Peripheral blood biomarkers for evaluating antitumor immune effects after radiation therapy

Abstract

The present invention provides a peripheral blood biomarker for evaluating the anti-tumor immune effect of radiation therapy. The present invention also provides a method for using the composition of cell subpopulations in a sample obtained from a subject as an indicator of immune activation by radiation therapy in the subject. CD4, which correlates with dendritic cell stimulation in anti-tumor immune responses, is also provided. + By comparing the amount of a T cell subpopulation or a dendritic cell subpopulation that correlates with dendritic cell stimulation in an anti-tumor immune response with a standard, the presence and / or degree of immune activation occurring in a subject due to radiation therapy can be determined.

Description

[Technical field]

[0001] The present invention relates to the field of cancer treatment, and in particular to evaluation of the effect of radiation therapy on immunity in cancer treatment. [Background technology]

[0002] It has been reported that radiation therapy for cancer can cause cancer outside the radiation field to shrink, a phenomenon known as the abscopal effect. The abscopal effect is thought to be a phenomenon that occurs through some kind of immune activation, but the detailed mechanism is largely unknown, and no biomarkers have been found to quantify or evaluate the abscopal effect. Summary of the Invention [Means for solving the problem]

[0003] The present invention provides a method for using the composition of cell subpopulations in a sample obtained from a subject as an indicator of the immune activation caused by radiation therapy in the subject.By comparing the amount of specific cell subpopulations described herein with a standard, it is possible to determine the presence and / or degree of immune activation caused by radiation therapy that occurs in the subject.

[0004] Cell subpopulations that can be used as indicators in the present invention include, but are not limited to, CD4 + CD8 T cell subpopulations, CD8 T cell subpopulations, and CD8 T cell subpopulations that correlate with dendritic cell stimulation in antitumor immune responses + CD4 T cell subpopulations correlate with dendritic cell stimulation in antitumor immune responses. + T cell subpopulations include, for example, CD62L low CD4 + A subpopulation of cells within the T cell population (e.g., CD62L low CD4 + T cell subpopulation itself, or ICOS+ CD62L low CD4 + Dendritic cell subpopulations that correlate with dendritic cell stimulation in anti-tumor immune responses include, for example, HLA-DR + CD141 + CD11c + CD8 correlates with dendritic cell stimulation in antitumor immune responses. + T cell subpopulations include, for example, CD137 + CD62L low CD8 + T cell subpopulations, etc.

[0005] In a further embodiment of the present invention, the immune activation caused by radiation therapy occurring in a subject can be shown to indicate whether or not to administer cancer immunotherapy to the subject, or when to administer cancer immunotherapy.It is considered to be advantageous to administer cancer immunotherapy that utilizes immune response against cancer at the time when immune activation caused by radiation therapy occurs, but there has been no biomarker to determine whether immune activation occurs due to radiation therapy.

[0006] The cancer immunotherapy preferably includes the cancer immunotherapy comprising the administration of an immune checkpoint inhibitor. The administration of the immune checkpoint inhibitor may be combined with other forms of treatment, or may be combined with different agents (e.g., different immune checkpoint inhibitors).

[0007] Examples of embodiments of the present invention are presented in the following sections. (Item 1) A method for determining whether a composition of a cell subpopulation in a sample obtained from a subject who has undergone radiation therapy is an indicator of immune activation caused by radiation therapy in the subject, comprising: analyzing the composition of cell subpopulations in said sample obtained from said subject; CD4 correlates with dendritic cell stimulation in the anti-tumor immune response in the sample. +A comparison of the amount of the T cell subpopulation to a reference indicates the presence or absence of immune activation in the subject. (Item 2) A method for determining whether a composition of a cell subpopulation in a sample obtained from a subject who has undergone radiation therapy is an indicator of immune activation caused by radiation therapy in the subject, comprising: analyzing the composition of cell subpopulations in said sample obtained from said subject; A comparison of the amount of a dendritic cell subpopulation in the sample that correlates with dendritic cell stimulation in an anti-tumor immune response to a reference indicates the presence or absence of immune activation in the subject. (Item 3) A method for determining whether a composition of a cell subpopulation in a sample obtained from a subject who has undergone radiation therapy is an indicator of immune activation caused by radiation therapy in the subject, comprising: analyzing the composition of cell subpopulations in said sample obtained from said subject; CD8 correlates with dendritic cell stimulation of the anti-tumor immune response in the sample + A comparison of the amount of the T cell subpopulation to a reference indicates the presence or absence of immune activation in the subject. (Item 4) CD4 correlates with dendritic cell stimulation of the anti-tumor immune response in the sample. + The abundance of T cell subpopulations and CD8 correlates with dendritic cell stimulation in antitumor immune responses. + The method according to any of the preceding paragraphs, wherein the presence or absence of immune activation in the subject is indicated by comparing the amount of at least two selected from the group consisting of the amount of T cell subpopulations with a reference. (Item 5) CD4 correlates with dendritic cell stimulation in the antitumor immune response + A subpopulation of T cells expressing CD62L low CD4 + The method according to any of the preceding items, wherein the T cell population is a cell subpopulation within the T cell population. (Item 6) CD4 correlates with dendritic cell stimulation in the antitumor immune response + A subpopulation of T cells expressing CD62Llow CD4 + T cell subpopulations or CCR7 + CD45RA - CD62L low CD4 + The method of any preceding claim, wherein the T cell subpopulation. (Item 7) CD4 correlates with dendritic cell stimulation in the antitumor immune response + T cell subpopulations are + CD62L low CD4 + T cell subpopulation, LAG3 + CD62L low CD4 + T cell subpopulations, PD-1 + CD62L low CD4 + T cell subpopulations or CD28 + CD62L low CD4 + The method of any preceding claim, wherein the T cell subpopulation. (Item 8) The dendritic cell subpopulation correlated with dendritic cell stimulation in the anti-tumor immune response is HLA-DR + CD141 + CD11c + The method according to any preceding claim, wherein the cell is a subpopulation. (Item 9) CD8 correlates with dendritic cell stimulation in the antitumor immune response + A subpopulation of T cells expressing CD62L low CD8 + The method according to any of the preceding items, wherein the T cell population is a cell subpopulation within the T cell population. (Item 10) CD8 correlates with dendritic cell stimulation in the antitumor immune response + A subpopulation of T cells expressing CD137 + CD62L low CD8 + The method of any preceding claim, wherein the T cell subpopulation. (Item 11) 2. The method of any of the preceding items, wherein the sample is a peripheral blood sample. (Item 12) 20. The method of any of the preceding items, wherein the reference is the amount of the cell subpopulation in a sample from the subject prior to the radiation therapy. (Item 13) The method of any of the preceding items, wherein an increase in the amount of the cell subpopulation in the sample compared to the reference indicates that radiation therapy-induced immune activation has occurred in the subject. (Item 14) The method of any of the preceding items, wherein the amount of the cell subpopulation in the sample is not increased from the reference, indicating that radiation therapy has not caused immune activation in the subject. (Item 15) 2. The method according to any of the preceding items, wherein the radiation therapy is administered to an area that includes a tumor. (Item 16) The method of any of the preceding items, wherein the occurrence of immune activation due to radiation therapy in the subject further indicates that the subject should be administered a cancer immunotherapy comprising an immune checkpoint inhibitor at this time. (Item 17) The method according to any of the preceding items, wherein the lack of immune activation due to radiation therapy in the subject further indicates that the subject should be administered radiation therapy again. (Item 18) The method according to any of the preceding items, further defined as a method for using the composition of cell subpopulations in samples obtained from a subject at multiple time points as an index for monitoring immune activation due to radiation therapy in the subject, comprising a step of analyzing the composition of cell subpopulations in samples obtained from the subject at multiple time points. (Item 19) 1. A composition comprising an immune checkpoint inhibitor for treating cancer in a subject, comprising: The composition is administered to a subject who has undergone radiation therapy, and in which immune activation has been shown to occur in the subject by a method according to any one of the preceding items. (Item 20) The composition described in the preceding item, wherein the immune checkpoint inhibitor is a PD-1 inhibitor and / or a PD-L1 inhibitor. (Item 21) The composition of any of the preceding items, further characterized in that it is administered in combination with one or more additional agents. (Item 22) The composition of any of the preceding items, wherein the additional agent comprises a second immune checkpoint inhibitor. (Item 23) A product comprising the composition described in any of the preceding items and a package insert stating that the composition is used in combination with radiation therapy. (Item 24) A kit for determining whether immune activation due to radiation therapy has occurred in a subject, comprising: (A) CD4 and CD62L; (B) (i) a marker selected from ICOS, PD-1, LAG-3 and CD28, (ii) CD4, and (iii) CD62L; (C) CD11c, CD141 and HLA-DR; (D) CD11c, CD123 and HLA-DR; or (E) CD8, CD62L and CD137 A kit comprising a detection agent for (Item 25) A composition comprising an immune checkpoint inhibitor for treating cancer, the composition being administered to a subject who has undergone radiation therapy, the subject having an immune cell composition that is not responsive to cancer immunotherapy prior to receiving the radiation therapy. (Item 26) The immune cell composition that is not responsive to the cancer immunotherapy is a CD4 + CD62L low The composition described in the preceding paragraph, which is an amount or proportion of a cell subpopulation. (Item 27) The composition of any of the preceding items, wherein the immune checkpoint inhibitor comprises an anti-PD-1 antibody and / or an anti-PD-L1 antibody. Effect of the Invention

