5 ⁺ . It is a figure which shows the result of having measured the variation | change_quantity of IL-13 ^<+ > / CD4 ^<+> ratio. In the figure, ● and ■ indicate each tick allergic patient in each group.

This determination method includes a peripheral blood mononuclear cell-containing sample (peripheral blood mononuclear cell fraction) obtained from an allergic rhinitis patient during or after allergen immunotherapy, or such peripheral blood mononuclear cells. ST2-positive CD4-positive T cells (i) contained in T-cell-containing samples (samples containing allergen-specific T cells) obtained by culturing the containing sample in the presence of allergen (hereinafter referred to as “CD4 positive after treatment”) T cells (sometimes referred to as “T cells (i)”) and CD27 negative, CD161 positive, IL-5 positive, and IL-13 positive contained in the peripheral blood mononuclear cell-containing sample or T cell-containing sample. A step of measuring the number of cells of one or more CD4 positive T cells (ii) selected from the group (hereinafter sometimes referred to as “post-treatment CD4 positive T cells (ii))” (a) And "post-treatment CD4" The number of “sex T cells (i)” is compared with a control value (hereinafter sometimes referred to as “control value (i)”), and the number of “post-treatment CD4 positive T cells (ii)” Is compared with a control value (hereinafter also referred to as “control value (ii)”), wherein the number of cells of “post-treatment CD4 positive T cells (i)” is “control value (i) ) ”And the number of“ post-treatment CD4 positive T cells (ii) ”is lower than“ control value (ii) ”, immunotherapy using the allergen is performed on the allergic rhinitis patient. The method is not particularly limited as long as it is a method comprising the steps (a) and (b) in the step (b), which shows that the possibility of being effective is high, and before the step (a), allergen immunotherapy Blood samples taken from later allergic rhinitis patients ( Liquid, serum, from plasma, etc.), may be further provided a method step (p) preparing peripheral blood mononuclear cell fraction.

The determination kit includes an antibody (hereinafter referred to as “anti-CD4”) that specifically binds to ST2-positive CD4 positive T cells (i) (hereinafter sometimes simply referred to as “CD4 positive T cells (i)”). Positive T cells (i) antibodies ”), and one or more CD4 positive T cells selected from the group consisting of CD27 negative, CD161 positive, IL-5 positive, and IL-13 positive (Ii) an antibody that specifically binds (hereinafter sometimes simply referred to as “CD4 positive T cell (ii)”) (hereinafter also referred to as “anti-CD4 positive T cell (ii) antibody”), and / or Or a kit for use in the determination of the therapeutic effect of allergen immunotherapy, comprising a label of “anti-CD4 positive T cell (i) antibody” and a label of “anti CD4 positive T cell (ii) antibody”. If there is no particular restriction, this judgment key Is a use invention relating to kits for determining (diagnosing) the therapeutic effect of allergen immunotherapy, and these kits generally include components, such as carriers, pH buffers, and stabilizers, generally used in this type of determination kit. In addition, package inserts such as instruction manuals and instructions for determining the therapeutic effect of allergen immunotherapy are usually included.

The biomarker for determination of this case is a biomarker for determination (diagnosis) of the therapeutic effect of allergen immunotherapy for allergic rhinitis, comprising “CD4 positive T cells (i)” and “CD4 positive T cells (ii)”. If it is, it will not be restrict | limited.

In the present specification, “allergic rhinitis” means that an antibody is produced in a living body by ingestion or contact of a certain exogenous substance, and an antigen is obtained by reuptake or recontact of the same exogenous substance (allergen [antigen]). Means inflammation of type I allergic (allergic to IgE antibodies) in the nasal mucosa, where an antibody reaction occurs and manifests symptoms of allergic rhinitis (nasal congestion, recurrent sneezing, and / or aqueous rhinorrhea) To do.

In the present specification, “allergen immunotherapy for allergic rhinitis” means allergic rhinitis caused by allergic rhinitis patients who are allergic rhinitis by administering allergens that cause allergic rhinitis. It means a treatment that reduces (improves) the symptoms. The form of allergen administration in allergen immunotherapy is not particularly limited, and includes oral administration, intravenous administration, intramuscular administration, subcutaneous administration, sublingual administration, eye drop administration, inhalation administration, transdermal administration, nasal administration, etc. Sublingual administration is preferred.

