Biomarker for predicting onset of chronic graft-versus-host disease, and assessment method

Abstract

To provide a biomarker for predicting onset of the chronic graft-versus-host disease and an assessment method.SOLUTION: A biomarker for predicting onset of the chronic graft-versus-host disease is provided, the biomarker being a combination of the Tim3 protein and PD-1 protein.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] The present invention relates to a biomarker for predicting the onset of chronic graft-versus-host disease and a determination method.

Background Art

[0002] Hematopoietic stem cell transplantation such as bone marrow transplantation is widely performed as a radical treatment for hematological malignancies. As a late complication after hematopoietic stem cell transplantation, it is known that chronic graft-versus-host disease (chronic graft-versus-host disease: chronic GVHD or cGVHD) occurs. In chronic GVHD, immune cells of the blood provider (donor) after transplantation attack the whole body target organs (skin, liver, lung, eye, etc.) of the recipient who receives the transplantation, causing an excessive immune response, resulting in collagen disease-like chronic inflammation in multiple organs, which is a major risk for treatment.

[0003] As a systemic treatment method for chronic GVHD, administration of biological agents and immunosuppressants is performed, but once the disease develops, the risk of exacerbation is high. For example, as eye symptoms, severe inflammation and fibrosis occur in the conjunctiva, cornea, lacrimal gland, etc., causing severe dry eye that can lead to visual impairment, which is a factor reducing the quality of life and visual quality of long-term survivors. Therefore, it is desired to prevent exacerbation by early intervention. However, there is a problem that it is difficult to predict the disease because no biomarker for chronic GVHD, that is, a biomarker that shows a difference only in the group causing chronic GVHD, has been reported so far.

[0004] So far, as new biomarkers and combinations of biomarkers for detecting or predicting gastrointestinal graft-versus-host disease (GI GVHD) and for predicting and analyzing the response to treatment for acute GVHD, pancreatic regenerating protein 3α (regenerating islet-derived protein 3-alpha; Reg3α) and amino acids having a specific sequence contained in blood or urine have been reported (see, for example, Patent Document 1).

[0005] In addition, prevention of graft-versus-host disease (GVHD) using cyclosporine (CSP), a calcineurin inhibitor, is known to be a standard treatment method in allogeneic hematopoietic stem cell transplantation (HCT). However, there is a problem that a considerable number of patients develop chronic GVHD and long-term tolerance cannot be induced. To address this problem, after HCT, CD8 + T cells of allogeneic reactive donors rapidly differentiate into PD-1 + TIGIT+ terminal exhausted T cells (terminal Tex), and prevention of GVHD by cyclosporine (CSP) suppresses the expression of TOX in donor CD8 + T cells and promotes the differentiation from transitory Tex, which expresses both inhibitory receptors and effector molecules, to terminal Tex, suppressing tolerance induction (see, for example, Non-Patent Document 1).

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Non-Patent Documents

[0007]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0008] An object of the present invention is to solve the above-described conventional problems and achieve the following objects. That is, an object of the present invention is to provide a biomarker and a determination method capable of predicting the onset of chronic graft-versus-host disease.