[0008] The present invention allows the quantification or evaluation of immune activation caused by radiation therapy. This allows the priming phase activation and effector T cell increase effect caused by radiation therapy to be monitored over time. Furthermore, the present invention allows the determination of the timing of immune checkpoint (e.g., PD-1) inhibitory therapy after radiation therapy. In addition, the biomarker of the present invention can be a biomarker for evaluating the effect of a treatment that transforms immune checkpoint inhibitor-ineffective to effective by radiation pseudovaccine therapy aimed at the T cell priming effect, and an index for selecting patients who require treatment for such transformation. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 shows the change in peripheral blood T cell composition before and after chest radiotherapy. The left panel shows the percentage of the CD62LlowCD4+ T cell subpopulation in the CD4+ T cell population. The middle panel shows the percentage of the FOXP3+CD25+CD4+ T cell subpopulation in the CD4+ T cell population. The right panel shows the percentage of the CD8+ T cell subpopulation in the T cell population. It can be seen that the percentage of the CD62LlowCD4+ cell subpopulation in the CD4+ cell population is significantly increased. [Diagram 2]FIG. 2 shows changes in peripheral blood CD4+ T cell composition before and after chest radiotherapy. The upper left shows the percentage of the CD4+ T cell subpopulation in the T cell population. The upper right shows the percentage of the CD62LlowCD4+ T cell subpopulation in the CD4+ T cell population. The lower left shows the percentage of the ICOS+CD62LlowCD4+ T cell subpopulation in the CD62LlowCD4+ T cell population. The lower right shows the percentage of the FOXP3+CD25+CD4+ T cell subpopulation in the CD4+ T cell population. It can be seen that the percentage of the CD62LlowCD4+ cell subpopulation in the CD4+ cell population and the percentage of the ICOS+CD62LlowCD4+ cell subpopulation in the CD62LlowCD4+ cell subpopulation are significantly increased. [Diagram 3] Figure 3 shows the change in the percentage of the CD62LlowCD4+ cell subpopulation in the CD4+ cell population for each type of treatment. It can be seen that the percentage of the CD62LlowCD4+ cell subpopulation in the CD4+ cell population tends to increase with radiation therapy in each type of treatment. Curative TRT: therapeutic chest radiation therapy, curative CRT: therapeutic chemotherapy combined with radiation therapy, palliative TRT: palliative chest radiation therapy. [Figure 4] 4 shows changes in peripheral blood myeloid DC cell composition before and after thoracic radiotherapy. The left panel shows the percentage of the HLA-DR+CD141+CD11c+ cell subpopulation in the CD141+CD11c+ cell population. The right panel shows the percentage of the CD141+CD11c+ cell subpopulation in the PBMC cell population. It can be seen that the percentage of the HLA-DR+CD141+CD11c+ cell subpopulation in the CD141+CD11c+ cell population is significantly increased. [Diagram 5] 5 is a graph showing the changes in the percentage of the CD62LlowCD4+ cell subpopulation in the CD4+ cell population and the percentage of the HLA-DR+CD141+CD11c+ cell subpopulation in the CD141+CD11c+ cell population up to about one month after radiation therapy. The percentages of the CD62LlowCD4+ cell subpopulation and the HLA-DR+CD141+CD11c+ cell subpopulation, which were temporarily increased, tended to return to the levels before radiation therapy. [Figure 6] 6 is a diagram showing the relationship between the percentage of CD62Llow CD4+ T cells among CD4+ T cells and the percentage of effector memory cells (CCR7-CD45RA-) CD4+ T cells among CD4+ T cells. It is understood that the amounts of these cell subpopulations are strongly correlated. [Figure 7] Figure 7 shows the change in peripheral blood T cell composition before and after radiation therapy. Figure 7a shows the change in the percentage of CD62LlowCD4+ T cell subpopulation in the CD4+ T cell population (P<0.0001, paired t-test). Figure 7b shows the change in the percentage of HLA-DRhighCD11c+CD123-mDC subpopulation (P=0.0008, paired t-test). Figure 7c shows the correlation between pre-treatment %CD62LlowCD4+ T cells and PFS after TRT or CRT in patients who did not receive durvalumab combination therapy (n=34). [Figure 8] FIG. 8 shows the change in expression of immune checkpoint molecules on CD62LlowCD4+ T cells before and after radiation therapy (paired t-test). [Figure 9] Figure 9 shows the changes in T cell subpopulations based on CD4+ T cells gated for CCR7 and CD45RA before and after radiation therapy. Naive: CCR7+CD45RA+, CM: CCR7+CD45RA-, EM: CCR7-CD45RA- (paired t-test). [Figure 10] FIG. 10 shows the dynamics of %mDC, %CD62LlowCD4+T cells, and immune checkpoint molecule expression before radiation therapy, at the completion of radiation therapy, and up to about one month after radiation therapy (one-way ANOVA with Tukey post-hoc analysis). [Figure 11] Figure 11 shows the changes in T cell subpopulations based on CD8+ T cells gated for CCR7 and CD45RA before and after radiation therapy. Naive: CCR7+CD45RA+, CM: CCR7+CD45RA-, EM: CCR7-CD45RA-, EMRA: CCR7-CD45RA+ (paired t-test). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The present invention will be described below while showing the best mode. Throughout this specification, the expression of the singular form should be understood to include the concept of the plural form, unless otherwise specified. Therefore, the singular article (for example, in the case of English, "a", "an", "the", etc.) should be understood to include the concept of the plural form, unless otherwise specified. In addition, it should be understood that the terms used in this specification are used in the sense commonly used in the field, unless otherwise specified. Therefore, unless otherwise defined, all technical terms and scientific and technical terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In case of conflict, this specification (including definitions) will take precedence.

[0011] The following provides definitions of terms particularly used in this specification and / or explains basic technical content as appropriate.

[0012] (definition) As used herein, the term "biomarker" refers to a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathological processes, or pharmacological responses to a therapeutic intervention.

[0013] As used herein, the terms "cancer" and "carcinoma" are used interchangeably and refer to a malignant tumor that is highly atypical, grows faster than normal cells, and can destructively invade surrounding tissues or cause metastasis, or a state in which such a malignant tumor exists. In the present invention, cancer includes, but is not limited to, solid cancers and hematopoietic tumors.

[0014] As used herein, the term "cancer immunotherapy" refers to a method for treating cancer using a living organism's defense mechanisms, such as the immune system.

[0015] As used herein, the term "anti-tumor immune response" refers to any immune response against a tumor in a living body.

[0016] As used herein, the term "dendritic cell stimulation in anti-tumor immune response" refers to any stimulation that activates dendritic cells, which occurs during the immune response against tumors in vivo. This stimulation can be one of the factors that directly or indirectly cause an anti-tumor immune response. Representatively, but not limited to, dendritic cell stimulation in anti-tumor immune response is CD4 + It can be activation of dendritic cells by T cells (e.g., effector T cells), whereby the activated dendritic cells become CD8 + Stimulates T cells and stimulates CD8 + T cells exert antitumor effects.

[0017] As used herein, "correlate" refers to two events having a statistically significant correlation. For example, "a relative amount of B correlated with A" refers to the occurrence of event A, whereby the relative amount of B is affected (e.g., increased or decreased) in a statistically significant manner.

[0018] As used herein, "flow cytometry" refers to a technique for measuring the number of cells, solids, and other biological particles suspended in a liquid, as well as the physical, chemical, and biological properties of each particle.

[0019] As used herein, "immune activation" refers to an increase in the immune system's ability to eliminate foreign substances in the body, and may be indicated by an increase in the amount of any factor (e.g., immune cells or cytokines) that acts positively on immune function.

[0020] As used herein, the term "cell subpopulation" refers to any collection of cells that share some common characteristic in a cell population that contains cells with diverse characteristics. If a specific name is known in the art, such a term can be used to refer to a specific cell subpopulation, or any property (e.g., expression of a cell surface marker) can be described to refer to a specific cell subpopulation.

[0021] As used herein, the "amount" of a cell subpopulation includes the absolute number of a cell and the relative amount of the cell in a cell population. LOW CD4 + The "amount of a T cell subpopulation" may be a relative amount to the amount of CD4+ cells. In the present specification, the "cell ratio" refers to the amount of the cell subpopulation, for example, "CD62L LOW CD4 + "T cell ratio" refers to CD62L LOW CD4 + This refers to the amount of a T cell subpopulation.

[0022] As used herein, the term "relative amount" with respect to cells is used interchangeably with "proportion." Typically, the terms "relative amount" and "proportion" refer to the amount of a particular cell population (e.g., CD4 + The number of cells forming a desired cell subpopulation (e.g., CD62L T cell population) relative to the number of cells forming the desired cell subpopulation (e.g., CD62L T cell population). low CD4 + This refers to the number of cells that form a T cell subpopulation.

[0023] As used herein, the term "standard" refers to a comparative amount for determining the increase or decrease of the amount of a marker described herein. When determining the increase or decrease of a certain amount before and after a certain treatment (e.g., radiation therapy), the "standard" may be, for example, the amount before the treatment or a value generally regarded as a guide in the art.

[0024] When used herein to modify a numerical value, the word "about" is used to mean a range of up to ±10% of the stated numerical value.

[0025] As used herein, "radiation" refers to the propagation of energy in space or matter in the form of waves or particles.

[0026] As used herein, "radiation therapy" refers to any treatment that employs radiation exposure.

[0027] (marker) In an embodiment of the present invention, a method is provided in which the composition of cell subpopulations in a subject that has undergone radiation therapy is an indicator of immune activation caused by radiation therapy in the subject. The method may include a step of analyzing the composition of cell subpopulations in a sample. The analysis of the composition of cell subpopulations can be performed by any method described herein or known to those skilled in the art. The method may be in vitro or in silico. In one embodiment of the present invention, the amount of the cell subpopulation is compared with a suitable standard to indicate the presence or absence of immune activation in the subject. In particular, the cell subpopulation may be a cell subpopulation that correlates with dendritic cell stimulation in anti-tumor immune response.

[0028] In one embodiment, the indicator cell subpopulation is CD4+, which correlates with dendritic cell stimulation in anti-tumor immune responses. + For example, in the Examples of the present specification, CD62L low CD4 + Increased abundance of T cell subpopulations has been observed. low CD4 + T cells are responsible for dendritic cell activation in antitumor immunity. CD4 T cells correlate with dendritic cell stimulation in antitumor immune responses. + T cell subpopulations may also be used as indicators of immune activation by radiation therapy.

[0029] CD4 correlates with dendritic cell stimulation in antitumor immune responses + T cell subpopulations include, for example, CD4 + T cell subpopulation, CD4 primed effector T cells + CD4 T cell subpopulation primed by antigen recognition + These include, but are not limited to, T cell subpopulations, and regulatory T cell subpopulations.

[0030] CD4 correlates with dendritic cell stimulation + Examples of T cell subpopulations include, for example, CD62L low CD4+ T cell subpopulations, CCR7 - CD4 + T cell subpopulation, LAG-3 + CD62L low CD4 + T cell subpopulation, ICOS + CD62L low CD4 + T cell subpopulation, CCR4 + CD25 + CD4 + T cell subpopulation, CD45RA - CD4 + T cell subpopulation, CD45RO + CD4 + T cell subpopulation, CD28 + CD62L low CD4 + T cell subpopulation, CD62L high CD25 + CD4 + T cell subpopulation, CD127 + CD25 + CD4 + T cell subpopulation, CD45RA - Foxp3 + CD4 + T cell subpopulations, and Foxp3 + CD25 + CD4 + These include, but are not limited to, T cell subpopulations.