In the present specification, as allergens, specifically, house dust (for example, mold, fungal spores, textile fibers, animal scales, mites [dust mites, claw mites, mites, etc.], insect dead bodies) antigens, pollen (For example, cedar pollen, cypress pollen, ragweed pollen, rice pollen, zelkova pollen, anemone pollen, birch pollen, Japanese oak pollen, alder pollen, and pine pollen).

The above allergic rhinitis is classified into year-round allergic rhinitis and seasonal allergic rhinitis from the beginning. Such perennial allergic rhinitis can develop year-round due to the house dust antigen and the like. On the other hand, the seasonal allergic rhinitis can develop at a specific time of the year due to allergens such as pollen.

As the allergic rhinitis and allergen immunotherapy, allergic rhinitis caused by mites (tick allergic rhinitis) and allergen immunotherapy using mite antigens are preferable.

In the present specification, “CD4 positive”, “ST2 positive” and “CD161 positive” mean that CD (cluster-of-differentiation) 4 antigen, ST2 antigen and CD161 antigen are expressed on the surface of T cells. In the present specification, “CD27 negative” means that the CD27 antigen is not expressed on the surface of T cells. In this specification, “IL-5 positive” and “IL-13 positive” mean that IL (Interleukin) -5 and IL-13 are expressed in T cells.

Specific examples of the “CD4 positive T cell (ii)” include CD27 negative CD4 positive T cell; CD161 positive CD4 positive T cell; IL-5 positive CD4 positive T cell; IL-13 positive CD4 CD27 positive and CD161 positive CD4 positive T cells; CD27 negative and IL-5 positive CD4 positive T cells; CD27 negative and IL-13 positive CD4 positive T cells; CD161 positive And IL-5 positive CD4 positive T cells; CD161 positive and IL-13 positive CD4 positive T cells; IL-5 positive and IL-13 positive CD4 positive T cells; CD27 negative, CD161 positive And IL-5 positive CD4 positive T cells; CD27 negative, CD161 positive and IL-13 positive CD4 positive T cells; CD161 positive, I -5-positive and IL-13-positive CD4-positive T cells; CD27-negative, CD161-positive, IL-5-positive and IL-13-positive CD4-positive T cells, and CD27-negative CD4-positive T cells Cells are preferred, CD27 positive, CD161 positive, IL-5 positive and IL-13 positive CD4 positive T cells are more preferred.

The peripheral blood mononuclear cell-containing sample may be any sample containing peripheral blood mononuclear cells (mononuclear cells) prepared from a patient with allergic rhinitis after allergen immunotherapy. The purity of peripheral blood mononuclear cells in the contained sample is usually at least 50%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%. A peripheral blood mononuclear cell-containing sample can be prepared from a blood sample (blood, serum, plasma, etc.) collected from a patient with allergic rhinitis after allergen immunotherapy by a known method such as density gradient centrifugation. it can. The period after the start of the allergen immunotherapy is not particularly limited, and for example, within the range of 2 months to 3 years (60 to about 1000 days) after the start, preferably 180 to 700 days, more preferably 300 to 400. Day.

In the step (a) of the determination method, the sample to be measured is a T cell from the viewpoint of increasing the detection sensitivity of “post-treatment CD4 positive T cell (i)” and “post treatment CD4 positive T cell (ii)”. Containing samples are preferred. The ratio of T cells to the total cells contained in the T cell-containing sample is not particularly limited, and is, for example, in the range of 20 to 60%, preferably 30 to 40%.