Means for Solving the Problems

[0009] Means for solving the above problems are as follows. That is, <1> A biomarker for predicting the onset of chronic graft-versus-host disease, which is characterized by being a combination of Tim3 protein and PD-1 protein. <2> A determination method for predicting the onset of chronic graft-versus-host disease, comprising the steps of detecting the presence or amount of T cells expressing a biomarker that is a combination of Tim3 protein and PD-1 protein in blood cells obtained from a subject, and determining that there is a risk of onset of chronic graft-versus-host disease when T cells expressing the biomarker are present. The determination method is characterized by including the above steps. <3> The determination method according to <2>, wherein the subject is a subject from after hematopoietic stem cell transplantation to before the onset of the chronic graft-versus-host disease. <4> The determination method according to <2> or <3>, wherein the T cells are at least one of CD4-positive T cells and CD8-positive T cells. <5> The determination method according to any one of <2> to <4>, wherein the subject is at least one of a human, a humanized mouse, and a mouse. <6> Comparing the presence or amount of T cells expressing the biomarker in the blood cells obtained from the subject with the presence or amount of T cells expressing the biomarker in the blood cells obtained from a comparison subject, and based on the comparison, when the T cells expressing the biomarker in the blood cells obtained from the subject are not detected or the amount is reduced, the risk of onset of chronic graft-versus-host disease is reduced, when there is no change in the amount of T cells expressing the biomarker in the blood cells obtained from the subject, there is no change in the risk of onset of chronic graft-versus-host disease, or When T cells expressing the biomarker in the blood cells obtained from the subject are detected or the amount is increased, determining that the risk of developing chronic graft-versus-host disease is enhanced The determination method according to any one of <3> to <5>, further comprising

Advantages of the Invention

[0010] According to the present invention, it is possible to solve the above-described various problems, achieve the above object, and provide a biomarker and a determination method capable of predicting the onset of chronic graft-versus-host disease.

Brief Description of the Drawings

[0011]

Figure 1

Figure 2

Figure 3

Figure 4

Modes for Carrying Out the Invention

[0012] (Biomarker) The biomarker of the present disclosure is a biomarker for predicting the onset of chronic graft-versus-host disease, and is a combination of Tim3 protein and PD-1 protein.

[0013] (Determination Method) The determination method of the present disclosure is a determination method for predicting the onset of chronic graft-versus-host disease, and includes a step of detecting the presence or amount of T cells expressing a biomarker that is a combination of Tim3 protein and PD-1 protein in blood cells obtained from a subject, and a step of determining that there is a risk of onset of chronic graft-versus-host disease when T cells expressing the biomarker are present, and further includes other steps as necessary.

[0014] The present inventors have previously reported that in the spleen cells of a chronic GVHD mouse model, the cell population of PD-1 + is increased compared to the control, but the details have been unclear. If a biomarker that shows a difference only in the group that causes chronic GVHD and can be detected prior to the onset of chronic GVHD can be found, it will be effective for predicting the onset of chronic GVHD, and at the same time, it will be effective as an index for searching for drugs that can reduce the risk of onset of chronic GVHD, and has great clinical significance.

[0015] As a result of intensive studies to solve the above object, the present inventors have obtained the following findings. That is, when analyzing the time-dependent changes after bone marrow transplantation of T cells expressing Tim3 protein and PD-1 protein in a chronic GVHD mouse model by flow cytometry, prior to the onset of chronic GVHD, PD-1 + Tim3 + CD4 + T cells and PD-1 + Tim3 + CD8 + T cells were found to be detectable. Therefore, it was found that Tim3 protein and PD-1 protein can be used as biomarkers for predicting the onset of chronic GVHD, and the present invention has been completed.

[0016] Previously, mainly in the field of tumor immunology such as cancer, the expression of PD-1 protein and Tim3 protein has been used mainly as a marker for exhausted T cells in CD8 + T cells. PD-1+ Tim3 + CD8 + It is known that inflammation is suppressed by a decrease in the production of IFN-γ, TNF-α, and IL-2 due to exhaustion of T cells, (Zhou Q et al., Coexpression of Tim-3 and PD-1 identifies a CD8 + T-cell exhaustion phenotype in mice with disseminated acute myelogenous leukemia. Blood. (2011) 117 (17): 4501-4510.).

[0017] Similarly, in the field of tumor immunology, PD-1 + Tim3 + CD8 + T cells are known to cause a decrease in cytotoxic ability and a decrease in the ability to produce inflammatory cytokines due to exhaustion, thereby reducing antitumor activity (Ando M et al., Memory T cell, exhaustion, and tumor immunity. Immunol Med. (2020) 43(1): 1-9.).