[0031] CD4 correlates with dendritic cell stimulation in antitumor immune responses + T cell subpopulations include, for example, CD62L low CD4 + CD4 T cells may be a subpopulation of T cells. + As a T cell subpopulation, CD62L low CD4 + T cell subpopulations (i.e., CD62L low CD4 + T cell population itself), ICOS + CD4 + T cell subpopulation, ICOS + CD62L low CD4+ T cell subpopulations, PD-1 + CD4 + T cell subpopulations, PD-1 + CD62L low CD4 + T cell subpopulation, LAG-3 + CD4 + T cell subpopulation, LAG-3 + CD62L low CD4 + T cell subpopulation, CD28 + CD4 + T cell subpopulations and CD28 + CD62L low CD4 + These include, but are not limited to, T cell subpopulations.

[0032] In one embodiment, CD4 correlates with dendritic cell stimulation in anti-tumor immune responses. + T cell subpopulations (preferably CD62L low CD4 + T cell population) can be an indicator of progression-free survival (PFS).

[0033] For the above cell subpopulations, instead of or in addition to using the amount of the cell subpopulation as an index, the expression amount of a suitable surface marker molecule in a suitable cell may be used as an index. For example, CD4 + The expression levels of ICOS, PD-1, LAG-3, CD28, and the like expressed in T cells may be used as indicators. More preferably, CD62L low CD4 + The expression levels of ICOS, PD-1, LAG-3, CD28, and the like expressed in T cells may also be used as indicators.

[0034] In one embodiment, the indicator cell subpopulation is a dendritic cell subpopulation that correlates with dendritic cell stimulation in anti-tumor immune responses. For example, in the Examples herein, HLA-DR + CD141 + CD11c +Expansion of a subpopulation of HLA-DR cells has been observed. + It mediates the activation of dendritic cells by T cells. Dendritic cell subpopulations that correlate with dendritic cell stimulation in antitumor immune responses may also be used as indicators of immune activation by radiation therapy. + CD141 + CD123 - This cell population is generally considered to be myeloid dendritic cells (mDCs, DCs important for antitumor immunity) with high Th1 induction ability. However, CD141 and CD123 are generally expressed exclusively. Therefore, in this specification, CD141 + CD11c + As a result, CD123 - CD11c + Note that both identify the same cell population.

[0035] Dendritic cell subpopulations that correlate with dendritic cell stimulation in anti-tumor immune responses include, for example, CD4 + A dendritic cell subpopulation, CD4, increases due to an increase in a cell subpopulation with reduced expression of homing molecules in the T cell population. + CD4 primed effector T cells in the T cell population + Dendritic cell subpopulations, which increase due to the expansion of T cell subpopulations, and CD4 + CD4 primed by antigen recognition in T cell populations + Dendritic cell subpopulations that increase due to an increase in a T cell subpopulation include, but are not limited to, those that increase due to an increase in a T cell subpopulation. + Dendritic cell subpopulation, CD80 + Dendritic cell subpopulation, CD86 + Dendritic cell subpopulations and PD-L1 + Dendritic cell subpopulations include, but are not limited to, myeloid dendritic cells (mDCs, CD141 + CD11c + dendritic cells) and plasmacytoid dendritic cells (pDCs, CD123 + CD11c + dendritic cells).

[0036] Dendritic cell subpopulations that correlate with dendritic cell stimulation in antitumor immune responses include HLA-DR + CD141 + CD11c + For the above-mentioned cell subpopulations, instead of or in addition to using the amount of the cell subpopulation as an index, the expression amount of a suitable surface marker molecule in a suitable cell may be used as an index. For example, CD141 + CD11c + The expression level of HLA-DR and the like expressed in cells may also be used as an index.

[0037] In one embodiment, the indicator cell subpopulation is CD8, which correlates with dendritic cell stimulation in anti-tumor immune responses. + T cell subpopulations. For example, CD62L low CD8 + The expression of CD137 on T cells changed significantly before and after radiation therapy (Table 4). + Dendritic cells activated by T cells express CD8 + Stimulates T cells and stimulates CD8 + T cells ultimately exert antitumor activity. CD8 + CD137 on T cells mediates CD8 expression by dendritic cells. + Mediates stimulation of T cells. CD8 correlates with dendritic cell stimulation in antitumor immune responses + T cell subpopulations may also be used as indicators of immune activation by radiation therapy.

[0038] CD8 correlates with dendritic cell stimulation in antitumor immune responses + T cell subpopulations include, for example, CD4 + Increased CD8 expression in T cell populations due to an increase in a subpopulation of cells with reduced expression of homing molecules + T cell subpopulation, CD4 + CD4 primed effector T cells in the T cell population + Increased CD8 due to expansion of T cell subpopulations +T cell subpopulation, CD4 + CD4 primed by antigen recognition in T cell populations + Increased CD8 due to expansion of T cell subpopulations + HLA-DR in T cell and dendritic cell subpopulations + Increased CD8 due to expansion of dendritic cell subpopulations + CD80 in T cell and dendritic cell subpopulations + Increased CD8 due to expansion of dendritic cell subpopulations + PD-L1 in T cell and dendritic cell subpopulations + Increased CD8 due to expansion of dendritic cell subpopulations + Additionally, CD8 T cell subpopulations, which correlate with dendritic cell stimulation in anti-tumor immune responses, are included, but are not limited to these. + T cell subpopulations include, for example, CD62L low CD8 + T cell subpopulation, CD137 + CD8 + T cell subpopulations, and CD28 + CD62L low CD8 + These include, but are not limited to, T cell subpopulations.

[0039] CD8 correlates with dendritic cell stimulation in antitumor immune responses + As a T cell subpopulation, CD137 + CD62L low CD8 + For the above cell subpopulations, instead of or in addition to using the amount of the cell subpopulation as an index, the expression amount of a suitable surface marker molecule in a suitable cell may be used as an index. For example, CD62L low CD8 + The expression levels of CD137, PD-1, CD28, and the like expressed on T cells may also be used as indicators.

[0040] Myeloid-derived immunosuppressive cells (MDSC, CD33 + CD14 + HLA-DR -Significant changes in the cell population (cell population) before and after radiation therapy have been observed, and the proportion of such cell population can also be used as an indicator.

[0041] The amount of the cell subpopulations described herein can be used as an index by combining multiple amounts. Combining the indices can make the prediction of responsiveness more accurate. In one embodiment, CD4+, which correlates with dendritic cell stimulation in the anti-tumor immune response in the sample, is used. + The abundance of T cell subpopulations and CD8 correlates with dendritic cell stimulation in antitumor immune responses. + The presence or absence of immune activation in a subject can be indicated by comparing the amount of at least two selected from the group consisting of the amount of T cell subpopulations and a reference. For example, in one embodiment, the amount of CD4 + CD62L on T cells low The percentage of cells (X) and CD4 + CD25 on T cells + FoxP3 + The proportion of (Y) combined with X 2 An index of / Y can be used. It is understood by those skilled in the art that, instead of or in addition to these indicators, further indicators that can indicate immune activation by radiation therapy can be used. Note that, in the present invention, instead of the amount of a desired cell subpopulation, or in addition to the amount of the desired cell subpopulation, the amount of a cell subpopulation contained in the desired cell subpopulation may be used.

[0042] For example, based on the results shown in the Examples of the present specification, the following factors can be used as markers. In the present specification, when the proportion of a cell subpopulation is expressed in the form of %XX / XX, for example, CD62L low / CD4 + T cells are CD4 + CD62L on T cells low CD4 + The numerator refers to the percentage of T cells, where the cells listed in the numerator have all the characteristics of the cells listed in the denominator. If no denominator is specified for a cell subpopulation, CD4 + T cell population or CD8+ Percentages relative to any cell population that includes the cell subpopulation, such as a T cell population, may be included.

[0043] Among the factors that are significantly changed by radiation therapy (RT) and are thought to be related to the T cell immune boost, the factors that increase immediately after the end of RT are: ·%CD62L low / CD4 + T cells (CD4 + CD62L on T cells low CD4 + percentage of T cells); %LAG3 + / CD62L low CD4 + T cells (LAG3 + / Effector Memory (CCR7 - CD45RA - )CD4 + (which may be T cells); ·%ICOS + / CD62L low CD4 + T cells (%ICOS + / Effector Memory (CCR7 - CD45RA - )CD4 + (which may be T cells); %CD28 + / CD62L low CD4 + T cells (%CD28 + / Effector Memory (CCR7 - CD45RA - )CD4 + T cells); and ·%HLA-DR + / CD11c + CD141 + Cells (HLA-DR high CD11c + CD123 - mDC, %mDC / CD3 - CD14 - CD19 - (Cells may also be used) %PD-1 + / CD62Llow CD4 + T cells ·%CD62L low CCR7 + CD45RA - / CD4 + T cells (CCR7 + CD45RA - CD4 + (Cells may also be used) ·%CCR7 - CD45RA - CD4 + T cells ·%CCR7 - CD45RA - CD8 + T cells (EM CD8 + T cells) ·%CCR7 + CD45RA - CD8 + T cells (CM CD8 + T cells) Factors that decrease immediately after RT are: ·%CCR7 + CD45RA + CD4 + Cells (naive CD4 + T cells) ·%CCR7 - CD45RA + CD8 + T cells (EMRA CD8 + T cells) Examples include:

[0044] In addition, factors that increase within one month after the end of RT include: %PD-1 + / CD62L low CD4 + T cells (%PD-1 + / Effector Memory (CCR7 - CD45RA - )CD4 + (which may be T cells); ·%CD137 + / CD62L low CD4 + T cells (%CD137+ / Effector Memory (CCR7 - CD45RA - )CD4 + (which may be T cells); %PD-1 + / CD62L low CD8 + T cells (%PD-1 + / Effector (CCR7 + CD45RA - )CD8 + T cells, %PD-1 + / Effector Memory (CCR7 - CD45RA - )CD8 + T cells); and ·%CD137 + / CD62L low CD8 + T cells (%CD137 + / Effector (CCR7 + CD45RA - )CD8 + T cells, %CD137 + / Effector Memory (CCR7 - CD45RA - )CD8 + T cells may also be used) Examples include:

[0045] Furthermore, factors that were significantly increased by radiation therapy and are thought to be related to T cell immunosuppression include: %CD25 + FoxP3 + / CD4 + T cells (regulatory T cells); and %CD33 + CD14 + HLA-DR low Cells (Myeloid-derived suppressor (MDSC)) Although not wishing to be bound by theory, these are considered to be negative feedback that suppresses activated cellular immunity. They are considered to be useful as factors for evaluating the immune status after radiation therapy.