In this specification, the T cell-containing sample is obtained by culturing a peripheral blood mononuclear cell-containing sample in the presence of an allergen (preferably, the same allergen used in allergen immunotherapy), and “CD4 positive T cell (i ) "And" CD4 positive T cells (ii) "can be prepared by proliferating them. The culture period of the peripheral blood mononuclear cell-containing sample is not particularly limited, and is, for example, 1 to 10 days, preferably 4 to 8 days. Further, the culture medium used for culturing the peripheral blood mononuclear cell-containing sample is not particularly limited. For example, a basic culture medium for animal cell culture containing 5-20% Fetal bovine serum (FBS) (Fetal bovine serum; FBS). DMEM, EMEM, RPMI-1640, α-MEM, F-12, F-10, M-199, AIM-V and the like. The culture solution used for culturing the peripheral blood mononuclear cell-containing sample may contain cytokines such as IL-2, IL-7, and IL-33, anti-CD3 antibody, PMA (Phorbol 12-Myristate 13), as necessary. -Acetate), T cell growth stimulating substances such as ionomycin, and antibiotics such as streptomycin and penicillin can also be added. The concentration (final concentration) of the added cytokine in the culture medium can be appropriately selected depending on the type of cytokine. For example, when the cytokine is IL-2, for example, 3 to 60 (JIU) / ML), preferably 10 to 20 (JIU / mL). When the cytokine is IL-33, for example, it is in the range of 10 to 600 (ng / mL), preferably 50 to 200 (ng / mL). The concentration of allergen in the culture solution is not particularly limited as long as it is a concentration that can proliferate allergen-specific T cells, and is usually in the range of 0.1 to 50 μg / mL, preferably 1 to 10 μg / mL. It is. The culture temperature is usually in the range of 30 to 40 ° C., preferably about 37 ° C. The CO ₂ concentration during the cultivation is usually within the range of about 1 to 10%, preferably about 5%. Further, the humidity during the cultivation is usually in the range of about 70 to 100%, preferably in the range of about 95 to 100%.

In the step (a) of the determination method, the number of “post-treatment CD4-positive T cells (i)” and “post-treatment CD4-positive T cells (ii)” can be measured using “CD4-positive T cells (i ) "And" CD4 positive T cells (ii) ", or components derived therefrom (eg, CD4, ST2, CD161, CD27, IL-5, and / or IL-13 protein, CD4, ST2, CD161, CD27, IL −5 and / or IL-13 gene mRNA) can be specifically detected and the increase or decrease in the number of “CD4 positive T cells (i)” and “CD4 positive T cells (ii)” can be measured. There is no particular limitation and, for example, using “anti-CD4 positive T cell (i) antibody” and “anti CD4 positive T cell (ii) antibody”, CD4, ST2, CD161, CD27, IL-5 in T cells, And / or Methods for analyzing the expression of IL-13 protein, specifically Western blotting method, flow cytometry, ELISA method, EIA method, RIA method, etc., CD4, ST2, CD161, CD27, IL- in T cells 5, and / or methods for analyzing mRNA expression of IL-13 gene, specifically, methods such as quantitative RT-PCR (Reverse Transcription Polymerase Chain Reaction) method, RT-PCR method, Southern blotting method, etc. be able to. Since CD4 and ST2 in “CD4 positive T cells (i)” and CD4, CD161, and CD27 in “CD4 positive T cells (ii)” are cell surface receptors, flow sites are considered in consideration of simplicity. It is preferable to use a meter.

In the present determination method, the “control value (i)” is used to determine (determine) whether or not the number of “CD4-positive T cells (i)” has decreased due to receiving allergen immunotherapy. The index (control) value is not particularly limited, for example, a peripheral blood mononuclear cell-containing sample obtained from an allergic rhinitis patient (preferably the same patient as the determination subject) before the start of allergen immunotherapy, or “CD4-positive T cells (i)” (hereinafter referred to as “pre-treatment CD4-positive T cells (i)”) contained in a T cell-containing sample obtained by culturing such a peripheral blood mononuclear cell-containing sample in the presence of allergen. ROC (Receiver Operating Characteristic) using statistical analysis software based on the number of cells and the number of “pretreatment CD4 positive T cells (i)” and “post treatment CD4 positive T cells (i)”. ) Song And the like threshold (cutoff value) calculated using the. As the number of “pre-treatment CD4 positive T cells (i)” used as a control value, the number of cells measured each time when the present determination method is carried out may be used, or one measured in advance may be used.