[0018] CD4 + Regarding the expression of Tim3 protein in CD4 + T cells, in experiments using experimental autoimmune encephalomyelitis (EAE) model mice, it is known that the Tim3 protein in the disease group is increased, and the EAE symptoms are alleviated by inducing apoptosis of Th1 cells via the Tim3 protein (Zhu C et al., The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity. Nat Immunol. (2005) 6(12): 1245-1252; and Guo Q. Tim-3 on CD4 +T cells are associated with pathology in experimental autoimmune encephalomyelitis of mouse. European Journal of Inflammation. (2021). https: / / doi.org / 10.1177 / 20587392211030149).

[0019] On the other hand, in chronic GVHD, the relationship between the expression of Tim3 protein and PD-1 protein and the onset of chronic GVHD has not yet been reported.

[0020] According to the biomarker and determination method of the present disclosure, the onset risk of chronic GVHD can be predicted, and it has the advantage of being able to predict by a simple, minimally invasive or non-invasive method of collecting blood from a subject. Therefore, before the onset of chronic GVHD, it is expected that the treatment policy can be determined and early intervention in treatment can be carried out to prevent exacerbation.

[0021] <Tim3 protein> Tim3 (T-cell immunoglobulin mucin-3) protein is a 60 kDa member of the TIM family of immune regulatory molecules. It is also an immune checkpoint receptor expressed on a variety of immune system cells, including cytotoxic T cells, regulatory T cells (Tregs), NK cells, and several antigen-presenting cells (APCs) such as dendritic cells (DCs). Mature human TIM-3 consists of an extracellular domain (ECD) of 181 amino acids (aa), a transmembrane segment of 21 amino acids, and a cytoplasmic tail of 78 amino acids. Within the ECD, human TIM-3 has 58% amino acid sequence identity with mouse TIM-3.

[0022] As one embodiment of the mouse Tim3 protein, it has the amino acid sequence (281 amino acids) shown in SEQ ID NO: 1 as registered under Genbank accession ID: AAL35776. Also, a plurality of Tim3 variants have been reported. For example, the variant registered as the same protein under UniProt ID: Q8VIM0 (https: / / www.uniprot.org / ) is also included in the mouse Tim3 protein of this embodiment.

[0023] As one embodiment of the human Tim3 protein, it has the amino acid sequence (301 amino acids) shown in SEQ ID NO: 2 as registered under Genbank accession ID: AAL65158. Also, a plurality of Tim3 variants have been reported. For example, the variant registered as the same protein under UniProt ID: Q8TDQ0 is also included in the human Tim3 protein of this embodiment.

[0024] The Tim3 protein is not particularly limited as long as it is a protein transcribed, translated, and optionally post-translationally modified from the Tim3 locus, and can be appropriately selected according to the purpose. However, the amino acid sequence of the Tim3 protein is preferably 90% or more identical to the amino acid sequences of SEQ ID NOs: 1 to 2, and more preferably 95% or more identical.

[0025] From the examples described below, it was found that the proportion of T cells expressing Tim3 protein in spleen cells is specifically detected prior to the onset of chronic GVHD in a chronic GVHD mouse model. Therefore, by detecting the presence or amount of T cells expressing Tim3 protein in blood cells, it is possible to determine the risk of developing chronic graft-versus-host disease. Thus, the Tim3 protein can be used as a biomarker for determining the risk of developing chronic GVHD. Here, in mice, the number of cells in peripheral blood alone is limited. As an alternative to the analysis of peripheral blood, evaluation can be performed using spleen cells from which a sufficient number of cells can be obtained.

[0026] <PD-1 protein> PD-1 (Programmed cell death 1) protein is a receptor expressed on the surface of activated T cells. In the field of tumor immunology such as cancer, when the PD-1 receptor is activated by binding to PD-L1 (Programmed cell Death 1- Ligand 1), a ligand molecule expressed on the surface of antigen-presenting cells (cancer cells), it is known that T cells stop attacking target cells.