[0046] In the present invention, the amount of cell subpopulation can be compared with a suitable standard, and the presence or absence of immune activation in the subject can be determined by comparison. The amount of cell subpopulation in the sample is increased from the standard, which can indicate that immune activation caused by radiation therapy has occurred in the subject. Alternatively, the amount of cell subpopulation in the sample is not increased from the standard, which can indicate that immune activation caused by radiation therapy has not occurred in the subject.

[0047] The standard may be, for example, the amount of the corresponding cell subpopulation in a sample of a subject before radiation therapy, but is not limited thereto. In addition, the standard may be a value experimentally calculated from a sample of a subject not receiving radiation therapy. The standard may be, for example, a value statistically calculated by regression analysis or the like based on data obtained from samples of multiple subjects. The standard may be calculated from data obtained from a subject by machine learning or artificial intelligence or the like.

[0048] The increase compared to the standard can be indicated by the amount of the cell subpopulation after radiation therapy being greater than the standard, or being increased by more than 1, 2, 3, 4, 5, 10, 15, 20, 30% of the standard, or being increased by more than 1.5, 2, 3, 5 times the standard. Typically, an increase compared to the standard is considered to occur if the value of the standard is exceeded. When the standard is experimentally calculated, an increase compared to the standard can be observed when an increase that exceeds an appropriate error from the standard value is observed. Suitable errors include, for example, 1 standard deviation, 2 standard deviations, 3 standard deviations, or more.

[0049] (Cell fractionation and separation) Samples for fractionation and separation of T cells can be appropriately collected from a subject by a conventional method. For example, samples can be collected from the subject's peripheral blood, bone marrow, tumor tissue, hematopoietic tissue, spleen, normal tissue, lymph, etc. Collection of samples from peripheral blood can be advantageous because it is non-invasive and simple.

[0050] The composition of T cells in a sample of a subject can be measured by a person skilled in the art using a conventional method. Usually, the number of cells positive for a marker (e.g., CD4) that defines a target cell subpopulation in a sample can be measured using flow cytometry or the like. The composition of a cell population is generally measured using flow cytometry, but may also be measured using immunostaining of a sample containing cells, a method using an antibody array, protein expression analysis in a sample containing cells (e.g., Western blot, mass spectrometry, HPLC, etc.), mRNA expression analysis in a sample containing cells (e.g., microarray, next-generation sequencing, etc.), etc.

[0051] CD62L low CD4 + To count the number of each cell subpopulation, such as a T cell subpopulation, cells other than the respective cell subpopulation may be experimentally removed from the total cells. Kits that allow this are available. For example, the CD4+ Effector Memory T cell Isolation Kit, Human (Militenyi Biotech) can be used to isolate cells corresponding to the CD4+CD62Llow T cell subpopulation from peripheral blood without using CD4 and CD62L antibodies. The total number of live cells is counted and recorded, and the number of cells obtained using this kit is also counted and recorded.

[0052] It is also not necessary to use antibodies. Antibodies can specifically recognize and bind to molecules expressed in individual cells, and when the antibodies bind to molecules expressed on the cell surface or inside the cell, they are colored to detect the number of colored cells. Here, the molecules expressed on the cell surface or inside the cell are proteins, so when the protein is expressed, the mRNA encoding it is also produced inside the cell. In other words, it is sufficient to examine the mRNA in each cell to check the presence or absence of the mRNA encoding the protein molecule of interest. This is made possible by single-cell gene expression analysis, that is, mRNA analysis at the single-cell level. Examples of single-cell gene expression analysis include 1) a method of performing next-generation sequencing using Quartz-Seq, 2) a method of isolating cells using the Fluidigm C1 System or ICELL8 SIngle-Cell System and preparing a library using SMART-Seq v4, 3) a method of separating cells using a cell sorter and measuring by quantitative PCR using the Ambion Single Cell-to-CT kit, and 4) a method using the CyTOF SYSTEM (Helios).

[0053] Blood is collected, the number of live cells is counted, and the cells are separated using a cell sorter or the like. The expression levels of specific genes in each of the separated cells can be measured using a quantitative PCR device, for example, using the Ambion Single Cell-to-CT kit. Based on the results, it can be determined whether each cell is CD62L low CD4 + Determine which subpopulations of T cells the cells belong to, and count the number of cells that belong to each subpopulation. Candidates for genes to be examined for expression include αβTCR, CD3, CD4, CD25, CTLA4, GITR, FoxP3, STAT5, FoxO1, FoxO3, IL-10, TGFbeta, IL-35, SMAD2, SMAD3, SMAD4, CD62Llow, CD44, IL-7R (CD127), IL-15R, CCR7low, BLIMP1, etc.

[0054] For example, CD62Llow CD4 + In T cells, CD62L high CD4 + Genes that are more highly expressed than T cells include AURAKA, CCL17, CD101, CD24, FOXF1, GZMA, GZMH, IL18RAP, IL21, IL5RA, ND2, SMAD5, SMAD7, and VEGFA (WO2018 / 147291, the entirety of which is incorporated herein by reference for all purposes). By examining the expression of these genes, it may be possible to determine which T cell subpopulation the obtained T cells belong to, and to measure the amount and / or proportion of the cell subpopulation.

[0055] Also, CD62L high CD4 + In T cells, CD62L low CD4 + Genes that are more highly expressed than T cells include BACH2, CCL28, CCR7, CD27, CD28, CD62L, CSNK1D, FOXP1, FOXP3, IGF1R, IL16, IL27RA, IL6R, LEF1, MAL, and TCF7 (WO2018 / 147291). By examining the expression of these genes, it is possible to determine which T cell subpopulation the obtained T cells belong to, and to measure the amount and / or proportion of the cell subpopulation.

[0056] In the present invention, the measurement of the proportion of a cell subpopulation or comparison with a threshold value may be performed using a standard sample with a specified signal.The signal between a standard (e.g., particles with fluorescent dye attached) prepared to generate a fluorescent signal corresponding to a specified cell subpopulation and a sample containing a cell population can be compared, and the amount or proportion of the cell subpopulation in the sample can be measured by comparing with the standard.In addition, the signal between a standard (e.g., particles with fluorescent dye attached) prepared to generate a fluorescent signal corresponding to a specified threshold and a sample containing a cell population can be compared, and the presence or absence or amount of the marker of the present invention in the T cell composition in the sample can be determined by comparing with the standard.

[0057] In the present invention, when determining whether a particular marker is high (high expression) or low (low expression), a person skilled in the art can use classification criteria for expression intensity commonly used in the art. For example, for CD62L, the signal intensity corresponding to the 10E2 signal when a PE-labeled anti-human CD62L antibody is used as the boundary, low and CD62L high It is possible to clearly separate the above (WO2018 / 147291).

[0058] (Radiation Therapy) In an embodiment of the present invention, an index of immune activation by radiation therapy is provided. In radiation therapy, radiation is applied to destroy DNA or RNA in cancer cells, inhibiting cell division and / or inducing apoptosis (cell death), thereby reducing the number of cancer cells. In general, tissues are irradiated with radiation in divided doses (approximately 2 Gy per day) up to the limit of the dose tolerated by normal cells (approximately 50 to 60 Gy). Normal cells survive by repairing the destruction of their genes, but cancer cells, whose self-repair function is slower than that of normal cells, are irradiated again before repairing the destroyed genes, and are unable to repair their genes, leading to the induction of cell death. This allows tumor regression to be achieved in the irradiation field.

[0059] In radiation therapy, in addition to tumor regression within the radiation field, tumor regression outside the radiation field has been reported, and is called the abscopal effect. The tumor regression outside the radiation field cannot be explained by the above-mentioned suppression of cancer cell proliferation and cell death due to radiation, and was thought to be an action mediated by some kind of immune system activation, but the detailed mechanism is unclear. It is thought that the effectiveness of cancer immunotherapy that utilizes anti-tumor immunity can be increased by activating the immune system through radiation therapy, but a biomarker for confirming whether the abscopal effect occurs in a subject who has received radiation therapy has not been found. In this specification, a biomarker that indicates immune activation (abscopal effect) that affects outside the radiation field in a subject who has received radiation therapy is provided.

[0060] Radiation can be broadly divided into two types: electromagnetic waves and particle beams. Electromagnetic waves include X-rays and gamma rays. Particle beams are material particles that flow with high kinetic energy, and examples of such beams include alpha rays, beta rays, neutron beams, proton beams, heavy ion beams, and meson beams.

[0061] Radiation therapy can be divided into two types: "external irradiation," which applies radiation from outside the body, and "internal irradiation," which applies radiation to the cancer and its surroundings from inside the body. It is also possible to combine external and internal irradiation.

[0062] External beam radiation is delivered from outside the body through the skin. The most common method is to deliver high-energy X-rays. There are various types of external beam radiation, including, but not limited to, X-ray radiation from a linac (linear accelerator), three-dimensional conformal radiation therapy (3D-CRT), intensity-modulated radiation therapy (IMRT), stereotactic radiation therapy (SRT), particle beam therapy (proton beam therapy and heavy particle beam therapy), and image-guided radiation therapy (IGRT).

[0063] Examples of internal radiation modalities include, but are not limited to, brachytherapy (interstitial irradiation, intracavitary irradiation) or treatment using unsealed radioisotopes (internal therapy).

[0064] The radiation therapy that can be the subject of the present invention is not limited in its form, so long as it is an irradiation that can cause immune activation. For example, the irradiation field in radiation therapy can be an irradiation range that includes tumor tissue. Although not wishing to be bound by theory, it is considered that the immunogenic cell death of tumor cells that have been subjected to radiation therapy is important for the increase of anti-tumor effector T cells. For example, radiation therapy can include chest radiation, radiation to bone metastasis sites, radiation to lymph node metastasis, radiation to adrenal metastasis, radiation to liver metastasis, and radiation to brain metastasis.