Moreover, in this determination method, as the “control value (ii)”, it is determined (determination) whether or not the number of “CD4 positive T cells (ii)” is decreased by receiving allergen immunotherapy. For example, a peripheral blood mononuclear cell-containing sample obtained from an allergic rhinitis patient (preferably the same patient as the determination target) before starting allergen immunotherapy Or “CD4-positive T cells (ii)” (hereinafter referred to as “pre-treatment CD4-positive T cells (ii)” contained in a T cell-containing sample obtained by culturing such a peripheral blood mononuclear cell-containing sample in the presence of allergen. ) ”, And the number of“ pre-treatment CD4 positive T cells (ii) ”and“ post-treatment CD4 positive T cells (ii) ”, ROC curves using statistical analysis software, etc. Calculated using Threshold (cutoff value) can be exemplified. As the number of “pre-treatment CD4 positive T cells (ii)” used as a control value, the number of cells measured each time when the present determination method is carried out may be used, or one measured in advance may be used.

In the step (a) of the determination method, when the number of cells is measured using a T cell-containing sample, the ratio of the number of “post-treatment CD4 positive T cells (i)” to the total number of CD4 positive T cells ( Ratio) (hereinafter also referred to as “post-treatment ratio (i)”); and the ratio of the number of cells after “treatment CD4 positive T cells (ii)” to the total number of CD4 positive T cells (hereinafter “treatment”) In the step (b) of the determination method, the “post-treatment ratio (i)” and the “post-treatment ratio (ii)” are respectively calculated as CD4 positive T The ratio of the number of "pre-treatment CD4 positive T cells (i)" to the total number of cells; and the ratio of the number of "pre-treatment CD4 positive T cells (ii)" to the total number of CD4 positive T cells; It is better to compare whether or not Arbitrariness.

As the “anti-CD4 positive T cell (i) antibody” in this determination kit, a substance (usually a protein) derived from “CD4 positive T cell (i)” contained in the peripheral blood mononuclear cell fraction is used as an epitope. The antibody is not particularly limited as long as it specifically binds, and specific examples include one or two antibodies selected from anti-ST2 antibody and anti-CD4 antibody. The “anti-CD4 positive T cell (ii) antibody” in this determination kit is a substance (usually a protein) derived from “CD4 positive T cell (ii)” contained in the peripheral blood mononuclear cell fraction. The antibody is not particularly limited as long as it is an antibody that specifically binds as an epitope. Specifically, the antibody is selected from the group consisting of an anti-CD27 antibody, an anti-CD161 antibody, an anti-IL-5 antibody, an anti-IL-13 antibody, and an anti-CD4 antibody. One or two or more types of antibodies may be mentioned.

The origin, type, class, form, etc. of the above-mentioned “anti-CD4 positive T cell (i) antibody” and “anti CD4 positive T cell (ii) antibody” are not particularly limited, and “anti CD4 positive T cell (i) antibody” And “anti-CD4 positive T cell (ii) antibody” include, for example, human-derived antibodies; antibodies derived from non-human animals such as mice and rats; polyclonal antibodies, oligoclonal antibodies (several to several tens of antibodies) Mixture), monoclonal antibody; chimeric antibody or humanized antibody in which a part of antibody region (for example, constant region) is substituted with a region derived from a different species, or F (ab ′) ₂ obtained by digesting monoclonal antibody with pepsin antibody fragments, F (ab ') antibody fragment ₂ Fab obtained antibody fragments are reduced', antibody fragments such as Fab obtained by digesting the monoclonal antibody with papain, Etc.; the weight (H) chain variable region and an antibody light (H) chain variable region, recombinant antibodies of the scFv and the like are linked via an amino acid bridge.

The labeling substance in the labeling product of the determination kit includes peroxidase (eg, horseradish peroxidase), alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malic acid dehydrase, Enzymes such as elementary enzyme, penicillinase, catalase, apoglucose oxidase, urease, luciferase or acetylcholinesterase; APC, PE, FITC, Alexa Fluor 488, Alexa Fluor 647, Alexa Fluor 700, PE-Texas Red, PE-Cy5, PE- Fluorescent substances such as Cy7; Green Fluorescence Protein (GFP), Cyan Fluorescence Protein (CFP), Blue Firefly Fluorescent proteins such as photoprotein (Blue Fluorescence Protein; BFP), yellow Fluorescence Protein (YFP), Red Fluorescence Protein (RFP), luciferase; ³ H, ¹⁴ C, ¹²⁵ I or Radioactive isotopes such as ¹³¹ I; biotin, avidin and the like.