[0027] As one aspect of mouse PD-1 protein, as registered in Genbank accession ID: AAI19180, it has the amino acid sequence shown in SEQ ID NO: 3 (288 amino acids). In addition, multiple variants of PD-1 have been reported. For example, the variant registered as the same protein in UniProt ID: Q02242 is also included in the mouse PD-1 protein of this embodiment.

[0028] As one aspect of human PD-1 protein, as registered in Genbank accession ID: AAH74740, it has the amino acid sequence shown in SEQ ID NO: 4 (288 amino acids). In addition, multiple variants of Tim3 have been reported. For example, the variant registered as the same protein in UniProt ID: Q15116 is also included in the human PD-1 protein of this embodiment.

[0029] The PD-1 protein is not particularly limited as long as it is a protein transcribed, translated, and optionally post-translationally modified from the PD-1 locus, and can be appropriately selected according to the purpose. However, the amino acid sequence of PD-1 is preferably 90% or more identical to the amino acid sequences of SEQ ID NOs: 3 to 4, and more preferably 95% or more identical.

[0030] From the following examples, it has been found that the ratio of T cells expressing Tim3 protein and PD-1 protein in splenocytes is specifically detected prior to the onset of chronic GVHD in a chronic GVHD mouse model. Therefore, by detecting the presence or amount of T cells expressing Tim3 protein and PD-1 protein in blood cells, it is possible to determine that there is a risk of developing chronic graft-versus-host disease. Accordingly, the combination of Tim3 protein and PD-1 protein can be used as a biomarker for determining the risk of developing chronic GVHD.

[0031] Hereinafter, each step in the determination method of the present embodiment will be described. <Detection step> This is a step of detecting the presence or amount of T cells expressing a biomarker in blood cells obtained from a subject. In one aspect, the biomarker is a combination of Tim3 protein and PD-1 protein.

[0032] The subject is generally a mammal, and examples include humans, non-human primates, dogs, cats, humanized mice, mice, rats, cows, horses, pigs, etc. Among these, at least one of humans, humanized mice, and mice is preferred.

[0033] The subject may be any of an adult, an infant, or a child, and a subject who has received a hematopoietic stem cell transplantation such as a bone marrow transplantation, a peripheral blood stem cell transplantation, or a cord blood transplantation is preferred, and a subject after hematopoietic stem cell transplantation and before the onset of the chronic graft-versus-host disease is preferred. Here, the period until the onset of chronic graft-versus-host disease varies depending on the subject. For example, in mice, it is known to develop about 3 to 6 weeks after hematopoietic stem cell transplantation, and in humans, it is known to develop about 5 to 24 months after hematopoietic stem cell transplantation. Therefore, in mice, a subject up to 6 weeks after hematopoietic stem cell transplantation is preferred, and in humans, a subject up to 24 months after hematopoietic stem cell transplantation is preferred.

[0034] The blood cells obtained from the subject may be pretreated as necessary and presented for the detection of biomarkers.

[0035] The method for detecting T cells expressing the biomarker is not particularly limited and can be appropriately selected according to the purpose. Specifically, a flow cytometer (e.g., CytoFLEX S, Beckman Coulter, USA) and flow cytometry using an antibody that binds to the target molecule can be mentioned.

[0036] The T cells to be detected are preferably at least one of CD4-positive T cells and CD8-positive T cells. The CD4-positive T cells are glycoproteins and are T cells (mainly helper T cells) that express CD4 (cluster of differentiation 4), a cell surface antigen, and can be detected by known flow cytometry. The CD8-positive T cells are T cells (mainly cytotoxic T cells) that express CD8 (cluster of differentiation 8), a transmembrane glycoprotein that acts as a co-receptor for the T cell receptor, and can be detected by known flow cytometry.