[0065] The biomarkers of the present invention can be used to consider the schedule of radiation therapy that is intended to cause immune activation.For example, the absence of immune activation caused by radiation therapy in a subject can indicate that the subject should be administered radiation therapy again.Alternatively, the presence of immune activation caused by radiation therapy in a subject can indicate that radiation therapy should be terminated.

[0066] Radiation therapy can be performed with a dose of about 1 to 3 Gy per session, about 1 to 2 times per day, for 3 to 8 weeks. However, when considering combination with cancer immunotherapy, multiple small doses of radiation can affect immune cells (e.g., T cells), so a hypofractionated radiation method (e.g., multiple large doses of radiation over 1 to 2 weeks) may be preferable.

[0067] In order to reduce the possibility of side effects occurring in radiation therapy, further radiation therapy can be avoided when immune activation is shown to occur. In particular, when the dose per session is increased, it is advantageous to cause immune activation without unnecessary irradiation. Conventionally, it is not possible to monitor when immune activation occurs, and radiation therapy is performed according to a schedule empirically determined in advance, but the biomarker of the present invention allows the appropriate timing for ending radiation therapy to be determined.

[0068] (Cancer immunotherapy) Cancer immunotherapy is a method of treating cancer using the biological defense mechanism of an organism. Cancer immunotherapy can be broadly divided into cancer immunotherapy that strengthens the immune function against cancer and cancer immunotherapy that inhibits the immune evasion function of cancer. Furthermore, cancer immunotherapy can be divided into active immunotherapy that activates the immune function in the body and passive immunotherapy that returns immune cells that have been activated or proliferated outside the body to the body. By showing immune activation caused by radiation therapy using the biomarker of the present invention, it is possible to know the appropriate timing for performing cancer immunotherapy that utilizes immune function.

[0069] Examples of cancer immunotherapy include non-specific immunostimulants, cytokine therapy, cancer vaccine therapy, dendritic cell therapy, adoptive immunotherapy, non-specific lymphocyte therapy, cancer antigen-specific T cell therapy, antibody therapy, and immune checkpoint inhibitor therapy.

[0070] A representative example of an immune checkpoint inhibitor is a PD-1 inhibitor. PD-1 inhibitors include the anti-PD-1 antibody nivolumab (Opdivo). TM These include, but are not limited to, pembrolizumab, spartalizumab, and cemiplimab (sold as serotoninib). In one preferred embodiment, nivolumab may be selected as the target.

[0071] In the present invention, PD-L1 inhibitors can also be used in the same manner as PD-1 inhibitors. Anti-PD-1 antibodies are thought to exert anti-cancer effects by releasing the inhibition of T cell activation by PD-1 signals. Anti-PD-L1 antibodies are also thought to exert anti-cancer effects by releasing the inhibition of T cell activation by PD-1 signals. Although the mechanism by which PD-1 inhibits T cell function has not been fully elucidated, it is believed that when PD-1 (programmed death 1) interacts with PD-L1 or PD-L2, SHP-1 and 2, a type of tyrosine phosphatase, are recruited to the cytoplasmic domain of PD-1, and T cell activation is suppressed by inactivating ZAP70, a T cell receptor signaling protein (Okazaki, T., Chikuma, S., Iwai, Y. et al.: A rheostat for immune responses: the unique properties of PD-1 and their advantages for clinical application. Nat. Immunol., 14, 1212-1218 (2013)). This is thought to be due to SHP-1 and 2 being recruited to the ITSM motif, which dephosphorylates the proximal signaling kinase of the nearby T cell receptor. In other words, it can be said that the memory of the antigen-stimulated T cell is erased.

[0072] PD-1 is expressed at high levels on killer T cells and natural killer cells infiltrating cancer tissues. It is also believed that PD-L1 on tumors attenuates the immune response mediated by PD-1 signals. Although PD-L1 attenuates the immune response mediated by PD-1 signals, blocking the interaction between PD-1 and PD-L1 and / or the signal transduction resulting from the interaction with anti-PD-1 antibodies enhances the antitumor immune response.

[0073] Other examples of immune checkpoint inhibitors include PD-L1 inhibitors (e.g., the anti-PD-L1 antibodies avelumab, durvalumab, or atezolizumab).

[0074] PD-L1 inhibitors bind to the PD-L1 side and inhibit the above-mentioned PD-1 pathway, inhibiting the interaction between PD-1 and PD-L1 and / or the signal transduction resulting from the interaction, thereby generating an anti-tumor immune response.

[0075] Other examples of immune checkpoint inhibitors include CTLA-4 inhibitors (e.g., anti-CTLA-4 antibodies ipilimumab or tremelilumab). CTLA-4 inhibitors activate T cells and generate antitumor immune responses. T cells are activated by the interaction of CD28 on the surface with CD80 or CD86. However, even once activated, it is believed that CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4) expressed on the surface of T cells preferentially interacts with CD80 or CD86 with a higher affinity than CD20, thereby suppressing activation. CTLA-4 inhibitors inhibit CTLA-4, thereby preventing the inhibition of the interaction between CD20 and CD80 or CD86, thereby generating antitumor immune responses.

[0076] In further embodiments, the immune checkpoint inhibitor may target immune checkpoint proteins such as TIM-3 (T-cell immunoglobulin and mucin containing protein-3), LAG-3 (lymphocyte activation gene-3), B7-H3, B7-H4, B7-H5 (VISTA), or TIGIT (T cell immunoreceptor with Ig and ITIM domain).

[0077] The immune checkpoints described above are thought to suppress immune responses to self-tissues, but when antigens such as viruses are present in the body for a long period of time, the immune checkpoints in T cells also increase. As for tumor tissues, they are also antigens that exist in the body for a long period of time, so it is thought that these immune checkpoints evade anti-tumor immune responses, and the immune checkpoint inhibitors described above disable such evasive functions and exert anti-tumor effects.

[0078] In the present invention, immune checkpoint inhibitors can be used in combination with other cancer treatments as appropriate.Other cancer treatments include, but are not limited to, other cancer immunotherapy (e.g., adoptive cell transfer), chemotherapy, hyperthermia, surgical procedures, etc., in addition to radiation therapy.Immune checkpoint inhibitors can be administered in combination with one or more additional agents.One or more additional agents can be any chemotherapy drug, or can include a second immune checkpoint inhibitor.

[0079] In one embodiment of the present invention, a composition comprising an immune checkpoint inhibitor is provided.The composition comprising the immune checkpoint inhibitor of the present invention is usually administered systemically or locally, orally or parenterally.It is believed that the composition comprising the immune checkpoint inhibitor of the present invention can exert a remarkable therapeutic effect by being administered to a subject who is shown to have immune activation caused by radiation therapy by the method described herein.

[0080] The dosage varies depending on age, body weight, symptoms, therapeutic effect, administration method, treatment time, etc., but is usually, for example, orally administered once to several times a day in the range of 0.1 mg to 100 mg per dose per adult, or parenterally administered (preferably intravenously) once to several times a day in the range of 0.01 mg to 30 mg per dose per adult, or continuously administered intravenously for 1 to 24 hours a day. Of course, the dosage varies depending on various conditions, so in some cases a smaller dosage than the above-mentioned dosage is sufficient, and in other cases a dosage exceeding the range is necessary.

[0081] The composition containing the immune checkpoint inhibitor may be administered in the form of a solid preparation for oral administration, a liquid preparation for oral administration, and an injection, an external preparation, a suppository, etc. for parenteral administration. The solid preparation for oral administration includes tablets, pills, capsules, powders, granules, etc. The capsule includes hard capsules and soft capsules.

[0082] The composition of the present invention may be used by mixing one or more active ingredients (e.g., antibodies against immune checkpoint proteins) as is or with excipients (lactose, mannitol, glucose, microcrystalline cellulose, starch, etc.), binders (hydroxypropylcellulose, polyvinylpyrrolidone, magnesium aluminometasilicate, etc.), disintegrants (calcium cellulose glycolate, etc.), lubricants (magnesium stearate, etc.), stabilizers, solubilizers (glutamic acid, aspartic acid, etc.), etc., according to a conventional method. If necessary, it may be coated with a coating agent (sucrose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, etc.), or may be coated with two or more layers. Furthermore, capsules of absorbable substances such as gelatin are also included.

[0083] When the composition of the present invention is formulated as an oral liquid for oral administration, it includes pharma- ceutically acceptable solutions, suspensions, emulsions, syrups, elixirs, etc. In such liquids, one or more active substances are dissolved, suspended, or emulsified in a commonly used diluent (such as purified water, ethanol, or a mixture thereof). In addition, the liquid may contain a wetting agent, a suspending agent, an emulsifying agent, a sweetener, a flavoring agent, an aromatic agent, a preservative, a buffer, etc.

[0084] Injectables for parenteral administration include solutions, suspensions, emulsions, and solid injectables that are dissolved or suspended in a solvent when used. Injectables are used by dissolving, suspending, or emulsifying one or more active substances in a solvent. As the solvent, for example, distilled water for injection, physiological saline, vegetable oil, propylene glycol, polyethylene glycol, alcohols such as ethanol, and combinations thereof are used. Furthermore, the injectables may contain stabilizers, dissolving aids (glutamic acid, aspartic acid, polysorbate 80 (registered trademark), etc.), suspending agents, emulsifying agents, soothing agents, buffering agents, preservatives, etc. These are sterilized in the final process or prepared by an aseptic operation method. Also, a sterile solid preparation, for example a lyophilized product, can be prepared and dissolved in sterilized or sterile distilled water for injection or other solvent before use.

[0085] The composition of the present invention may be packaged in a box or the like together with a package insert that describes that the composition is used in combination with radiation therapy. The package insert may describe that the composition of the present invention is preferably administered within a predetermined period after radiation therapy. The package insert may explicitly indicate that the composition is used in combination with radiation therapy, or may simply mention the possibility of use in combination.

[0086] (cancer) Examples of cancers that are the subject of the present invention include, but are not limited to, melanoma (malignant melanoma), non-small cell lung cancer, renal cell carcinoma, malignant lymphoma (Hodgkin's lymphoma or non-Hodgkin's lymphoma), head and neck cancer, urological cancer (bladder cancer, urothelial carcinoma, prostate cancer), small cell lung cancer, thymic cancer, gastric cancer, esophageal cancer, gastroesophageal junction cancer, liver cancer (hepatocellular carcinoma, intrahepatic cholangiocarcinoma), primary brain tumors (glioblastoma, primary central nervous system lymphoma), malignant pleural mesothelioma, gynecological cancer (ovarian cancer, cervical cancer, uterine cancer), soft tissue sarcoma, biliary tract cancer, multiple myeloma, breast cancer, and colorectal cancer.