Hereinafter, the present invention will be described more specifically by way of examples. However, the technical scope of the present invention is not limited to these examples.

[Preparation of peripheral blood mononuclear cell fraction from patients with tick allergic rhinitis]
For patients with tick allergic rhinitis (see Table 1), sublingual immunotherapy (actual drug group) using sublingual sublingual tablets (Stallergenes) and sublingual using placebo (Stallergenes) as a control Immunotherapy (placebo group) was performed in a double-blind comparative study. Peripheral blood was collected from patients in the active drug group and placebo group before and 1 year after the treatment, and a peripheral blood mononuclear cell fraction was prepared according to a conventional method and stored frozen at -80 ° C.

[Preparation of mite antigen-specific peripheral blood mononuclear cells 1]
The peripheral blood mononuclear cell fraction prepared according to the method described in the above item [Preparation of peripheral blood mononuclear cell fraction derived from mite allergic rhinitis patients] was thawed, and the culture solution (RPMI-1640, manufactured by WAKO) After culturing at 37 ° C. under 5% CO ₂ for 4 hours, the cells were stained with Cell Trace cell proliferation assay reagent (Thermo Fisher Scientific), and the culture solution was treated with 6.5 μg / mL mite antigen ( INDOOR biotechnologies), 20 JIU / mL human IL-2 (Immunes [manufactured by Shionogi Pharmaceutical Co., Ltd.)], and 100 ng / mL human IL-33 (manufactured by R & D Systems). and cultured for 7 days under 5% CO _2.

[Preparation of tick antigen-specific peripheral blood mononuclear cells 2]
The peripheral blood mononuclear cell fraction prepared according to the method described in the above item [Preparation of peripheral blood mononuclear cell fraction derived from tick allergic rhinitis patients] was thawed, and the culture solution (RPMI-1640, manufactured by WAKO) ) At 37 ° C. under 5% CO ₂ condition for 4 hours, and then stained with Cell Trace cell proliferation assay reagent (Thermo Fisher Scientific), and the culture solution was 6.5 μg / mL mite antigen. The culture medium was replaced with a culture solution containing INDOOR biotechnologies and 20 JIU / mL human IL-2 (Immunes [Shionogi Pharmaceutical Co., Ltd.]), and cultured at 37 ° C. under 5% CO ₂ for 7 days. Thereafter, the culture solution was replaced with a culture solution containing 50 ng / mL PMA (Phorbol 12-Myristate 13-Acetate) (manufactured by SIGMA) and 1 μM ionomycin (manufactured by Calbiochem), at 37 ° C. under 5% CO ₂ conditions. For 4 hours.

[Cell sorting and flow cytometry analysis 1]
Mononuclear cells (mononuclear cells) prepared according to the method described in the above item [Preparation of mite antigen-specific peripheral blood mononuclear cells 1], two types of cell surface markers (CD4 and Antigen-antibody reaction was performed under the condition of 4 ° C. for 30 minutes using an antibody against ST2) (see Table 2). Thereafter, the CD4 ⁺ T cell population and a CD4 ⁺ T cell population of ST2 ^+, and analyzed using a flow cytometer (FACSCantII, manufactured by BD Biosciences, Inc.), the ST2 ⁺ of CD4 ⁺ T cells to CD4 ⁺ T cells The ratio (ST2 ⁺ / CD4 ⁺ ratio) was measured. In addition, the amount of change in ST2 ⁺ / CD4 ⁺ ratio before and after treatment with sublingual immunotherapy was calculated using the formula (“ST2 ⁺ / CD4 ⁺ ratio after treatment with sublingual immunotherapy” − “before sublingual immunotherapy treatment”. The calculation was performed based on “ST2 ⁺ / CD4 ⁺ ratio” = “ST2 ⁺ / CD4 ⁺ change in ratio”) (see FIG. 1A).