[0037] As the antibody that binds to the target molecule, antibodies capable of detecting target molecules such as biomarkers such as Tim3 protein and PD-1 protein; differentiation antigens such as CD4 and CD8 by flow cytometry can be used, and a fluorescently labeled antibody is preferably used. There are no particular restrictions on the fluorescent label of the fluorescently labeled antibody, and known fluorescent labels can be appropriately selected according to the purpose. For example, fluorescein (FITC), allophycocyanin (APC), Brilliant Violet 421 (registered trademark) (Jackson, BV421), Brilliant Violet 510 (registered trademark) (Jackson, BV510), PE-Cy7 (Becton, Dickinson and Company) composed of R-phycoerythrin (PE) donor and acceptor dye Cy 781, and modified products thereof can be mentioned.

[0038] By the above detection, the presence, absence, amount, or effective amount of T cells expressing the biomarker in blood cells obtained from the subject can be detected or measured. For example, the ratio of T cells expressing the biomarker can be detected or measured.

[0039] <Determination step> This is a step of determining that there is a risk of developing chronic graft-versus-host disease when T cells expressing the biomarker are present. The detection limit value of the presence or amount of the biomarker can be set according to the antibody used and the detection limit value of flow cytometry. However, a reference value or cut-off value may be provided according to the biomarker.

[0040] [Screening method] As one aspect of the determination method, by comparing the subject with a subject serving as a comparative control (comparative subject), for example, the therapeutic effect in a subject administered with a therapeutic drug or a candidate drug can be evaluated, and screening of a therapeutic drug or a candidate drug can be performed. In the present embodiment, the determination method further includes a comparison step in addition to the detection step and the determination step.

[0041] <Comparison step> The comparison step is a step of comparing the presence or amount of T cells expressing the biomarker in the blood cells obtained from the subject with the presence or amount of T cells expressing the biomarker in the blood cells obtained from the comparison subject. The amount of the biomarker may be an absolute value or a relative value, as long as it is a comparable value.

[0042] The Tim3 protein and PD-1 protein that contribute as the biomarker, and the T cells expressing the biomarker transiently increase from after hematopoietic stem cell transplantation to the onset of chronic graft-versus-host disease and then decrease. Therefore, as the measured value (comparative measured value) of T cells expressing the biomarker in the blood cells obtained from the comparison subject, it is preferable to use measured values from a plurality of comparison subjects that have been plotted or statistically processed over time after hematopoietic stem cell transplantation. Also, when screening for the administration (treatment) of a therapeutic drug or a candidate drug, as the measured value of T cells expressing the biomarker in the blood cells obtained from the subject, it is preferable to use measured values from a plurality of subjects who have undergone the same administration (treatment) and have been plotted or statistically processed over time after hematopoietic stem cell transplantation.

[0043] <Determination step> The determination step of the present embodiment is based on the comparison, (1) When T cells expressing the biomarker in the blood cells obtained from the subject are no longer detected or the amount is reduced, the risk of onset of chronic graft-versus-host disease is reduced, (2) When there is no change in the amount of T cells expressing the biomarker in the blood cells obtained from the subject, there is no change in the risk of onset of chronic graft-versus-host disease, or (3) When T cells expressing the biomarker in the blood cells obtained from the subject are detected or the amount is increasing, it is a step of determining that the risk of onset of chronic graft-versus-host disease is enhanced. Thus, for example, it is possible to evaluate the therapeutic effect in a subject administered with a therapeutic drug or a candidate drug, and to perform evaluations such as screening of a therapeutic drug or a candidate drug.

Example

[0044] Hereinafter, the present invention will be described more specifically based on examples, but the present invention is not limited to the following examples.

[0045] <Materials and Methods> <<Mouse>> B10.D2 / nSnSlc at 7 - 9 weeks of age and BALB / cCrSlc were purchased from Sankyo Labo Service Co., Ltd. and used.