[0087] (Combination treatment of radiation therapy and immunotherapy) The subject may be administered radiotherapy, and a sample may be collected from the subject after radiotherapy. The composition of cell subpopulations in the sample may be analyzed to determine whether or not the subject has immune activation due to radiotherapy, as described herein. If necessary, a sample may be collected from the subject before radiotherapy. The composition of cell subpopulations in the sample may be used as a standard for comparison.

[0088] The time point for collecting the sample is not particularly limited. Whether or not immune activation by radiation therapy occurs and how long it lasts varies from subject to subject, and samples can be collected at any time point to examine immune activation at that time point. Changes in cell subpopulations, for example, dendritic cells (CD11c + CD141 + HLA-DR positivity and CD62L low CD4 + The change in ICOS positivity of T cells may continue for 2-3 months. low CD4 +In some cases, the T cell ratio can be maintained for more than a year. The period during which Durvalumab (anti-PD-L1 antibody) can be administered after chemotherapy and radiation therapy and covered by insurance is set as the period from the first day after the end of chemotherapy and radiation therapy as long as there is no progression of the disease, and it is believed that within this period, it is possible to detect changes in immune activation status after radiation therapy and determine whether or not to perform cancer immunotherapy.

[0089] Changes in dendritic cells or T cell subpopulations peaked immediately after radiation therapy, followed by CD62L low CD4 + The proportion of cell subpopulations, such as T cell ratio, tends to return to normal. Therefore, although not essential, by obtaining a sample at a certain time after the time of radiation therapy, the possibility of detecting immune activation for cancer immunotherapy can be increased. For example, the sample can be obtained at a time point, such as, but not limited to, about 1 year, about 6 months, about 3 months, about 2 months, about 4 weeks, or about 14 days after radiation therapy, or immediately after radiation therapy.

[0090] When radiation therapy is administered over a period of time, samples can be obtained from the subject at multiple time points to monitor the immune activation caused by radiation therapy in the subject. For example, if radiation therapy does not cause immune activation, the subject can be administered radiation therapy again. If radiation therapy causes immune activation, radiation therapy can be terminated and cancer immunotherapy can be administered. The multiple time points can be after each radiation dose, after several radiation doses (e.g., 2, 3, 4, or 5 or more), or after several days (e.g., 2, 3, 4, 5, 1, or 2 weeks or more) independent of radiation dose.

[0091] When the biomarker of the present invention indicates that immune activation has occurred, the subject may be administered cancer immunotherapy, but the timing of administering the cancer immunotherapy is not particularly limited as long as the immune activation state is maintained. Since the immune activation state may return to the original state as time passes from the time of radiation therapy, it is not essential, but when immune activation has occurred, the subject may be administered cancer immunotherapy for a certain period from the time of radiation therapy, for example, up to about 4 weeks after the time of radiation therapy or up to about 14 days after the time of radiation therapy. This can increase the possibility of enjoying the benefit of the abscopal effect. In one embodiment, the subject may be administered cancer immunotherapy including an immune checkpoint inhibitor at that time due to the immune activation caused by radiation therapy in the subject.

[0092] In one embodiment, the timing of administering cancer immunotherapy may be determined by further using an immunosuppressive factor. For example, regulatory T cells and MDSCs may increase about one month after radiation therapy, and before the increase in these cell subpopulations (i.e., when the proportion of these cell subpopulations is below a certain level), the subject may be administered cancer immunotherapy containing an immune checkpoint inhibitor.

[0093] The present inventors have demonstrated that radiation therapy induces the production of specific cell subpopulations in immune cells in subjects (e.g., CD4 + CD8 T cell subpopulation correlates with dendritic cell stimulation in antitumor immune responses + It has been found that the amount or proportion of CD4 T cell subpopulations or dendritic cell subpopulations that correlate with dendritic cell stimulation in anti-tumor immune responses is altered. Thus, in one aspect of the present invention, there is provided a method for regulating the proportion of cell subpopulations in a subject by administering radiation therapy to the subject. In particular, radiation therapy can be used to regulate the amount or proportion of CD4 + CD62L low Cell subpopulations within a cell population (e.g., CD4 + CD62L lowThe amount or percentage of a particular cell subpopulation (e.g., CD4 + CD62L low It is believed that certain cell subpopulations (cell subpopulations) responsible for anti-tumor immunity in a subject, and since the amount or proportion of a specific cell subpopulation responsible for such anti-tumor immunity can be changed by radiation therapy, it is believed that radiation therapy can change a subject to a state responsive to cancer immunotherapy (e.g., immune checkpoint inhibitors).

[0094] In one embodiment of the present invention, immune cell compositions that are not responsive to cancer immunotherapy (e.g., low CD4 + CD62L low The present invention provides a method for treating cancer in a subject having a cancer subpopulation (e.g., a tumor cell population) that is not responsive to cancer immunotherapy, the method comprising administering radiation therapy to the subject. The method optionally comprises administering cancer immunotherapy to the subject. The method may also comprise measuring the immune cell composition in the subject. This allows confirmation that the subject has become responsive to cancer immunotherapy through radiation therapy, and allows administration of cancer immunotherapy. The present invention provides a composition for treating cancer comprising an immune checkpoint inhibitor, the composition comprising an immune checkpoint inhibitor for determining whether the immune cell composition is not responsive to cancer immunotherapy (e.g., a tumor cell population) that is not responsive to cancer immunotherapy (e.g., a tumor cell population that is not responsive to cancer immunotherapy). + CD62L low Also provided is a composition for administering to a subject who has had a tumor (amount or percentage of a cell subpopulation) and has undergone radiation therapy. For example, the immune cell composition may be a peripheral blood immune cell composition.

[0095] The immune cell composition that is not responsive (or responsive) to cancer immunotherapy can be determined by those skilled in the art with reference to WO2018 / 147291, etc. For example, the immune cell composition can be determined by measuring the CD4 + CD62L on T cells low When the proportion of T cells is lower than the threshold (non-response group threshold), it is considered that the patient is not responsive to cancer immunotherapy. The threshold can be appropriately determined by those skilled in the art in consideration of sensitivity and specificity. An example of the threshold is CD62Llow CD4 + The ratio is 19.4%. The immune cell composition is CD62L low CD4 + The ratio and regulatory T cells (e.g., CD25 + Foxp3 + CD4 + For example, the CD62L low CD4 + Ratio x CD25 + Foxp3 + CD4 + Let Y be the cell ratio, and X / Y or X 2 In this case, as an example, the ratio (X / Y) may be thresholded at 7.35, and X 2 A threshold of 192 can be set for / Y.

[0096] (kit) In one embodiment of the present invention, a kit is provided for determining whether radiation therapy-induced immune activation occurs in a subject.The kit can include one or more detection agents for molecules suitable for detecting cell subpopulations as described herein.The combination of such detection agents can be used to determine the T cell composition of a subject.Such a kit can be used to measure the proportion of a specific cell subpopulation in a subject as a novel biomarker as described herein.

[0097] In one embodiment of the invention, the kit comprises: (A) CD4 and CD62L; (B) (i) a marker selected from ICOS, PD-1, LAG-3 and CD28, (ii) CD4, and (iii) CD62L; (C) CD11c, CD141 and HLA-DR; (D) CD11c, CD123 and HLA-DR; or (E) CD8, CD62L and CD137 The marker may include a detection agent for the marker. In one embodiment, the detection agent is an antibody. Preferably, the antibody is appropriately labeled to facilitate detection of the marker. EXAMPLES

[0098] Example 1: Indicators of immune activation by radiation therapy (overview) In this example, it is demonstrated that the composition of specific cell subpopulations in a subject is a parameter (ie, a biomarker) that is significantly altered in subjects before and after radiation therapy.

[0099] (material and method) [Subject] In this example, peripheral blood was collected from the following subjects (20 individuals) before and after radiation therapy (and after, if applicable), and the composition of cell populations in the peripheral blood was examined. [Table 1]

[0100] [PBMC analysis] Sampling and PBMC analysis at each time point were performed as follows. BD Vacutina CPT™ blood collection tubes for isolating mononuclear cells were used for PBMC analysis. FCM analysis was performed on PBMC. [Table 2]

[0101] During analysis 1.Myeloid cell analysis After thawing, the cells were stained with antibodies such as CD11c, CD141, HLA-DR, CD33, and CD14, and then analyzed by flow cytometry. 2.T cell analysis 1) Suspend in RPMI1640 + 10% FCS and leave at 37℃ in a 5% CO2 incubator for approximately 36 hours (32-48 hours). 2) Antibody staining for T cell surface markers was performed and flow cytometry analysis was performed.

[0102] (result) The t-statistics were calculated for the proportions of each cell population before and after radiation therapy in 20 subjects using a paired two-tailed Student's t-test. The results are shown in Table 3 below. t-statistics of 0.05 or less are shown in bold and underlined. [Table 3]

[0103] The changes in some cell subpopulations before and after radiation therapy are shown in the figure. + CD62L in cell populations low CD4 + Percentage of cell subpopulations, and CD62L low CD4 + ICOS in cell subpopulations + CD62L low CD4 + The proportion of CD4 cell subpopulations was significantly increased (Figures 1 and 2). + CD62L in cell populations low CD4 + The increase in the proportion of cell subpopulations was observed in each type of radiation therapy (curative TRT: therapeutic chest radiotherapy, curative CRT: therapeutic chemotherapy combined with radiotherapy, palliative TRT: palliative chest radiotherapy) (Figure 3). + CD11c + HLA-DR in cell populations + CD141 + CD11c + The proportion of cell subpopulations was significantly increased (Figure 4).

[0104] For subjects (n=11) who underwent three samplings, t-statistics were calculated for the difference in the proportion of each cell population before, at the completion of, and after radiation therapy using a paired two-tailed Student's t-test. The results are shown in Table 4 below. t-statistics of 0.05 or less are shown in bold and underlined. [Table 4]

[0105] The figure shows the changes in some cell subpopulations before and after radiation therapy. Approximately 30 days after radiation therapy, CD62L low CD4 + Percentage of cell subpopulations and HLA-DR + CD141 + CD11c + The proportions of cell subpopulations tended to return to pre-radiotherapy levels (Figure 5).