[Cell sorting and flow cytometry analysis 2]
Three types of cell surface markers (CD4, CD27, and CD161) obtained by labeling mononuclear cells prepared according to the method described in the above item [Preparation 2 of mite antigen-specific peripheral blood mononuclear cells] with a fluorescent substance. Antigen-antibody reaction was carried out for 30 minutes under the condition of 4 ° C. using the antibody against (see Table 2). Next, in order to detect two types of markers (IL-5 and IL-13) present in the cells, the mononuclear cells were labeled with two types of intracellular markers (IL-5 and IL-13) labeled with a fluorescent substance. ) And an intracellular staining kit (Transcription Buffer set, manufactured by BD Pharmingen), and immunofluorescent staining was performed according to the protocol attached to the product. Thereafter, a CD4 ⁺ T cell group and a CD27 ⁻ , CD161 ⁺ , IL-5 ⁺ (production), and IL-13 ⁺ (production) CD4 ⁺ T cell group were divided into flow cytometers (FACSCantII, manufactured by BD Biosciences). ) And the ratio of CD4 ⁺ T cells to CD27 ⁻ , CD161 ⁺ , IL-5 ⁺ and IL-13 ⁺ to CD4 ⁺ T cells (CD27 ⁻ .CD161 ⁺ IL-5 ⁺ .IL-13 ⁺ / CD4 ⁺ ratio) and CD27 ⁻ .. Before and after treatment with sublingual immunotherapy. CD161 ⁺ IL-5 ⁺ . The amount of change in the IL-13 ⁺ / CD4 ⁺ ratio was expressed by the formula (“CD27 ⁻ .CD161 ⁺ IL-5 ⁺ .IL-13 ⁺ / CD4 ⁺ ratio after treatment with sublingual immunotherapy” − “sublingual immunotherapy CD27 ⁻ .CD161 ⁺ IL-5 ⁺ .IL-13 ⁺ / CD4 ⁺ ratio before treatment ”=“ CD27 ⁻ .CD161 ⁺ IL-5 ⁺ .IL-13 ⁺ / CD4 ⁺ ratio change ”) Calculated (see FIG. 1B).

[result]
The change in the ST2 ⁺ / CD4 ⁺ ratio did not decrease in the placebo group (1.1 ± 0.95), but decreased in the active drug group as −5.0 ± 0.82 ( (See FIG. 1A). In addition, CD27 ⁻ . CD161 ⁺ IL-5 ⁺ . Similarly, the amount of change in the IL-13 ⁺ / CD4 ⁺ ratio was not decreased in the placebo group (0.15 ± 0.22), whereas it was −1.2 ± 0.28 in the active drug group. It decreased (see FIG. 1B).

Therefore, the threshold value (cutoff value) of the amount of change in ST2 ⁺ / CD4 ⁺ ratio is set to 0, and the percentage of true positive patients in which the amount of change decreases (below 0) in the active drug group (sensitivity) And the ratio (specificity) of true negative patients in which the amount of change does not decrease (becomes greater than 0) in the placebo group, and the ratio of the total true positive patients and true negative patients to the entire active drug group and placebo group ( (Correction rate). As a result, it was shown that the active drug group and the placebo group in sublingual immunotherapy can be determined with high accuracy (sensitivity, specificity, and correct diagnosis rate) by using the decrease in ST2 ⁺ / CD4 ⁺ ratio as an index. (Refer to “ST2 ⁺ ” in Table 4).

In addition, CD27 ⁻ . CD161 ⁺ IL-5 ⁺ . The cut-off value of the change amount of the IL-13 ⁺ / CD4 ⁺ ratio is similarly set to 0, and the ratio (sensitivity) of true positive patients in which the above change amount decreases (becomes less than 0) in the active drug group The percentage of true negative patients (specificity) in which the amount of change does not decrease (greater than 0) in the placebo group, and the ratio of the true positive patients and true negative patients to the total of the active drug group and the placebo group (positive) (Diagnosis rate) was calculated. As a result, CD27 ⁻ . CD161 ⁺ IL-5 ⁺ . It was shown that the active drug group and the placebo group in sublingual immunotherapy can be determined with high accuracy by using a decrease in the change in the IL-13 ⁺ / CD4 ⁺ ratio as an index (see “CD27 ⁻ .CD161 ⁺ IL in Table 4). −5 ⁺ .IL-13 ⁺ ”).