[0046] <<Bone Marrow Transplantation>> In allogeneic mice (disease group) that develop GVHD, bone marrow transplantation (BMT) was performed between allogeneic individuals with major histocompatibility antigen identity and minor antigen non - identity. Specifically, male B10.D2 / nSnSlc at 8 - 9 weeks of age was used as a donor, and female BALB / cCrSlc at 8 - 9 weeks of age was used as a recipient. In syngeneic mice (control group) that do not develop GVHD, bone marrow transplantation was performed between syngeneic individuals with major histocompatibility antigen identity and minor antigen identity. Specifically, bone marrow transplantation was performed in the same manner as in the disease group, except that male BALB / cCrSlc at 8 - 9 weeks of age, which was syngeneic with the recipient, was used as a donor. In addition, bone marrow transplantation experiments were performed using n = 4 - 6 individuals under each condition, and the same experiment was independently performed 2 times (n = 8 - 11 for each condition in each of the 2 experiments).

[0047] As shown in Figure 1, first, recipient mice were irradiated with 7 Gy of X - rays. Next, a suspension of 1×10 6 bone marrow cells from the donor and 2×10 6 spleen cells was transplanted into recipient mice by tail vein injection. As a solvent for the suspension, RPMI1640 medium (Thermofisher) was used.

[0048] <<Flow Cytometry>> Two weeks, three weeks, four weeks, five weeks, and six weeks after bone marrow transplantation, cells were collected from the spleens of mice at five time points, and a suspension was prepared. Analysis was performed using a flow cytometer (CytoFLEX S, Beckman Coulter, USA). The following antibodies were used as antibodies. Anti-CD4 antibody-negative cells were evaluated as CD8-positive cells. ·FITC conjugated anti-Tim3 (ebio science: 11-5870-82), ·APC conjugated anti-PD-1 (Biolegend: 109112), ·BV421 conjugated anti-CD4 (Biolegend: 100437), ·BV510 conjugated anti-CD45 (Biolegend: 103138), and ·PE-Cy7 conjugated anti-CD3e (Biolegend: 100320)

[0049] The following antibodies were used as isotype controls for each antibody. ·FITC conjugated anti-Rat IgG2a κ (ebio science: 11-4321-42), and ·APC conjugated anti-Rat IgG2b, κ (Biolegend: 400612)

[0050] <<Statistical Analysis>> Statistical analysis was performed by two-sided unpaired one-way ANOVA. A P-value of less than 0.05 was considered to indicate a significant difference. Statistical analysis and graph creation were performed using GraphPad Prism 9.1.2.226, a medical statistical analysis software.

[0051] <Results> The spleen cells were analyzed by flow cytometry from 2 weeks to 6 weeks after bone marrow transplantation to examine whether there were changes in the temporal profiles of T cell fractions expressing Tim3 protein and PD-1 protein between allogeneic mice (disease group) and syngeneic mice (control group). In addition, the temporal profile changes of T cell fractions overexpressing CD44 were examined.

[0052] The results are shown in Figs. 2 to 4. Fig. 2 is a graph showing the flow cytometry analysis detecting the profiles of PD-1 + Tim3 + CD4 + T cells after bone marrow transplantation, Fig. 3 is a graph showing the flow cytometry analysis detecting the profiles of PD-1 + Tim3 + CD8 + T cells after bone marrow transplantation, and Fig. 4 is a graph showing the flow cytometry analysis detecting the profiles of CD44 high PD-1 + T cells after bone marrow transplantation.

[0053] In Figs. 2 to 4, 2w to 6w indicate the number of weeks elapsed since bone marrow transplantation, "Syn" indicates syngeneic mice (control group), and "Allo" indicates allogeneic mice (disease group). *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001 respectively indicate, and "ns" indicates no significant difference.

[0054] Also, the vertical axis of Fig. 2 indicates the percentage (%) of PD-1 + Tim3 + CD4 + CD4 + T cells with respect to the total number of CD4-positive T cells (CD4 + T cells), the vertical axis of Fig. 3 indicates the percentage (%) of PD-1 + Tim3 + CD8 + T cells with respect to the total number of CD8-positive T cells (CD8 +CD44 relative to the total number of high PD-1 + Percentage of T cells (%).