[0106] (Consideration) CD62L low CD4 + The T cell subpopulations with strong positive correlation were type 1 helper CD4 + T cells (Th1), effector memory CD4 + T cells, CD8 + T cells, effector CD8 + T cells. These are cell subpopulations that play an important role in cell-mediated immunity, while type 2 helper CD4 + T cells (Th2), regulatory T cells. These are known as cell subpopulations that suppress cellular immunity. low CD4 + The increase in the cell subpopulation is believed to indicate activation of antitumor cellular immunity. + CD141 + CD11c + Dendritic cell subpopulations and CD62L low CD4 + The cell subpopulations are positively correlated. This is because activated dendritic cells express MHC class II-restricted antigens, and CD62L, which recognizes MHC class II-restricted antigens, is expressed by the dendritic cells. low CD4 + This is thought to result in an increase in a subpopulation of CD62L low CD4 + CD4 cell subpopulation correlates with dendritic cell stimulation in tumor immune responses +It is believed to be a T cell subpopulation. + CD141 + CD11c + The LAG3 dendritic cell subpopulation is thought to be a dendritic cell subpopulation that correlates with dendritic cell stimulation in tumor immune responses. + CD62L low CD4 + T cell subpopulation, ICOS + CD62L low CD4 + T cell subpopulation, CD28 + CD62L low CD4 + T cell subpopulations, PD-1 + CD62L low CD4 + T cell subpopulation, CD137 + CD62L low CD4 + T cell subpopulations, PD-1 + CD62L low CD8 + T cell subpopulation, CD137 + CD62L low CD8 + A T cell subpopulation is also thought to be a T cell subpopulation that correlates with dendritic cell stimulation in tumor immune responses (WO2018 / 147291).

[0107] In addition, the results of peripheral blood cell fraction analysis one month after the end of radiation therapy showed that HLA-DR + CD141 + CD11c + CD62L along with a decrease in dendritic cell subpopulations low CD4 + T cell subpopulation, CD28 + CD62L low CD4 + A decrease in the T cell subpopulation was observed. This was due to the disappearance of immunogenic cancer cell death induced by radiation therapy, which led to the suppression of dendritic cell activation, and the increase in CD62L low CD4 + This is understood to be due to a decrease in CD28 expression, which was necessary for interaction with T cell subpopulations and dendritic cells. However, CD62L expression, which was stimulated by cancer antigens from dendritic cells during radiation therapy, was reduced.low CD4 + A subpopulation of T cells expressed CD137 and ICOS and remained in an activated state. + Foxp3 + CD4 + Regulatory T cell subpopulations and CD33 + CD14 + HLA-DR - An increase in the MDSC cell subpopulation is observed. It is well known that regulatory T cells and MDSCs are cells that suppress T cell immunity, and this can be understood as a negative feedback to control activated T cell subpopulations. In the first place, CD62L is an indicator of the T cell immune state that shows the effect of PD-1 inhibitors. low CD4 + It is shown that the T cell subpopulation can be used as the numerator and the regulatory T cell subpopulation can be used as the denominator.

[0108] The above results suggest that immune activation by radiation therapy can be evaluated by using T cell subpopulations that correlate with dendritic cell stimulation in antitumor immune responses and / or dendritic cell subpopulations that correlate with dendritic cell stimulation in antitumor immune responses.

[0109] (Example 2: Application of immune activation by radiation therapy) CD4 in peripheral blood + CD62L low Immune checkpoint inhibitors are administered to subjects with a certain or higher T cell subpopulation ratio. In the responder group, CD4 + CD62L low The proportion of T cell subpopulations may be decreased. + CD62L low The subject's responsiveness to immune checkpoint inhibitors is monitored by monitoring the proportion of T cell subpopulations. + CD62L low If the proportion of T cell subpopulations is decreased, the subject is administered radiation therapy. + CD62L lowThe proportion of the T cell subpopulation is increased, restoring responsiveness to immune checkpoint inhibitors in the subject.

[0110] By the procedure described in WO2018 / 147291, CD62L in peripheral blood low CD4 + A subject whose T cell subpopulation ratio is less than the ineffective group threshold and who is determined to be non-responsive to cancer immunotherapy is administered radiation therapy. Then, an immune checkpoint inhibitor is administered to the subject. The CD62L low CD4 + A proportion of T cell subpopulations are expanded and become responsive to immune checkpoint inhibitors.

[0111] Example 3: CD62L low CD4 + Correlation between T cells and effector memory cells Fortessa analysis was performed on 84 samples (20 before and after surgery, 22 before and after EGFR-TKI) from 42 stage I to IV lung cancer patients. + CD62L in T cells low CD4 + The proportion of T cells and CD4 + Effector memory cells in T cells (CCR7 - CD45RA - )CD4 + The relationship with the proportion of T cells was analyzed.

[0112] The results are shown in Figure 6. low CD4 + T cells and CCR7 - CD45RA - CD4 + It is understood that there is a strong correlation between effector memory cells (CCR7 - CD45RA - )CD4 + T cell counts increased by CD62L low CD4 + It is suggested that it can be used as a surrogate for the amount of T cells.

[0113] Example 4: Examination of indicators of immune activation by radiation therapy in different patient populations (overview) Further to the discussion in Example 1, we investigated whether the composition of specific cell subpopulations in different patient populations were parameters (ie, biomarkers) that were significantly altered in subjects before and after radiation therapy.

[0114] (material and method) [Subject] This study was conducted at Saitama Medical University International Medical Center in 56 consecutive patients with locally advanced NSCLC who had received curative chemoradiotherapy (CRT) or thoracic radiotherapy. Twenty-two patients received durvalumab combination therapy after CRT. Peripheral blood samples were collected before and after radiotherapy and, in some cases, approximately 1 month after radiotherapy. PBMCs were cultured in LSR Fortessa TM Sampling at each time point was performed according to the same procedure as in Example 1. The patients and the treatments performed are as follows: [Table 5] [Table 6]

[0115] (result) The results are shown in Figures 7 to 11. "Before RT" in Figures 7 to 11 corresponds to "pre-TRT" in Figures 1 to 6 and indicates the time immediately before radiation therapy (RT). "Post-RT" in Figures 7 to 11 and "post-TRT", "at TRT completion", and "TRT complete" in Figures 1 to 6 all indicate the time immediately after the end of radiation therapy (RT).

[0116] Consistent with the results in Example 1, CD62L was detected in peripheral blood after thoracic radiotherapy. low CD4 + T cells and HLA-DR +A significant increase in the percentage of myeloid dendritic cells was observed (Figure 7a, b). low CD4 + A significant correlation was observed between T cells and progression-free survival (PFS) (Figure 7c). high CD11c + CD123 - mDC" is the HLA-DR high CD11c + CD141 + This is a cell population that corresponds to mDC.

[0117] Figure 8 shows the CD62L expression levels before and after radiation therapy. low CD4 + FIG. 1 shows changes in the expression of immune checkpoint molecules on T cells (paired t-test). Consistent with the results of Example 1 (Table 3), CD62L low CD4 + The expression of LAG-3 and ICOS on T cells was significantly increased (Fig. 8b and c). low CD4 + A significant difference was also observed in the increase in PD-1 expression on T cells (Figure 8a), which may be due to the increased sample number.

[0118] Figure 9 shows the CD45RA-gated CCR7 and CD45RA expression profiles before and after radiation therapy. + Figure 1. Changes in T cell subpopulations based on T cells. CCR7 and CD45RA are generally expressed in naive T cells (CCR7 + CD45RA + ), central memory (CM) T cells (CCR7 + CD45RA - ), effector memory T cells (CCR7 - CD45RA - ) is a cell surface marker that can be used to fractionate

[0119] As shown in Figure 9a, naive CD4 cells were found to decrease before and after radiation therapy. As shown in Figure 9b, central memory cells (CMs), CCR7 + CD45RA - CD4 + A significant increase in T cells was observed. Since central memory cells are cells that once functioned and then became dormant because there was no target to attack, the increase in central memory cells after radiation therapy does not match the logic of the present invention. Therefore, we actually further subpopulated the central memory cell population with CD62L low and CD62L high When we examined the cells separately, we found that CD62L, a cell surface marker characteristic of central memory cells, high CD62L, a characteristic of effector memory cells (EM), is expressed in the IL-16 / IL-2 cell population. low We found that the cell population of CD62L low CCR7 + The group in (Figure 9e) appeared to be CM due to transient expression of CCR7, but the cell group that actually increased was EM. Figures 9c and 9e show that EM increased due to radiation therapy. This result is consistent with Example 3 above.

[0120] Figure 10 shows the %mDC and %CD62L before radiation therapy, at the completion of radiation therapy, and up to about one month after radiation therapy. low CD4 + This figure shows the dynamics of T cell and immune checkpoint molecule expression (one-way ANOVA with Tukey post-hoc analysis). The P value at the top of each figure is the result of the ANOVA analysis. In addition, the p value between two points where significant differences were found by post-hoc analysis is shown with a bar. CD62L low CD4 + T cells began to decrease in half of the patients by 4 weeks after radiation therapy. Although there was no statistically significant difference between the time of completion of radiation therapy and about one month after radiation therapy, the number of T cells decreased over time in about half of the patients. As with the results in Table 4 in Example 1, the number of mDCs and CD62L increased after radiation therapy.low CD4 + T cells, CD62L low CD4 + The ICOS and PD-1 expression on T cells tended to decrease over the next month, indicating that the immune function against tumors weakens after activation. It is therefore advisable to start administering immune checkpoint inhibitors before the decrease in these cells.

[0121] Figure 11 shows the CD8+ / CD45RA-gated CCR7+ / CD45RA+ cell population before and after radiation therapy. + FIG. 1 shows changes in T cell subpopulations based on T cells.

[0122] Figure 1 shows the CD8 + It was shown that there was no significant change in the amount of cell population, and that the cells that actually have the ability to kill cancer cells are CD8 + In radiation therapy, CD8 + We analyzed whether there were changes in specific subpopulations of T cell populations by gating on CCR7 and CD45RA. As mentioned above, CCR7 and CD45RA are generally expressed in naive T cells (CCR7 + CD45RA + ), central memory (CM) T cells (CCR7 + CD45RA - ), effector memory T cells (CCR7 - CD45RA - ), a cell surface marker that can be used to differentiate - CD45RA + These cells are classified as terminally differentiated (EMRA) cells.