Furthermore, the sensitivity, specificity, and correct diagnosis rate when the above two types of changes were combined were calculated. That is, the amount of change in ST2 ⁺ / CD4 ⁺ ratio in the active drug group decreases (becomes less than 0), and CD27 ⁻ . CD161 ⁺ IL-5 ⁺ . The percentage of true positive patients (sensitivity) in which the amount of change in IL-13 ⁺ / CD4 ⁺ ratio decreases (below 0) and the amount of change in ST2 ⁺ / CD4 ⁺ ratio in the placebo group does not decrease (over 0) to become), or CD27 ^-. CD161 ⁺ IL-5 ⁺ . Proportion of true negative patients (specificity) showing either or both of IL-13 ⁺ / CD4 ⁺ ratio change does not decrease (becomes greater than 0) and the above true value for the active drug group and the placebo group as a whole. When the percentage of positive patients and true negative patients (correction rate) is calculated, the active drug group and the placebo group in sublingual immunotherapy are more accurately compared to the case where the above two types of changes are used alone. It was shown that it can be determined (see “ST2 ⁺ and CD27 ⁻ .CD161 ⁺ IL-5 ⁺ .IL-13 ⁺ ” in Table 4). This accuracy is based on the conventional average adjusted symptom score (AASS) (see reference "Allergy. 2016 Jul 29. doi: 10.1111 / all.12996.") Decrease in change rate (less than -30%). Decision accuracy (see “AASS” in Table 5) and single change amount other than the above two types, that is, decrease in change amount of IL-5 ⁺ / CD4 ⁺ ratio (see “IL-5 ⁺ ” in Table 5) ), Decrease in CD27 ⁻ / CD4 ⁺ ratio change (see “CD27 ⁻ ” in Table 5), IL-5 ⁺ . Decrease in the amount of change in the IL-13 ⁺ / CD4 ⁺ ratio (see “IL-5 ⁺ .IL-13 ⁺ ” in Table 5), CD27 ⁻ . It was shown that it is superior to the determination accuracy based on the decrease in the change amount of the CD161 ⁺ / CD4 ⁺ ratio (see “CD27 ⁻ .CD161 ⁺ ” in Table 5).

In general, when the symptom change rate is −30% or more (symptom improvement rate is 30% or less), the placebo effect cannot be distinguished and is not judged to be effective (reference “J. Allergy Clin. Immunol. 2010 126”). 969-75 "). Therefore, in the active drug group, the cut-off value of the AASS change rate is set to -30%, the effective (Responder; R) group showing symptom improvement with the AASS change rate of less than -30%, and the AASS change rate of -30 It was examined whether it was possible to determine an ineffective (Non-responder; NR) group that did not show improvement in symptoms of at least%, and the ST2 ⁺ / CD4 ⁺ ratio change decreased (less than −2.5) and CD27 ⁻ . CD161 ⁺ IL-5 ⁺ . When judged in combination with a decrease in IL-13 ⁺ / CD4 ⁺ ratio (less than −0.74) (see “ST2 ⁺ and CD27 ⁻ .CD161 ⁺ IL-5 ⁺ .IL-13 ⁺ ” in Table 6) , Decrease in single change, ie, decrease in ST2 ⁺ / CD4 ⁺ ratio change (see “ST2 ⁺ ” in Table 6), CD27 ⁻ . CD161 ⁺ IL-5 ⁺ . Reduced change in IL-13 ⁺ / CD4 ⁺ ratio (see “CD27 ⁻ .CD161 ⁺ IL-5 ⁺ .IL-13 ⁺ ” in Table 6), decreased change in IL-5 ⁺ / CD4 ⁺ ratio (Table 7) (See “IL5 ⁺ ”), CD27 ⁻ / CD4 ⁺ ratio change decrease (see “CD27 ⁻ ” in Table 7), or CD27 ⁻ . Compared to the case of using a decrease in CD161 ⁺ / CD4 ⁺ ratio change (see “CD27 ⁻ .CD161 ⁺ ” in Table 7), R group and NR group are judged more accurately (sensitivity, specificity, and correct diagnosis rate). It was shown that it can be done. Even when the decrease in ST2 ⁺ / CD4 ⁺ ratio change (less than −2.5) is combined with the decrease in CD27 ⁻ / CD4 ⁺ ratio change (less than 1), the R group and the NR group are accurately determined. It was shown that it can be determined (see “ST2 ⁺ and CD27 ⁻ ” in Table 7).
The above results indicate that the number of ST2 ⁺ / CD4 ⁺ T cells in all CD4 ⁺ T cells before and after the start of immunotherapy using allergens, CD27 ⁻ , CD161 ⁺ , IL-5 ⁺ , or IL-13 ⁺ CD4 ⁺ When the number of T cells is measured and both are decreased, it indicates that immunotherapy using allergen can be accurately determined to be effective for allergic rhinitis patients.