[0055] From the results in Figure 2, in the CD4 + T cell fraction of splenocytes from allogeneic mice (disease group), which is a chronic GVHD mouse model, at each time point 2 weeks, 3 weeks, and 4 weeks after bone marrow transplantation, T cells expressing Tim3 protein and PD-1 protein (PD-1 + Tim3 + T cells) were found to increase significantly.

[0056] CD8 + From the results in Figure 3 evaluating the CD8 + T cell fraction of splenocytes from allogeneic mice (disease group), at each time point 2 weeks, 3 weeks, and 4 weeks after bone marrow transplantation, PD-1 + Tim3 + T cells were found to increase significantly.

[0057] In allogeneic mice (disease group), it has been confirmed that chronic graft-versus-host disease (chronic GVHD) develops at the stage from 3 weeks to 6 weeks after bone marrow transplantation, using the changes in body weight, the decrease in tear production, and the significant increase in the GVHD score on the body surface compared to syngeneic mice as indicators (Sato S, et al., Positive Effects of Oral Antibiotic Administration in Murine Chronic Graft-Versus-Host Disease. Int J Mol Sci, (2021) 22(7):3745.). Also, in terms of tissue changes, it has been confirmed by evaluating the development of fibrosis and the significant increase in the infiltration of immune cells into GVHD target organs compared to the control (He J, et al., Ocular Surface and Tear Film Characteristics in a Sclerodermatous Chronic Graft-Versus-Host Disease Mouse Model. Cornea. 2018 Apr;37(4):486-494.; Yamane M, et al., Senescence-associated secretory phenotype promotes chronic ocular graft-vs-host disease in mice and humans. FASEB J. 2020 Aug;34(8):10778-10800.; and Ogawa Y, et al., MHC-compatible bone marrow stromal / stem cells trigger fibrosis by activating host T cells in a scleroderma mouse model. Elife. 2016 Jan 26;5:e09394.).

[0058] In this example, specifically, in addition to the appearance such as diarrhea, hair loss, deterioration of posture, and skin sclerosis, fibrosis and infiltration of inflammatory cells in the lacrimal gland were evaluated, and it was evaluated that chronic GVHD had developed. In contrast, PD-1 +Tim3 + CD4 + T cells and PD-1 + Tim3 + CD8 + Since the cell ratios of all of these T cells increase 2 weeks after bone marrow transplantation (i.e., 2 to 4 weeks prior to the onset of chronic GVHD), it was found that they can be used as biomarkers for predicting the onset of chronic graft-versus-host disease.

[0059] Among them, PD-1 + Tim3 + CD4 + The ratio of T cells is highest 2 weeks after bone marrow transplantation and decreases by the onset of chronic GVHD (Figure 2). Also, since this feature is only seen in allogeneic (allogeneic transplantation where the major histocompatibility antigens match and the minor antigens do not match), it was suggested that it is specifically expressed before the onset of chronic GVHD and can be used as a biomarker for predicting the onset of chronic GVHD.

[0060] Also, PD-1 + Tim3 + CD8 + The ratio of T cells increases 2 weeks after bone marrow transplantation and gradually decreases towards the onset of chronic GVHD (Figure 3). Therefore, it was suggested that by evaluating the biomarker in at least either CD4-positive T cells or CD8-positive T cells, the risk of onset of chronic GVHD can be predicted, and the risk of onset of chronic GVHD can also be predicted by combining the two.

[0061] Furthermore, the population of CD44 high PD-1 + T cells, which are considered senescent T cells, increased relatively after 3 to 4 weeks from bone marrow transplantation (Figure 4).