[0123] The increase in EM (Fig. 11c) and CM (Fig. 11d) and the decrease in EMRA (Fig. 11a) indicate an increase in young EMs and a decrease in senescent EMs. These results indicate that CD8+ cells are also activated by RT.

[0124] (Consideration) During radiation therapy, the immune system becomes activated and immune checkpoint blockade becomes more effective, but over time, the immune system returns to its original state. In an activated immune system, a specific cell population (CD62L low CD4 + The number of cells that differentiate into HLA-DR1, HLA-DR2, HLA-DR3, HLA-DR4, HLA-DR5, HLA-DR6, HLA-DR7, HLA-DR8, HLA-DR9, HLA-DR10, HLA-DR12, HLA-DR13, HLA-DR14, HLA-DR15, HLA-DR16, HLA-DR17, HLA-DR18, HLA-DR19, HLA- high CD11c + CD123 - The number of mDCs (e.g., mDCs) increases, and young EMs in the CD8+ cell population increases. By observing the changes in either of these cell populations, it is possible to determine whether the patient is in a state where immune checkpoint blockade is more effective after radiation therapy. In addition, since this state naturally returns to normal over time, it is also possible to determine whether or not it is necessary to perform radiation therapy again to make the patient in a state where immune checkpoint blockade is more effective.

[0125] In addition, when the analysis was performed after chemotherapy alone, not radiation therapy, (n=60, data not shown), the effector memory type (CCR7 - CD45RA - ) was significantly decreased. - CD45RA + CD8 + (EMRA) cell fraction was slightly increased, although the difference was not significant. low There was no change in the proportion of EM, but there was a slight downward trend. There was no change in dendritic cells. From the above, it appears that with chemotherapy alone, the T cell fraction tends to change in the opposite pattern to that after radiation therapy (with anticancer drug treatment alone, the young EM decreased, and the proportion of the EMRA fraction, which is an older effector, appears to have increased).

[0126] Even when analyzing a group of patients who had only received radiation therapy, the results were similar to those of the chemoradiotherapy (chemotherapy + radiation therapy) group. Therefore, the changes in immune cell composition before and after radiation therapy in this example are thought to be due to radiation therapy.

[0127] As an interpretation of the results of this example: Radiotherapy induces localized immunogenic cancer cell death, activates dendritic cells, and induces proliferation of effector T cells In anticancer drug treatments using chemotherapy agents, the chemotherapy agents cause immunogenic cancer cell death and dendritic cells are activated, but at the same time, the chemotherapy agents and steroids used as antiemetics have a dendritic cell reduction effect, and even if effector T cell proliferation occurs due to dendritic cell activation, T cells that have entered the cell cycle are cytotoxicized by the chemotherapy agents and die (EM fraction reduction effect), and the EMRA population, which has already lost the ability to proliferate, does not enter the cell cycle and is not cytotoxic even when presented with antigen, and is subject to the survival time extension effect of antigen stimulation (EMRA fraction temporary increase effect). The following mechanism is assumed.

[0128] Therefore, it is expected that repeated chemotherapy treatment will eventually exhaust EMRA, which are terminally differentiated effector cells, due to the reduction in young EMs, and that anti-tumor T cell immunity will disappear. For example, Keynote062, a clinical trial for gastric cancer, has shown that long-term treatment with anti-PD-1 antibodies in combination with chemotherapy agents is less effective than treatment with anti-PD-1 antibodies alone. In contrast, radiation therapy is thought to boost anti-tumor T cell immunity by increasing the number of young EMs. This boosting effect may also occur when any of the cell populations associated with the above-mentioned mechanisms (e.g., mDCs or CD62L low It is thought that it will be possible to quantify this by observing the amount of T cell proliferation, etc. [Industrial Applicability]

[0129] The present invention can be used in cancer treatment. The present invention makes it possible to evaluate immune activation caused by radiation therapy, and can be used in cancer immunotherapy (e.g., immune checkpoint inhibitors) in combination with radiation therapy.

[0130] (Note) As described above, the present invention has been illustrated using preferred embodiments of the present invention, but the present invention should not be interpreted as being limited to this embodiment. It is understood that the scope of the present invention should be interpreted only by the claims. It is understood that a person skilled in the art can implement an equivalent scope based on the description of the present invention and technical common sense from the description of specific preferred embodiments of the present invention. It is understood that the patents, patent applications and literature cited in this specification should be incorporated by reference to this specification in the same manner as the contents themselves are specifically described in this specification. This application claims priority to Japanese Patent Application No. 2019-028502 (filed on February 20, 2019) and Japanese Patent Application No. 2019-175852 (filed on September 26, 2019), the entire contents of which are incorporated by reference in this application.

Claims

1. A method for determining whether a composition of a cell subpopulation in a sample obtained from a subject who has undergone radiation therapy is an indicator of immune activation caused by radiation therapy in the subject, comprising: analyzing the composition of cell subpopulations in said sample obtained from said subject; CD4 correlates with dendritic cell stimulation in the anti-tumor immune response in the sample. ＋ Comparing the amount of the T cell subpopulation to a reference indicates the presence or absence of immune activation in the subject; CD4 correlates with dendritic cell stimulation in the anti-tumor immune response ＋ T cell subpopulations are CD62L ｌｏｗ CD4 ＋ A subpopulation of cells within a T cell population, if the amount of the cell subpopulation in the sample is increased from the baseline, this indicates that radiation therapy-induced immune activation has occurred in the subject; if the amount of the cell subpopulation in the sample is not increased from the baseline, this indicates that the subject has not experienced immune activation due to radiation therapy; It is further indicated that if immune activation due to radiation therapy occurs in the subject, the subject should be administered cancer immunotherapy including an immune checkpoint inhibitor at that time; The method, wherein the immune checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, and / or a CTLA-4 inhibitor.

2. The method of claim 1, further comprising indicating that the subject should be administered radiation therapy again if it is shown that radiation therapy has not caused immune activation in the subject.

3. Analyzing the composition of said cell subpopulations in a sample obtained from said re-radiotherapy-treated subject; The method of claim 2, further indicating that if immune activation due to radiation therapy occurs in the subject who has been subjected to radiation therapy again, the subject should be administered a cancer immunotherapy comprising the immune checkpoint inhibitor at that time.

4. Furthermore, the amount of dendritic cell subpopulations correlated with dendritic cell stimulation in anti-tumor immune responses and CD8 correlated with dendritic cell stimulation in anti-tumor immune responses. ＋ The method of claim 1, wherein comparison of an amount selected from the group consisting of the amount of a T cell subpopulation to a reference indicates the presence or absence of immune activation in the subject.

5. CD4 correlates with dendritic cell stimulation in the anti-tumor immune response ＋ T cell subpopulations are CCR7 ＋ CD45RA － CD62L ｌｏｗ CD4 ＋ T cell subpopulation, ICOS ＋ CD62L ｌｏｗ CD4 ＋ T cell subpopulation, LAG3 ＋ CD62L ｌｏｗ CD4 ＋ T cell subpopulation, PD-1 ＋ CD62L ｌｏｗ CD4 ＋ T cell subpopulations or CD28 ＋ CD62L ｌｏｗ CD4 ＋ The method according to any one of claims 1 to 4, which is a T cell subpopulation.

6. The dendritic cell subpopulation that correlates with dendritic cell stimulation in the anti-tumor immune response is HLA-DR ＋ CD141 ＋ CD11c ＋ The method of claim 4, which is a cell subpopulation.

7. CD8 correlates with dendritic cell stimulation in the anti-tumor immune response ＋ T cell subpopulations are CD62L ｌｏｗ CD8 ＋ The method of claim 4, which is a cell subpopulation within a T cell population.

8. CD8 correlates with dendritic cell stimulation in the anti-tumor immune response ＋ T cell subpopulations expressing CD137 ＋ CD62L ｌｏｗ CD8 ＋ The method of claim 7, which is a T cell subpopulation.

9. The method of any one of claims 1 to 8, wherein the sample is a peripheral blood sample.

10. The method of any one of claims 1 to 9, wherein said criterion is the amount of said cell subpopulation in a sample from said subject before said radiotherapy.

11. The method according to any one of claims 1 to 10, wherein the radiation therapy is administered to an area including a tumor.

12. 12. The method of any one of claims 1 to 11, wherein the immune checkpoint inhibitor is a PD-1 inhibitor, a PD-L1 inhibitor, or a combination of a PD-1 inhibitor or a PD-L1 inhibitor with a CTLA-4 inhibitor.

13. The method according to any one of claims 1 to 12, further characterized in that the composition of cell subpopulations in samples obtained from a subject at multiple time points is used as an index for monitoring immune activation due to radiation therapy in the subject, the method comprising a step of analyzing the composition of cell subpopulations in samples obtained from the subject at multiple time points.

14. 1. A composition for treating cancer in a subject, comprising a first immune checkpoint inhibitor, The composition is characterized in that it is administered to a subject who has undergone radiation therapy and in which immune activation has been shown to occur in the subject by the method of any one of claims 1 to 13.

15. The composition of claim 14, wherein the first immune checkpoint inhibitor is a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.

16. 16. The composition of claim 14 or 15, further characterized in that it is administered in combination with one or more additional agents.

17. 17. The composition of claim 16, wherein the additional agent comprises a second immune checkpoint inhibitor, and wherein the first immune checkpoint inhibitor and the second immune checkpoint inhibitor are different.

18. A kit for use in the method according to any one of claims 1 to 13, comprising the composition according to any one of claims 14 to 17 and an attached document stating that the composition is used in combination with radiation therapy.

19. A kit for use in the method according to any one of claims 1 to 13, comprising: (A) CD4 and CD62L and determining whether immune activation due to radiation therapy has occurred in the subject is based on a detection agent for CD4+, which correlates with dendritic cell stimulation in an anti-tumor immune response in a sample obtained from the subject. ＋ A kit, performed by comparing the amount of a T cell subpopulation with a standard.

20. The composition according to any one of claims 14 to 17, wherein the subject had an immune cell composition that was not responsive to cancer immunotherapy before receiving radiation therapy.

21. The immune cell composition that is not responsive to the cancer immunotherapy has a lower CD62L ｌｏｗ CD4 ＋ The composition of claim 20, which is an amount or percentage of a T cell subpopulation.

22. 18. The composition of claim 17, wherein the second immune checkpoint inhibitor comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA4 antibody.

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