The present invention contributes to predicting the early effects of allergen immunotherapy such as sublingual immunotherapy, reducing the burden on allergen immunotherapy patients, and reducing medical costs.

Claims

A method for determining the therapeutic effect of allergen immunotherapy for allergic rhinitis, comprising the following steps (a) and (b):
(A) In a peripheral blood mononuclear cell-containing sample obtained from a patient with allergic rhinitis after the start of allergen immunotherapy, or a T cell-containing sample obtained by culturing the peripheral blood mononuclear cell-containing sample in the presence of allergen Measuring the number of the following CD4-positive T cells (i) and CD4-positive T cells (ii) contained in
(I) ST2-positive CD4-positive T cells (ii) one or more CD4-positive T cells selected from the group consisting of CD27-negative, CD161-positive, IL-5-positive, and IL-13-positive (b) A step of comparing the number of cells of the CD4 positive T cells (i) and the number of cells of the CD4 positive T cells (ii) with respective control values, when both are lower than the respective control values, The step (b), which shows that immunotherapy using the allergen is likely to be effective for the allergic rhinitis patient;
The determination method according to claim 1, wherein the number of CD4 positive T cells (i) and CD4 positive T cells (ii) contained in the T cell-containing sample is measured in step (a).
3. The determination method according to claim 1, wherein the allergic rhinitis is tick allergic rhinitis, and the allergen immunotherapy is an allergen immunotherapy using a tick antigen.
The determination method according to any one of claims 1 to 3, wherein the CD4-positive T cells (ii) are CD27-negative.
The determination method according to claim 4, wherein the CD4 positive T cells (ii) are CD27 negative, CD161 positive, IL-5 positive and IL-13 positive CD4 positive T cells.
An allergenicity comprising an antibody that specifically binds to the following CD4 positive T cells (i), an antibody that specifically binds to the following CD4 positive T cells (ii), or a label thereof: A kit for determining the therapeutic effect of allergen immunotherapy for rhinitis.
(I) ST2-positive CD4-positive T cells (ii) one or more CD4-positive T cells selected from the group consisting of CD27-negative, CD161-positive, IL-5-positive, and IL-13-positive
The kit according to claim 6, wherein the allergic rhinitis is mite allergic rhinitis, and the allergen immunotherapy is an allergen immunotherapy using a tick antigen.
The kit according to claim 6 or 7, wherein the CD4 positive T cells (ii) are CD27 negative.
The kit according to claim 8, wherein the CD4 positive T cells (ii) are CD27 negative, CD161 positive, IL-5 positive and IL-13 positive CD4 positive T cells.
A biomarker for judging the therapeutic effect of allergen immunotherapy for allergic rhinitis, comprising the following CD4 positive T cells (i) and CD4 positive T cells (ii).
(I) ST2-positive CD4-positive T cells (ii) one or more CD4-positive T cells selected from the group consisting of CD27-negative, CD161-positive, IL-5-positive, and IL-13-positive
The biomarker according to claim 10, wherein the allergic rhinitis is tick allergic rhinitis and the allergen immunotherapy is an allergen immunotherapy using a tick antigen.
The biomarker according to claim 10 or 11, wherein the CD4 positive T cell (ii) is CD27 negative.
The biomarker according to claim 12, wherein the CD4-positive T cells (ii) are CD27-negative, CD161-positive, IL-5-positive and IL-13-positive CD4-positive T cells.