[0062] <Discussion> In the field of tumor immunology such as mainly cancer, the expression of PD-1 protein and Tim3 protein is mainly in CD8 +It has been reported that it is used as a marker for exhausted T cells in T cells. From the results of FIGS. 2 to 3, it was first shown that in allogeneic mice (disease group), which is a chronic GVHD mouse model, exhausted T cells expressing PD-1 protein and Tim3 protein are generated during the process of the onset of chronic GVHD and prior to the onset. Also, in addition to CD8 + T cells, in CD4 + T cells, it was also revealed that exhausted T cells expressing PD-1 protein and Tim3 protein are generated prior to the onset of chronic GVHD.

[0063] During the period from after hematopoietic stem cell transplantation to the onset of chronic GVHD, it is considered that a strong immune response by highly reactive T cells occurs. At that time, a population of exhausted T cells associated with strong reactivity is considered to be PD-1 + Tim3 + CD4 + exhausted T cells and PD-1 + Tim3 + CD8 + exhausted T cells. Excessive inflammation by highly reactive T cells is harmful to the individual, so inflammation is terminated by cell death such as activation-induced cell death (AICD).

[0064] The PD-1 + Tim3 + CD4 + exhausted T cells and PD-1 + Tim3 + CD8 + exhausted T cells, which were clarified in this example, are also considered to be heading in the direction of causing apoptosis and terminating inflammation, as the cell count decreases with a peak at 2 weeks after bone marrow transplantation. On the other hand, a part of pathological cell populations including the population of CD44 high PD-1 + T cells (FIG. 4), which are senescent cells of T cells generated during strong inflammation induction, survive, and these are considered to be involved in the onset of chronic GVHD.

[0065] As described above, in allogeneic mice (disease group), inflammation associated with the Allo reaction (an immune reaction caused between allogeneic individuals that do not cross species) occurs, and some inflammation subsides along with T cell exhaustion. On the other hand, it is thought that some pathological T cells survive and cause chronic GVHD by inducing inflammation in the target organs.

[0066] According to the biomarker and determination method of the present embodiment, the risk of developing chronic GVHD can be predicted using the biomarker, and it has the advantage of being able to predict by a simple, minimally invasive or non-invasive method of collecting blood from a subject. As a result, before the onset of chronic GVHD, it is possible to determine the treatment policy and perform early intervention in treatment, and it is expected to prevent exacerbation.

Claims

Claim 1 A biomarker for predicting the onset of chronic graft-versus-host disease, characterized in that it is a combination of Tim3 protein and PD-1 protein. A biomarker characterized by being a combination of Tim3 protein and PD-1 protein. Claim 2 A determination method for predicting the onset of chronic graft-versus-host disease, comprising: detecting the presence or amount of T cells expressing a biomarker that is a combination of Tim3 protein and PD-1 protein in blood cells obtained from a subject; when T cells expressing the biomarker are present, determining that there is a risk of onset of chronic graft-versus-host disease; A determination method characterized by including the above steps. Claim 3 The determination method according to claim 2, wherein the subject is a subject after hematopoietic stem cell transplantation and before the onset of the chronic graft-versus-host disease. Claim 4 The determination method according to claim 2 or 3, wherein the T cells are at least one of CD4-positive T cells and CD8-positive T cells. Claim 5 The determination method according to claim 2 or 3, wherein the subject is at least one of a human, a humanized mouse, and a mouse. Claim 6 comparing the presence or amount of T cells expressing the biomarker in the blood cells obtained from the subject with the presence or amount of T cells expressing the biomarker in the blood cells obtained from a comparison subject; and based on the comparison, when the T cells expressing the biomarker in the blood cells obtained from the subject are not detected or the amount is reduced, determining that the risk of onset of chronic graft-versus-host disease is reduced; when there is no change in the amount of T cells expressing the biomarker in the blood cells obtained from the subject, determining that there is no change in the risk of onset of chronic graft-versus-host disease, or when the T cells expressing the biomarker in the blood cells obtained from the subject are detected or the amount is increased, determining that the risk of onset of chronic graft-versus-host disease is enhanced; The determination method according to claim 2 or 3, further including the above steps.

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